Preparation method of cell membrane bionic drug delivery system for targeting atherosclerotic lesion as well as product and application thereof

Abstract

The invention relates to the technical field of cell membrane bionic drug delivery, in particular to a preparation method of a cell membrane bionic drug delivery system targeting atherosclerotic lesion and a product and application of the cell membrane bionic drug delivery system targeting atherosclerotic lesion, and the preparation method comprises the following steps: culturing macrophages, extracting and purifying macrophage membranes, adding an enzyme inhibition compound, and storing; repeatedly extruding the stored macrophage membrane by using a micro extruder with a nano membrane to obtain a macrophage membrane microcapsule; preparing drug-loaded nanoparticles based on PEG-PLGA by using a solvent evaporation method; and finally, respectively injecting the macrophage membrane microcapsules and the drug-loaded nanoparticles from two inlets of the microfluidic electroporation chip, applying a two-stage alternating electric field, treating to obtain a uniformly dispersed mixed solution, and co-extruding the mixed solution by adopting a micro extruder with a nano-membrane to obtain the drug-loaded macrophage membrane micro-capsule micro-fluidic electroporation chip. The cell membrane bionic drug delivery system prepared by the invention has the advantages of biodegradability, drug sustained release, long-acting circulation, stable structure and active and passive dual efficient targeting, and can effectively inhibit the development of atheromatous plaques.

Description

Technical Field

[0001] The present invention relates to the technical field of cell membrane bionic drug delivery, in particular to a preparation method of a cell membrane bionic drug delivery system targeting atherosclerotic lesions, and products and applications thereof. Background Art

[0002] Atherosclerosis is a common cardiovascular disease. It is mainly caused by the deposition of fat, cholesterol and other substances in the arterial walls, forming atherosclerotic plaques, which harden and thicken the arterial walls and narrow the lumen. These plaques are like scale in water pipes, which hinder the normal flow of blood. If it occurs in the coronary arteries of the heart, it may cause angina pectoris and myocardial infarction; if it occurs in the blood vessels of the brain, it may cause serious consequences such as insufficient blood supply to the brain and cerebral infarction. The causes of its formation include bad living habits and disease factors such as high blood pressure, high blood lipids, high blood sugar, and smoking. The various diseases caused by atherosclerosis have kept its mortality rate at a high level, posing a serious threat to human life and health.

[0003] Traditional anti-inflammatory and lipid-lowering drugs do play a certain role in the treatment of atherosclerosis, but they also have some disadvantages, including:

[0004] 1. Muscle damage:

[0005] Some lipid-lowering drugs, especially statins, can cause symptoms of muscle damage such as muscle pain, tenderness, weakness or cramping. In rare cases, they can also cause rhabdomyolysis, a serious adverse reaction.

[0006] 2. Abnormal liver function:

[0007] Some patients may experience a mild increase in transaminase after using lipid-lowering drugs, which may even lead to liver damage in severe cases. Although this situation is relatively rare, it still needs attention.

[0008] 3. Abnormal blood sugar metabolism:

[0009] Long-term or high-dose use of statins may increase the risk of new-onset diabetes, especially in the elderly, which may be related to the potential effect of statins on blood glucose metabolism.

[0010] 4. Gastrointestinal discomfort:

[0011] Some lipid-lowering drugs such as bile acid binding resins may cause gastrointestinal reactions such as nausea, vomiting, constipation, etc., thereby affecting patients' medication compliance.

[0012] In recent years, the bionic strategy of cell membrane-coated nanoparticles has made great progress in the research of targeted drug delivery for various diseases. Cell membrane-coated nanoparticles are a new type of drug delivery carrier and bionic nanomaterial prepared by coating the surface of nanoparticles with cell membranes. Currently, there are two methods for coating nanoparticles with cell membranes: physical extrusion and ultrasonic treatment. Although the above methods can effectively prepare membrane bionic nanoparticles, the physical extrusion process is time-consuming and labor-intensive, and the ultrasonic treatment method will destroy the core nanoparticles. Therefore, how to study an efficient method for preparing membrane bionic nanoparticles in the preparation process of cell membrane bionic drug delivery system shows excellent research value and broad development prospects. Summary of the invention

[0013] In view of the problems existing in the prior art, the present invention provides a preparation method of a cell membrane bionic drug delivery system targeting atherosclerotic lesions, and a product and application thereof.

[0014] To achieve the above object, the present invention provides the following technical solutions:

[0015] A method for preparing a cell membrane bionic drug delivery system targeting atherosclerotic lesions, characterized in that it comprises the following steps:

[0016] a. Cultivate macrophages, extract and purify macrophage membranes, add enzyme inhibitory complexes and store at 2-4°C;

[0017] b. repeatedly extruding the stored macrophage membranes using a micro extruder with a nano-membrane to obtain macrophage membrane microcapsules;

[0018] c. Preparation of PEG-PLGA-based drug-loaded nanoparticles using solvent evaporation method;

[0019] d. Finally, the macrophage membrane microcapsules and drug-loaded nanoparticles are injected from the two inlets of the microfluidic electroporation chip at a mass ratio of (9-10):1, and a two-stage alternating electric field is applied. After treating for 90-120 seconds, a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles is obtained. The mixture is then co-extruded using a micro-extruder with a nanomembrane to finally obtain a well-dispersed cell membrane bionic drug delivery system targeting atherosclerotic lesions.

[0020] As a further technical solution, the specific method for extracting and purifying macrophage membranes in step a is:

[0021] Collect macrophages and prepare a resuspension;

[0022] The resuspension contains 0.012 mol / L Tris, 0.002 mol / L calcium chloride, and the pH is 7.5;

[0023] The collected macrophages were added to the resuspension solution and resuspended to adjust the number of macrophages to 3.0×10 7 / mL, and then add the resuspended solution to a micro homogenizer for homogenization for 10 minutes, and then add sucrose solution to the resuspended solution, stir and mix for 10 minutes to obtain a mixed solution;

[0024] The sucrose concentration in the mixed solution is 0.3 mol / L;

[0025] The mixed solution is subjected to multi-stage centrifugation to finally obtain macrophage membranes;

[0026] The multi-stage centrifugal treatment includes one-stage centrifugal treatment, two-stage centrifugal treatment, three-stage centrifugal treatment, and four-stage centrifugal treatment;

[0027] The first stage of centrifugation is as follows: centrifugation at 4°C, 1800×g for 10 min, taking the supernatant to obtain a first stage of supernatant;

[0028] The first supernatant was subjected to a second centrifugation treatment: 4°C, 2500×g for 10 min, and the supernatant was taken to obtain the second supernatant;

[0029] The second stage supernatant was subjected to three stages of centrifugation: 4°C, 3500×g for 25 min, the supernatant was discarded, and the precipitate was retained;

[0030] Four-stage centrifugation treatment: add the precipitate to the resuspension at a mass ratio of 1:50, centrifuge at 4°C, 3800×g for 30 min, discard the supernatant, retain the precipitate, and obtain the macrophage membrane.

[0031] As a further technical solution, the enzyme inhibition complex in step a is prepared by mixing a protease inhibitor and a phosphatase inhibitor in a mass ratio of (2-3):1.

[0032] As a further technical solution, the preparation method of the PEG-PLGA drug-loaded nanoparticles in step c is as follows: 10 to 15 mL of ultrapure water containing 2 to 2.5% (v / v) Tween 80 is placed in a constant temperature magnetic stirrer and stirred to obtain an aqueous solution; then 100 mg of PEG-PLGA and 5 mg of rapamycin are added to 5 to 7 mL of acetone, and the mixture is continuously shaken by a vortex instrument to be evenly mixed to obtain an organic phase mixed solution; the organic phase mixed solution is added dropwise to the above aqueous solution, and then stirred continuously, and then transferred to an ultrasonic cell disruptor for ultrasonic dispersion, and the acetone is completely removed by a rotary evaporator to obtain a colloidal solution, and finally the colloidal solution is centrifuged to obtain a supernatant solution to remove free drugs, and then the colloidal solution is washed and concentrated three times by ultracentrifugation using an ultrafiltration tube to obtain the obtained solution.

[0033] As a further technical solution, the rotation speed of the rotary evaporator is 120-150 rpm, the evaporation temperature is 40-43° C., and the processing time is 100-120 min.

[0034] As a further technical solution, the two-stage alternating electric field in step d includes a first stage alternating electric field and a second stage alternating electric field;

[0035] The frequency of the first alternating electric field is 30-35kHz, and the field strength is 2.0-2.2kV / cm;

[0036] The frequency of the second alternating electric field is 40-45kHz, and the field strength is 2.5-2.8kV / cm;

[0037] The duration of the first alternating electric field treatment is the same as that of the second alternating electric field treatment.

[0038] As a further technical solution, the nanofilm inside the micro extruder in step b and step d are respectively 1 μm, 350 nm and 180 nm.

[0039] As a further technical solution, a cell membrane bionic drug delivery system targeting atherosclerotic lesions is prepared by the preparation method.

[0040] As a further technical solution, the cell membrane bionic drug delivery system is used in the preparation of anti-atherosclerosis drug delivery system.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The cell membrane bionic drug delivery system targeting atherosclerotic lesions of the present invention uses macrophage membrane, a natural cell component, as a wrapping material, which is highly compatible with the physiological environment of the human body; because the membrane structure, protein and sugar derived from the cell are used, the drug delivery system of the present invention can escape the excessive recognition of the immune system, reduce immune rejection and potential toxic side effects. The surface of the macrophage membrane extracted and purified by the present invention retains specific receptors or ligands, and these molecules can bind to the corresponding receptors at the atherosclerotic lesions, can achieve accurate positioning, greatly improve the utilization rate of the drug, and can promote the drug to act more directly on the lesion through an active targeting mechanism, reduce the distribution in non-target tissues, and thus improve the utilization efficiency and therapeutic effect of the drug. Because the extracted macrophage membrane-coated nanoparticles have the ability to escape immune clearance, they can stably exist in the blood and circulate for a long time. The cell membrane bionic drug delivery system prepared by the present invention can achieve the slow release of the drug, so that the drug maintains an effective concentration for a long time, thereby enhancing the therapeutic effect and reducing the frequency of administration.

[0043] The cell membrane bionic drug delivery system prepared by the present invention can completely inherit the membrane surface function of the source macrophages, has the advantages of biodegradability, sustained drug release, long-term circulation, stable structure, and active and passive dual efficient targeting, and can effectively inhibit the development of atherosclerotic plaques, thereby achieving safe, stable and efficient treatment of atherosclerosis.

[0044] In the preparation process of the present invention, an enzyme inhibition complex is added after the macrophage membrane is extracted and purified, thereby maintaining the biological activity of the macrophage membrane protein and prolonging its storage time; and the cell membrane-coated nanoparticles utilize microfluidic electroporation technology to cause recoverable small holes in the macrophage membrane under the action of an external electric field, thereby achieving the introduction of exogenous molecules, thereby effectively promoting the entry of PEG-PLGA drug-loaded nanoparticles into cell membrane vesicles, and the particle size of the cell membrane bionic drug delivery in the aqueous solution is relatively uniform and maintains good dispersion characteristics, and no obvious aggregation reaction occurs between the particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a statistical chart of the encapsulation efficiency of each group of samples in the experiment. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The following are specific embodiments:

[0048] Example 1

[0049] A method for preparing a cell membrane bionic drug delivery system targeting atherosclerotic lesions comprises the following steps:

[0050] a. Cultivate macrophages, extract and purify macrophage membranes, add enzyme inhibition complexes and store at 3°C; RAW264.7 macrophage cell line is used as the source of macrophage membranes, and the culture medium is a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody;

[0051] The specific method for extracting and purifying macrophage membrane is as follows:

[0052] Collect macrophages and prepare a resuspension;

[0053] The resuspension contains 0.012 mol / L Tris, 0.002 mol / L calcium chloride, and the pH is 7.5;

[0054] The collected macrophages were added to the resuspension solution and resuspended to adjust the number of macrophages to 3.0×10 7 / mL, and then add the resuspended solution to a micro homogenizer for homogenization for 10 minutes, and then add sucrose solution to the resuspended solution, stir and mix for 10 minutes to obtain a mixed solution;

[0055] The sucrose concentration in the mixed solution is 0.3 mol / L;

[0056] The mixed solution is subjected to multi-stage centrifugation to finally obtain macrophage membranes;

[0057] The multi-stage centrifugal treatment includes one-stage centrifugal treatment, two-stage centrifugal treatment, three-stage centrifugal treatment, and four-stage centrifugal treatment;

[0058] The first stage of centrifugation is as follows: centrifugation at 4°C, 1800×g for 10 min, taking the supernatant to obtain a first stage of supernatant;

[0059] The first supernatant was subjected to a second centrifugation treatment: 4°C, 2500×g for 10 min, and the supernatant was taken to obtain the second supernatant;

[0060] The second stage supernatant was subjected to three stages of centrifugation: 4°C, 3500×g for 25 min, the supernatant was discarded, and the precipitate was retained;

[0061] Four-stage centrifugation treatment: add the precipitate to the resuspension at a mass ratio of 1:50, centrifuge at 4°C, 3800×g for 30 min, discard the supernatant, keep the precipitate, and obtain the macrophage membrane;

[0062] b. repeatedly extruding the stored macrophage membranes using a micro extruder with a nano-membrane to obtain macrophage membrane microcapsules;

[0063] c. Preparation of PEG-PLGA-based drug-loaded nanoparticles using solvent evaporation method;

[0064] d. Finally, the macrophage membrane microcapsules and drug-loaded nanoparticles were injected from the two inlets of the microfluidic electroporation chip at a mass ratio of 10:1, and a two-stage alternating electric field was applied. After 110 seconds of treatment, a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles was obtained. The mixture was then co-extruded using a micro-extruder with a nanomembrane to finally obtain a well-dispersed cell membrane bionic drug delivery system targeting atherosclerotic lesions.

[0065] The enzyme inhibition complex is formed by mixing a protease inhibitor and a phosphatase inhibitor in a mass ratio of 3:1; the preparation method of the PEG-PLGA drug-loaded nanoparticles in step c is as follows: 12 mL of ultrapure water containing 2.3% (v / v) Tween 80 is placed in a constant temperature magnetic stirrer and stirred to obtain an aqueous solution; then 100 mg of PEG-PLGA and 5 mg of rapamycin are added to 6 mL of acetone, and the mixture is continuously shaken by a vortexer to mix them evenly to obtain an organic phase mixed solution; the organic phase mixed solution is added dropwise to the above aqueous solution, and then stirred continuously, and then transferred to an ultrasonic cell disruptor for ultrasonic dispersion, and the acetone is completely removed by a rotary evaporator to obtain a colloidal solution, and finally the colloidal solution is centrifuged to obtain a supernatant solution to remove free drugs, and then the colloidal solution is concentrated three times by ultrafiltration tube ultracentrifugation washing; the rotation speed of the rotary evaporator is 130 rpm, the evaporation temperature is 42° C., and the treatment time is 110 min; the two-stage alternating electric field in step d includes a first alternating electric field and a second alternating electric field;

[0066] The frequency of the first alternating electric field is 30kHz and the field strength is 2.0kV / cm;

[0067] The frequency of the second alternating electric field is 40kHz and the field strength is 2.5kV / cm;

[0068] The duration of the first alternating electric field treatment is the same as that of the second alternating electric field treatment.

[0069] Example 2

[0070] Example 2 is basically the same as Example 1, except that in step a, the enzyme inhibition complex is formed by mixing a protease inhibitor and a phosphatase inhibitor in a mass ratio of 2:1, and the enzyme inhibition complex is stored at 2°C; in step c, the preparation method of PEG-PLGA drug-loaded nanoparticles is as follows: 10 mL of ultrapure water containing 2% (v / v) Tween 80 is placed in a constant temperature magnetic stirrer and stirred to obtain an aqueous solution; then 100 mg of PEG-PLGA and 5 mg of rapamycin are added to 5 mL of acetone, and the mixture is continuously shaken by a vortex instrument to obtain an organic phase mixture; the organic phase mixture is added dropwise to the above aqueous solution. After that, continue stirring, and then transfer to an ultrasonic cell disruptor for ultrasonic dispersion, use a rotary evaporator to completely remove acetone to obtain a colloidal solution, finally centrifuge the colloidal solution to obtain a supernatant solution to remove free drugs, and then use an ultrafiltration tube to wash and concentrate three times to obtain the product; the speed of the rotary evaporator is 120rpm, the evaporation temperature is 40°C, and the treatment time is 100min; in step d, the macrophage membrane microcapsules and the drug-loaded nanoparticles are respectively injected from the two inlets of the microfluidic electroporation chip at a mass ratio of 9:1, and then a two-stage alternating electric field is applied for 90s; the frequency of the two-stage alternating electric field is 40kHz, and the field strength is 2.2kV / cm.

[0071] Example 3

[0072] Example 3 is basically the same as Example 1, except that in step a, the enzyme inhibition complex is formed by mixing a protease inhibitor and a phosphatase inhibitor in a mass ratio of 3:1, and the enzyme inhibition complex is stored at 4°C; in step c, the preparation method of PEG-PLGA drug-loaded nanoparticles is as follows: 15 mL of ultrapure water containing 2.5% (v / v) Tween 80 is placed in a constant temperature magnetic stirrer and stirred to obtain an aqueous solution; then 100 mg of PEG-PLGA and 5 mg of rapamycin are added to 7 mL of acetone, and the mixture is continuously shaken by a vortex instrument to obtain an organic phase mixture; the organic phase mixture is added dropwise to the above aqueous solution. After that, continue stirring, and then transfer to an ultrasonic cell disruptor for ultrasonic dispersion, use a rotary evaporator to completely remove acetone to obtain a colloidal solution, finally centrifuge the colloidal solution to obtain a supernatant solution to remove free drugs, and then use an ultrafiltration tube to wash and concentrate three times to obtain the product; the speed of the rotary evaporator is 150 rpm, the evaporation temperature is 43°C, and the treatment time is 120 min; in step d, the macrophage membrane microcapsules and the drug-loaded nanoparticles are respectively injected from the two inlets of the microfluidic electroporation chip at a mass ratio of 10:1, and then a two-stage alternating electric field is applied for 120 s; the frequency of the two-stage alternating electric field is 45 kHz, and the field strength is 2.8 kV / cm.

[0073] Embodiment 4:

[0074] Embodiment 4 is substantially the same as embodiment 1, except that: in step d, the two-stage alternating electric field comprises a first stage alternating electric field and a second stage alternating electric field;

[0075] The frequency of the first alternating electric field is 35kHz and the field strength is 2.2kV / cm;

[0076] The frequency of the second alternating electric field is 45kHz and the field strength is 2.8kV / cm;

[0077] The first alternating electric field treatment time is the same as the second alternating electric field treatment time

[0078] Comparative Example 1: This comparative example is basically the same as Example 1, except that the macrophage membrane microcapsules and drug-loaded nanoparticles in step d are ultrasonically treated (42kHz, 100W) in ice water for 2 minutes at a mass ratio of 10:1 using an ultrasonic instrument to obtain a mixed solution in which the macrophage membrane microcapsules and drug-loaded nanoparticles are evenly dispersed, and the mixed solution is then co-extruded using a micro-extruder with a nanomembrane.

[0079] Comparative Example 2: This comparative example is basically the same as Example 1, except that, in the macrophage membrane extraction and purification process in step a, only one, two and four centrifugation processes are performed.

[0080] Comparative Example 3: This comparative example is basically the same as Example 1, except that only the first stage of alternating electric field treatment is used in step d.

[0081] Test: In order to verify the size, uniformity and surface potential characteristics of the cell membrane bionic drug delivery system, the average particle size, PDI dispersion coefficient and Zeta potential of the bionic nanoparticles were measured using a dynamic light scattering instrument. The results are shown in Table 1.

[0082] Table 1

[0083]

[0084]

[0085] It can be seen from Table 1 that the cell membrane biomimetic drug delivery targeting atherosclerotic lesions prepared in the present invention has a relatively uniform particle size in aqueous solution and maintains good dispersion characteristics, and no obvious aggregation reaction occurs between the particles.

[0086] The drug loading rate and encapsulation rate were determined by referring to the existing technology:

[0087] The drug loading rate and encapsulation rate of the examples and comparative samples were determined by ultraviolet spectrophotometry. Anhydrous ethanol was used as a solvent to prepare a RAP drug mother solution with a concentration of 0.1 mg / mL. After dilution, RAP standard samples with concentrations of 1, 5, 10, 15, and 20 μg / mL were obtained. Anhydrous ethanol solution was used as a blank control, and the absorbance values ​​of different RAP standard solution samples at 278 nm were recorded. Repeat three times to determine the fitting standard curve and equation. In order to determine the encapsulation rate and drug loading rate of the examples and comparative samples, appropriate small samples were taken from the colloidal solution before low-speed centrifugation and the final target nanoparticle solution, and anhydrous ethanol was added in a certain proportion. After vortexing for 30s, ultrasound (80W, 30min) was performed, and the obtained solution was centrifuged (5000rpm, 4°C, 15min) and the supernatant solution to be tested was taken. The nanoparticles without drug loading were treated in the same way as above as a control, and the absorbance of the supernatant solution at 278nm after centrifugation was detected, and W and W0 were calculated according to the standard curve and the dilution ratio. A certain volume of RAP nanodrug aqueous solution was transferred into a plate and freeze-dried using a freeze dryer. The sample was taken out after 48 hours and the mass was measured. Wtotal was calculated based on the volume ratio. Three parallel experimental groups (n=3) were set up and light was avoided throughout the process.

[0088] The calculation formulas of encapsulation efficiency (EE) and drug loading rate (DL) are as follows:

[0089] EE(%)=W / W0×100%;

[0090] DL(%)=W / W 总 ×100%;

[0091] Where W is the RAP content in the nanoparticles, W0 is the RAP content before centrifugation, and W 总 is the total mass of the nanoparticles;

[0092] Table 2

[0093]

[0094]

[0095] It can be seen from Table 2 that the technical solution of the present invention has higher encapsulation efficiency and drug loading rate, which can improve the treatment effect and shorten the treatment cycle.

[0096] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all details in detail, nor do they limit the invention to only the specific embodiments described. Obviously, many modifications and changes can be made according to the content of this specification.

Claims

1. A method for preparing a cell membrane bionic drug delivery system targeting atherosclerotic lesions, characterized in that: The following steps are involved: a. Cultivate macrophages, extract and purify macrophage membranes, add enzyme inhibitory complexes and store at 2-4°C; b. repeatedly extruding the stored macrophage membranes using a micro extruder with a nano-membrane to obtain macrophage membrane microcapsules; c. Preparation of PEG-PLGA-based drug-loaded nanoparticles using solvent evaporation method; d. Finally, the macrophage membrane microcapsules and drug-loaded nanoparticles are injected from the two inlets of the microfluidic electroporation chip at a mass ratio of (9-10):1, and a two-stage alternating electric field is applied. After treating for 90-120 seconds, a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles is obtained. The mixture is then co-extruded using a micro-extruder with a nanomembrane to finally obtain a well-dispersed cell membrane bionic drug delivery system targeting atherosclerotic lesions.

2. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that: The specific method of extracting and purifying macrophage membrane in step a is: Collect macrophages and prepare a resuspension; The resuspension contains 0.012 mol / L Tris, 0.002 mol / L calcium chloride, and the pH is 7.5; The collected macrophages were added to the resuspension solution and resuspended to adjust the number of macrophages to 3.0×10 7 / mL, and then add the resuspended solution to a micro homogenizer for homogenization for 10 minutes, and then add sucrose solution to the resuspended solution, stir and mix for 10 minutes to obtain a mixed solution; The sucrose concentration in the mixed solution is 0.3 mol / L; The mixed solution is subjected to multi-stage centrifugation to finally obtain macrophage membranes; The multi-stage centrifugal treatment includes one-stage centrifugal treatment, two-stage centrifugal treatment, three-stage centrifugal treatment, and four-stage centrifugal treatment; The first stage of centrifugation is as follows: centrifugation at 4°C, 1800×g for 10 min, taking the supernatant to obtain a first stage of supernatant; The first supernatant was subjected to a second centrifugation treatment: 4°C, 2500×g for 10 min, and the supernatant was taken to obtain the second supernatant; The second stage supernatant was subjected to three stages of centrifugation: 4°C, 3500×g for 25 min, the supernatant was discarded, and the precipitate was retained; Four-stage centrifugation treatment: add the precipitate to the resuspension at a mass ratio of 1:50, centrifuge at 4°C, 3800×g for 30 min, discard the supernatant, retain the precipitate, and obtain the macrophage membrane.

3. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that: In the step a, the enzyme inhibition complex is prepared by mixing a protease inhibitor and a phosphatase inhibitor in a mass ratio of (2-3):

1.

4. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that: The preparation method of the PEG-PLGA drug-loaded nanoparticles in step c is as follows: 10-15 mL of ultrapure water containing 2-2.5% (v / v) Tween 80 is placed in a constant temperature magnetic stirrer and stirred to obtain an aqueous solution; then 100 mg of PEG-PLGA and 5 mg of rapamycin are added to 5-7 mL of acetone, and the mixture is continuously shaken by a vortexer to be evenly mixed to obtain an organic phase mixed solution; the organic phase mixed solution is added dropwise to the aqueous solution, and the mixture is continuously stirred, and then transferred to an ultrasonic cell disruptor for ultrasonic dispersion, and the acetone is completely removed by a rotary evaporator to obtain a colloidal solution, and finally the colloidal solution is centrifuged to obtain a supernatant solution to remove free drugs, and then the colloidal solution is washed and concentrated three times by ultracentrifugation by an ultrafiltration tube to obtain the obtained solution.

5. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 4, characterized in that: The rotation speed of the rotary evaporator is 120-150 rpm, the evaporation temperature is 40-43° C., and the processing time is 100-120 min.

6. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that: The two-stage alternating electric field in step d includes a first stage alternating electric field and a second stage alternating electric field; The frequency of the first alternating electric field is 30-35kHz, and the field strength is 2.0-2.2kV / cm; The frequency of the second alternating electric field is 40-45kHz, and the field strength is 2.5-2.8kV / cm; The duration of the first alternating electric field treatment is the same as that of the second alternating electric field treatment.

7. The method for preparing the cell membrane bionic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that: In the steps b and d, the nano-films inside the micro extruder are 1 μm, 350 nm and 180 nm respectively.

8. A cell membrane bionic drug delivery system targeting atherosclerotic lesions prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the cell membrane bionic drug delivery system according to claim 8 in the preparation of an anti-atherosclerosis drug delivery system.

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