Method for preparing cell membrane biomimetic drug delivery system targeting atherosclerotic lesion, and product and use thereof
By using microfluidic electroporation technology and a micro extruder to prepare a cell membrane biomimetic drug delivery system that targets atherosclerotic lesions, the problems of short drug circulation and time-consuming and labor-intensive preparation in traditional methods have been solved, achieving efficient and safe treatment of atherosclerosis.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY (GUANGZHOU SEVERE MATERNAL TREATMENT CENTER GUANGZHOU ROUJI HOSPITAL)
- Filing Date
- 2025-02-21
- Publication Date
- 2026-07-30
AI Technical Summary
Traditional small molecule drugs have short circulation times and significant toxic side effects when treating atherosclerosis. Existing methods for preparing cell membrane biomimetic nanoparticles are time-consuming, labor-intensive, or may damage the core, resulting in low drug utilization.
By employing microfluidic electroporation technology and a micro extruder, combined with enzyme inhibitor complex treatment of macrophage membranes, a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions was prepared. The macrophage membrane was mixed with drug-loaded nanoparticles through an alternating electric field to form a stable targeted drug delivery system.
It achieves biodegradability, sustained drug release, long-lasting circulation, and dual active and passive targeting, effectively inhibiting atherosclerotic plaques and improving treatment efficacy and safety.
Smart Images
Figure PCTCN2025078516-FTAPPB-I100001
Abstract
Description
Preparation method, products and applications of cell membrane biomimetic drug delivery system targeting atherosclerotic lesions Technical Field
[0001] This invention relates to the field of cell membrane biomimetic drug delivery technology, specifically to the preparation method, products, and applications of a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions. Background Technology
[0002] Atherosclerosis is a typical chronic inflammatory disease of blood vessel walls. The rupture of its vulnerable plaques is a major cause of stroke, heart failure, peripheral vascular disease, ischemic heart disease, and other diseases. The mortality rate remains high, posing a serious threat to human life and health.
[0003] Traditional anti-inflammatory and lipid-lowering drugs are commonly used in clinical practice to treat atherosclerosis. However, these small-molecule drugs often have short circulation times in the body and exhibit toxic side effects, resulting in low drug utilization and poor therapeutic efficacy. In recent years, the application of a biomimetic strategy of cell membrane-encapsulated nanoparticles has made significant progress in targeted drug delivery research for various diseases. Cell membrane-encapsulated nanoparticles are novel drug delivery carriers and biomimetic nanomaterials prepared by coating the surface of nanoparticles with cell membranes. Currently, methods for coating nanoparticles with cell membranes include physical extrusion and ultrasonic treatment. While both methods can effectively prepare membrane-inspired biomimetic nanoparticles, physical extrusion is time-consuming and labor-intensive, and ultrasonic treatment can damage the core nanoparticles. Therefore, researching an efficient method for preparing membrane-inspired biomimetic nanoparticles in the fabrication of cell membrane-inspired biomimetic drug delivery systems shows great research value and broad development prospects. Summary of the Invention
[0004] To address the problems existing in the prior art, this invention provides a method for preparing a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions, as well as its products and applications.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] A method for preparing a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions, characterized by comprising the following steps:
[0007] a. Culture macrophages, extract and purify macrophage membranes, add enzyme inhibitory complex and store at 2-4℃;
[0008] b. Macrophage membrane microcapsules are obtained by repeatedly extruding the stored macrophage membranes using a micro extruder with nanomembranes.
[0009] c. Prepare drug-loaded nanoparticles based on PEG-PLGA using a solvent evaporation method;
[0010] d. Finally, macrophage membrane microcapsules and drug-loaded nanoparticles are injected from two inlets of the microfluidic electroporation chip at a mass ratio of (9-10):1. An alternating electric field is then applied, and the mixture is treated for 90-120 seconds to obtain a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles. This mixture is then co-extruded using a micro extruder with a nanomembrane to finally obtain a well-dispersed cell membrane biomimetic drug delivery system targeting atherosclerotic lesions.
[0011] As a further technical solution, the enzyme inhibition complex in step a is composed of a mixture of a protease inhibitor and a phosphatase inhibitor in a mass ratio of (2-3):1.
[0012] As a further technical solution, the preparation method of 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 with a vortex mixer to obtain an organic phase mixture; the organic phase mixture is added dropwise to the above aqueous solution, and stirring is continued, 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. Finally, the colloidal solution is centrifuged to obtain the supernatant to remove free drug, and then washed and concentrated three times by ultrafiltration centrifugation to obtain the final product.
[0013] As a further technical solution, the rotary evaporator has a rotation speed of 120-150 rpm, an evaporation temperature of 40-43℃, and a processing time of 100-120 min.
[0014] As a further technical solution, the frequency of the alternating electric field in step d is 40-45kHz, and the field strength is 2.2-2.8kV / cm.
[0015] As a further technical solution, the nanofilms inside the micro extruder in steps b and d are respectively 1μm, 350nm and 180nm.
[0016] As a further technical solution, a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions was prepared by the preparation method.
[0017] As a further technical solution, a cell membrane biomimetic drug delivery system is applied in the preparation of anti-atherosclerotic drug delivery systems.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] The cell membrane biomimetic drug delivery system prepared by this invention can completely inherit the membrane surface function of the source macrophages. It has the advantages of biodegradability, sustained drug release, long-term circulation, structural stability, and dual active and passive high-efficiency targeting. It can effectively inhibit the development of atherosclerotic plaques, thereby achieving safe, stable and efficient treatment of atherosclerosis.
[0020] In the preparation process of this invention, an enzyme inhibitor complex is added after the macrophage membrane is extracted and purified, thereby maintaining the bioactivity of macrophage membrane proteins and extending their shelf life. Furthermore, the cell membrane-coated nanoparticles are produced using microfluidic electroporation technology, which creates reversible pores in the macrophage membrane under an applied electric field, enabling the introduction of exogenous molecules. This effectively promotes the entry of PEG-PLGA drug-loaded nanoparticles into cell membrane vesicles. The cell membrane biomimetic drug delivery system exhibits relatively uniform particle size in aqueous solution and maintains good dispersion characteristics, with no significant aggregation reaction between particles. Detailed Implementation
[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] The following are specific examples:
[0023] Example 1
[0024] A method for preparing a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions includes the following steps:
[0025] a. Culture macrophages, extract and purify macrophage membranes, add enzyme inhibitory complexes and store at 3°C; RAW264.7 macrophage cell line is used as the source of macrophage membranes, and the culture medium is high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics.
[0026] b. Macrophage membrane microcapsules are obtained by repeatedly extruding the stored macrophage membranes using a micro extruder with nanomembranes.
[0027] c. Prepare drug-loaded nanoparticles based on PEG-PLGA using a solvent evaporation method;
[0028] d. Finally, macrophage membrane microcapsules and drug-loaded nanoparticles were injected from two inlets of the microfluidic electroporation chip at a mass ratio of 10:1. An alternating electric field was then applied, and after treatment for 110 s, a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles was obtained. This mixture was then co-extruded using a micro extruder with a nanomembrane to finally obtain a well-dispersed cell membrane biomimetic drug delivery system targeting atherosclerotic lesions.
[0029] Wherein: the enzyme inhibition complex is composed of a protease inhibitor and a phosphatase inhibitor mixed at a mass ratio of 3:1; the preparation method of 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 with a vortex mixer to obtain an organic phase mixture; the organic phase mixture is added dropwise to the above aqueous solution, and stirring is continued, 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. Finally, the colloidal solution is centrifuged to obtain the supernatant to remove free drug, and then washed and concentrated three times by ultrafiltration tube ultracentrifugation to obtain the final product; the rotation speed of the rotary evaporator is 130 rpm, the evaporation temperature is 42℃, and the processing time is 110 min; the frequency of the alternating electric field in step d is 43 kHz, and the field strength is 2.6 kV / cm.
[0030] Example 2
[0031] Example 2 is basically the same as Example 1, except that in step a, the enzyme inhibition complex is composed of a protease inhibitor and a phosphatase inhibitor mixed at a mass ratio of 2:1, and the enzyme inhibition complex is stored at 2°C; the preparation method of the PEG-PLGA drug-loaded nanoparticles in step c 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 with a vortex mixer to obtain an organic phase mixture; this organic phase mixture is then added dropwise to the above water. After adding the drug to the solution, continue stirring, then transfer to an ultrasonic cell disruptor for ultrasonic dispersion. After completely removing acetone using a rotary evaporator, a colloidal solution is obtained. Finally, the colloidal solution is centrifuged to remove free drug, and then washed and concentrated three times by ultrafiltration tube ultracentrifugation to obtain the final product. The rotary evaporator speed is 120 rpm, the evaporation temperature is 40℃, and the treatment time is 100 min. In step d, macrophage membrane microcapsules and drug-loaded nanoparticles are injected into the two inlets of the microfluidic electroporation chip at a mass ratio of 9:1, and then an alternating electric field is applied for 90 s. The frequency of the alternating electric field is 40 kHz and the field strength is 2.2 kV / cm.
[0032] Example 3
[0033] Example 2 is basically the same as Example 1, except that in step a, the enzyme inhibition complex is composed of a protease inhibitor and a phosphatase inhibitor mixed at a mass ratio of 3:1, and the enzyme inhibition complex is stored at 4°C; the preparation method of the PEG-PLGA drug-loaded nanoparticles in step c 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 with a vortex mixer to obtain an organic phase mixture; this organic phase mixture is then added dropwise to the above-mentioned water. After adding the drug to the solution, continue stirring, then transfer to an ultrasonic cell disruptor for ultrasonic dispersion. After completely removing acetone using a rotary evaporator, a colloidal solution is obtained. Finally, the colloidal solution is centrifuged to remove free drug, and then washed and concentrated three times by ultrafiltration tube ultracentrifugation to obtain the final product. The rotary evaporator speed is 150 rpm, the evaporation temperature is 43℃, and the treatment time is 120 min. In step d, macrophage membrane microcapsules and drug-loaded nanoparticles are injected into the two inlets of the microfluidic electroporation chip at a mass ratio of 10:1, and then an alternating electric field is applied for 120 s. The frequency of the alternating electric field is 45 kHz and the field strength is 2.8 kV / cm.
[0034] Comparative Example 1: This comparative example is basically the same as Example 1, except that in step d, the macrophage membrane microcapsules and drug-loaded nanoparticles are ultrasonically treated in ice water (42kHz, 100W) for 2 minutes at a mass ratio of 10:1 to obtain a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles. The mixture is then co-extruded using a micro extruder with a nanofilm.
[0035] Testing: To verify the size, uniformity, and surface potential characteristics of the cell membrane biomimetic drug delivery system, the average particle size, PDI dispersion coefficient, and Zeta potential of the biomimetic nanoparticles were measured using a dynamic light scattering instrument. The results are shown in Table 1.
[0036] Table 1
[0037] As shown in Table 1, the cell membrane biomimetic drug delivery system for targeting atherosclerotic lesions prepared in this invention has a relatively uniform particle size in aqueous solution and maintains good dispersion characteristics, with no obvious aggregation reaction between particles.
[0038] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.
Claims
1. A method for preparing a cell membrane biomimetic drug delivery system targeting atherosclerotic lesions, characterized in that, Includes the following steps: a. Culture macrophages, extract and purify macrophage membranes, add enzyme inhibitory complex and store at 2-4℃; b. Macrophage membrane microcapsules are obtained by repeatedly extruding the stored macrophage membranes using a micro extruder with nanomembranes. c. Prepare drug-loaded nanoparticles based on PEG-PLGA using a solvent evaporation method; d. Finally, macrophage membrane microcapsules and drug-loaded nanoparticles are injected from two inlets of the microfluidic electroporation chip at a mass ratio of (9-10):
1. An alternating electric field is then applied, and the mixture is treated for 90-120 seconds to obtain a uniformly dispersed mixture of macrophage membrane microcapsules and drug-loaded nanoparticles. This mixture is then co-extruded using a micro extruder with a nanomembrane to finally obtain a well-dispersed cell membrane biomimetic drug delivery system targeting atherosclerotic lesions.
2. The method for preparing the cell membrane biomimetic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that, In step a, the enzyme inhibition complex is composed of a mixture of a protease inhibitor and a phosphatase inhibitor in a mass ratio of (2-3):
1.
3. The method for preparing the cell membrane biomimetic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that, The preparation method of 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 with a vortex mixer to obtain an organic phase mixture; the organic phase mixture is added dropwise to the above aqueous solution, and stirring is continued. Then, it is transferred to an ultrasonic cell disruptor for ultrasonic dispersion, and the acetone is completely removed by a rotary evaporator to obtain a colloidal solution. Finally, the colloidal solution is centrifuged to obtain the supernatant to remove free drug, and then washed and concentrated three times by ultrafiltration centrifugation to obtain the final product.
4. The method for preparing the cell membrane biomimetic drug delivery system targeting atherosclerotic lesions according to claim 3, characterized in that, The rotary evaporator has a rotation speed of 120-150 rpm, an evaporation temperature of 40-43℃, and a processing time of 100-120 min.
5. The method for preparing the cell membrane biomimetic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that, In step d, the frequency of the alternating electric field is 40–45 kHz, and the field strength is 2.2–2.8 kV / cm.
6. The method for preparing the cell membrane biomimetic drug delivery system targeting atherosclerotic lesions according to claim 1, characterized in that, In steps b and d, the nanofilms inside the micro extruder are successively 1μm, 350nm, and 180nm.
7. A cell membrane biomimetic drug delivery system for targeting atherosclerotic lesions prepared by the preparation method according to any one of claims 1 to 6.
8. The application of the cell membrane biomimetic drug delivery system according to claim 7 in the preparation of anti-atherosclerotic drug delivery systems.