Self-driven micro-robot double-drug-loading system as well as preparation method and application thereof

Abstract

The invention relates to a self-driven micro-robot double-drug-loading system and a preparation method and application thereof, and belongs to the technical field of medicine. The self-driven micro-robot double-drug-loading system is composed of a PLGA microneedle and an effervescent base covering the PLGA microneedle. Wherein a connector of self-driven eggshell microparticles and azidation exosome is embedded in a needle cavity of the PLGA microneedle, and vascular endothelial growth factor lipid particles are wrapped in the effervescent base. The self-driven micro-robot double-drug-loading system provided by the invention can skillfully utilize an energy source in a medium to autonomously move, so that direct loading, transportation and delivery of therapeutic drugs at disease parts become possible, the therapeutic effect is remarkably improved, and the whole-body side effects of highly toxic drugs are greatly reduced.

Description

Technical Field

[0001] The invention relates to a self-driven micro-robot dual-drug loading system, a preparation method and application thereof, and belongs to the technical field of medicine. Background Art

[0002] Myocardial infarction, as the leading cause of death from cardiovascular disease worldwide, is accompanied by a sudden decrease in blood supply, followed by myocardial cell loss, ventricular structural remodeling, and the threat of heart failure. In the treatment of acute myocardial infarction, although drugs, catheters, and surgery can significantly improve myocardial blood perfusion, they are not enough to completely prevent the reduction of myocardial cells, left ventricular remodeling, or the worsening of heart failure. In view of this, it is particularly urgent to explore new treatment approaches to minimize cardiac remodeling and prevent cardiac function damage. The transplantation of stem cells and progenitor cells, as an emerging strategy for the treatment of cardiovascular diseases (CVDs), shows great potential. This therapy can replace damaged myocardial cells and enhance the heart's contractile ability, mainly due to the excellent regenerative ability of stem cells and the ability to transform into multiple cell lineages. In particular, the transplantation of mesenchymal stem cells (MSCs) can effectively control the expansion of infarct area, prevent left ventricular remodeling, and maintain key indicators of cardiac function. This discovery undoubtedly brings new hope for the treatment of cardiovascular diseases.

[0003] Although the idea that mesenchymal stem cells (MSCs) can cross lineage boundaries and mimic the morphology and function of cardiomyocytes to achieve myocardial regeneration is widely circulated, many research teams have questioned it because the efficiency of MSCs in the process of transdifferentiation into cardiomyocytes is low, and the survival rate after cell transplantation is also not optimistic.

[0004] As a major mediator of intercellular communication, exosomes are nanoscale membrane vesicles with diameters ranging from 30 to 150 nanometers. The effective information in exosomes mainly contains proteins, lipids and nucleic acids. Increasing evidence shows that active exosomes derived from stem cells act as important communicators between cardiac cells by delivering specific substances to adjacent or distant target cells. In addition, exosomes are stable in target tissues, have a long duration, and can deliver extracellular signaling molecules into cells. Among these exosomes, exosomes derived from mesenchymal stem cells (MSCs) may represent a new type of cell-free therapy, which, in addition to the advantages of anti-cardiac remodeling and restoration of cardiac function, also has no potential risk of pulmonary embolism and low immunogenicity. Even so, it is essential to develop a new drug delivery system to fully realize the potential of exosomes in the treatment of myocardial infarction (MI). As nanoscale carriers, exosomes are easily retained in non-specific organs, especially in the lungs and liver, resulting in insufficient targeting of myocardial ischemic areas. Therefore, how to accurately target exosomes to receptor cells remains a problem.

[0005] Microneedle-assisted drug delivery technology, as an innovative alternative to intramyocardial injection, is being widely used in local and minimally invasive treatment of myocardial infarction (MI). This technology achieves sustained and controlled release of active ingredients by precisely delivering therapeutic materials such as DNA vaccines and subunit antigens to the myocardial infarction area, effectively avoiding many of the difficulties faced by traditional injection drug delivery systems. Not only that, microneedle technology can also penetrate deep into tissues in a minimally invasive manner, providing the possibility of local drug delivery for the treatment of cardiac fibrosis. More strikingly, biocompatible microneedles can also serve as protective patches for damaged hearts, providing mechanical support to prevent heart rupture. It can be seen that microneedle technology provides a unique and effective strategy for local and minimally invasive drug delivery for the treatment of MI, heralding a new chapter in the treatment of cardiovascular diseases in the future. However, although microneedles can be inserted into myocardial tissue by relying on their own characteristics, the depth of insertion is limited by their own length, that is, the depth of insertion into myocardial tissue is the same as the size of the microneedle itself, and there is no way to further extend the depth and width of drug delivery. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In order to solve the above problems in the prior art, the present invention provides a self-driven micro-robot dual-drug loading system, a preparation method and application thereof.

[0008] (II) Technical solution

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] A self-propelled microrobot dual drug delivery system, which consists of a PLGA microneedle and an effervescent base covering the PLGA microneedle, wherein a connector of self-propelled eggshell microparticles and azidated exosomes is embedded in the needle cavity of the PLGA microneedle, and vascular endothelial growth factor lipid particles are wrapped in the effervescent base.

[0011] Furthermore, the self-propelled eggshell microparticles are eggshell microparticles containing shell membranes modified by dibenzocyclooctyne-N-hydroxysuccinimide ester.

[0012] The size of eggshell microparticles is less than 5 microns.

[0013] Among them, the shell membrane is the protein membrane that is tightly attached to the eggshell inside the eggshell.

[0014] Furthermore, the eggshell is a chicken eggshell, a bird eggshell, a duck eggshell, a black chicken eggshell, an ostrich eggshell, a pigeon eggshell or a goose eggshell.

[0015] In the self-propelled microrobot dual drug delivery system as described above, preferably, the exosomes are exosomes of mesenchymal stem cells, exosomes derived from bone marrow mesenchymal stem cells, exosomes derived from endothelial cells or exosomes derived from cardiomyocytes, and further, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0016] In the self-propelled microrobot dual drug delivery system as described above, preferably, the vascular endothelial growth factor lipid particles are vascular endothelial growth factor lipid particles activated by carboxylation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0017] A method for preparing a self-driving micro-robot dual-drug loading system comprises the following steps:

[0018] S1, mesenchymal stem cells were treated with N-azidoacetyl-mannosamine tetraacylation, and then azidated exosomes were extracted;

[0019] S2, drying and grinding the eggshell containing the shell membrane to obtain eggshell microparticles, and modifying the eggshell microparticles with dibenzocyclooctyne-N-hydroxysuccinimide ester to obtain functionalized eggshell microparticles;

[0020] S3, co-incubating the azidated exosomes with the functionalized eggshell microparticles to obtain a connector of the functionalized eggshell microparticles and the azidated exosomes;

[0021] S4, after the vascular endothelial growth factor and the lipid preparation are mixed, the mixture is frozen and thawed, the lipid solution is squeezed out through an extruder, and DLC-VEGF particles are obtained after ultrafiltration, and the DLC-VEGF particles are incubated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to obtain carboxylated vascular endothelial growth factor lipid particles;

[0022] S5. Add the connector of functionalized eggshell microparticles and azidated exosomes into the microneedle casting liquid and add it into the needle cavity of the microneedle mold. After drying, mix the carboxylated vascular endothelial growth factor lipid particles with the effervescent base casting liquid and cover the dried microneedle mold base. After the second drying, take out the microneedle mold to obtain a self-driven microrobot dual drug delivery system.

[0023] In the preparation method as described above, preferably, the final concentration of the N-azidoacetyl-mannosamine tetraacylation is 15-2000 μM, and the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, endothelial cells or cardiomyocytes; and the azidated exosomes are extracted after treating the cells for 1-4 days.

[0024] According to the preparation method as described above, preferably, the particle size of the eggshell microparticles is less than 5 microns, the eggshell microparticles are mixed with water to obtain an eggshell microparticle emulsion, and the modification is carried out using dibenzocyclooctyne-N-hydroxysuccinimide ester, DBCO-PEG-NHS or diphenylcyclooctyne polyethylene glycol active ester and the eggshell microparticle emulsion, wherein the final concentration of dibenzocyclooctyne-N-hydroxysuccinimide ester, DBCO-PEG-NHS or diphenylcyclooctyne polyethylene glycol active ester is 1 to 1000 μM, and the modification is incubated at a temperature of 35 to 40°C for 1 to 3 hours, wherein the molecular weight of PEG can be 400, 600, 1K, 2K, 3.4K, 4K, 5K or 10K.

[0025] In the preparation method as described above, preferably, the mass ratio of the azid exosomes to the functionalized eggshell microparticles is 1:10-50; the concentration of the azid exosomes is 30-500 mg / ml, and the incubation conditions are incubated at a temperature of 35-40° C. for 1-3 hours.

[0026] In the preparation method as described above, preferably, the microneedle casting liquid is a polylactic acid-glycolic acid copolymer solution with a mass concentration of 15 to 50%, wherein the solvent is diethylene glycol dimethyl ether;

[0027] The effervescent base casting solution is a mixture of 13% polyvinylpyrrolidone (PVP), 4% citric acid and 5% sodium bicarbonate in anhydrous ethanol at a final concentration by mass volume;

[0028] The first drying condition is vacuum drying at 50-80°C for 15-60 minutes;

[0029] The second drying condition is 35-45°C for 1-3 days.

[0030] The self-driving microrobot dual-drug loading system and the self-driving microrobot dual-drug loading system obtained by the preparation method thereof as described above are used in the preparation of drugs for myocardial infarction, cerebral infarction and thrombosis.

[0031] (III) Beneficial effects

[0032] The beneficial effects of the present invention are:

[0033] The self-driving microrobot dual drug delivery system provided by the present invention comprises self-propelled eggshell microparticles, which are loaded with exosomes. Furthermore, exosomes derived from MSCs for the treatment of myocardial infarction (MI) can be accurately released and effectively treat myocardial infarction, and can further extend the depth and width of drug delivery. In addition, the bottom of the self-driving microrobot dual drug delivery system is designed as an effervescent base, which releases lipid particles containing VEGF, which can effectively reduce the burden on the heart and activate endothelial cells in the infarcted tissue.

[0034] The self-driving microrobot dual drug-carrying system provided by the present invention can cleverly use the energy source in the medium to move autonomously. This feature makes it possible to directly load, transport and deliver therapeutic drugs at the disease site, significantly improving the therapeutic effect and greatly reducing the systemic side effects of highly toxic drugs; especially after myocardial infarction (MI), the external pH value drops rapidly from 7.4 to 5.9. In this case, the self-driving eggshell microparticles based on calcium carbonate (CaCO 3 ) become an ideal choice for delivering drugs to the infarct area and improving the living environment of myocardial cells. Therefore, the present invention combines self-propelled particles with microneedles to form a powerful and effective drug delivery system for the treatment of infarction, which can accurately target the lesions. This innovation not only broadens the possibilities of drug delivery, but also brings new hope for the treatment of myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of the overall structure of the self-driving micro-robot dual-drug loading system provided by the present invention;

[0036] Figure 2 This is the identification result of the azide exosome preparation in Example 1;

[0037] Figure 3 Identification of functionalized eggshell microparticles and their connection with azide exosomes;

[0038] Figure 4 This is the result of VEGF lipid particle identification;

[0039] Figure 5 The identification results of the microneedles prepared by the present invention;

[0040] Figure 6 This is a picture showing the connection between the microneedle tip and the base;

[0041] Figure 7 The results were proved for the motility of eggshell microparticles;

[0042] Figure 8 This is the result of characterizing the mechanical properties of the microneedle patch;

[0043] Fig. 9 This is a graph showing the degradation characteristics of the microneedle patch;

[0044] Fig.10 This is a photo of microneedles inserted into rat heart tissue;

[0045] Fig.11 Validation results of microneedle insertion into rat heart tissue;

[0046] Fig.12 This is a picture of minimally invasive delivery of MN patch into pig heart using thoracoscopic technique;

[0047] Fig.13 This is a picture of the minimally invasive delivery of the MN patch into the rabbit heart using thoracoscopic technology. DETAILED DESCRIPTION

[0048] The self-driving microrobot dual drug delivery system provided by the present invention consists of two parts: ① PLGA microneedles and ② effervescent base, wherein the PLGA microneedles are embedded with a connector of self-driving eggshell microparticles and exosomes derived from mesenchymal cells; and the effervescent base is wrapped with VEGF lipid particles. The schematic diagram of its construction is shown in the figure below. Figure 1 As shown, azidated exosomes metabolize azidomonosaccharides through mesenchymal stem cells (a), and use protein glycosylation modification to complete azidation modification during the intracellular synthesis of exosomes (b), and are secreted outside the cell to obtain azidated exosomes (N3-exosomes). Eggshell microparticles are modified with DBCO-NHS (d) and connected to N3-exosomes through click chemistry reaction (e). VEGF is packaged in lipid particles. By doping the exosome and eggshell microparticle connector into the microneedle casting liquid (f), and VEGF into the effervescent base casting liquid (g), a dual-drug self-driven microneedle patch is obtained. Specifically, it includes the following steps:

[0049] S1. Construction of azido exosomes: a) azido monosaccharide GLC enters mesenchymal stem cells through cell metabolism, b) mesenchymal stem cells use azido monosaccharide to synthesize azido exosomes intracellularly, and c) azido exosomes are secreted to the extracellular space through exocytosis, thus obtaining azido exosomes.

[0050] S2. Construction of functionalized eggshell microparticles: d) cross-linking the protein membrane surface of the eggshell microparticles with DBCO-NHS to obtain DBCO-eggshell microparticles.

[0051] S3. Connection of azid exosomes to functionalized eggshell microparticles: e) The azid exosomes were connected to DBCO-eggshell microparticles via a click chemistry cycloaddition reaction.

[0052] S4. Construction of vascular endothelial growth factor (VEGF) lipid particles: g) Liposomes were generated from a lipid preparation consisting of 1,2-dioleoyl-SN-glycero-3-phosphocholine (DOPC): phospholipid-polyethylene glycol-carboxyl.

[0053] S5. Construction of self-driven dual-drug-loaded microneedle patch: f) Preparation of microneedle patch, adding the connector of azidated exosomes and functionalized eggshell microparticles into PLGA casting liquid to form PLGA microneedles; adding VEGF lipid particles into the effervescent base casting liquid, and covering the effervescent base casting liquid on the PLGA microneedles, thereby obtaining a self-driven microrobot dual-drug loading system.

[0054] The present invention is the first to modify the exosomes by azidation and connect them with the prepared eggshell microparticles to achieve drug loading of the microrobot. Studies have found that the metabolic modification method of azidomonosaccharides used in the present invention has great advantages: 1. Simple operation; 2. Reduced unnecessary functional interference, which is mainly compared with the in vitro modification of exosomes. Although the exosomes can be extracted first and then chemically cross-linked to obtain azido exosomes, this increases the number of operating steps and the position of the chemical cross-linking is uncontrollable, which is likely to cover the functional area of ​​the exosomes. The metabolic method used in the present invention is the result of active cell selection, which can basically avoid the disadvantages of exosome functional destruction.

[0055] The present invention prepares functionalized eggshell microparticles for the first time, which are loaded with exosomes (MI) derived from MSCs for the treatment of infarction, greatly improving its mechanical permeability and precise release in the infarct tissue. The specific therapeutic effect of eggshell microparticles loaded with exosomes on myocardial infarction is reflected in two aspects: 1. Eggshell microparticles use the weak acid microenvironment of the myocardial infarction site for self-driving while increasing the local pH value, improving the survival microenvironment of myocardial cells; 2. Self-driven microparticles deliver exosomes to deeper and wider areas of myocardial infarction on the basis of microneedles, thereby promoting exosomes to save more myocardial cells and promote vascular reconstruction in a wider area to restore regional blood supply.

[0056] The present invention has developed a self-propelled eggshell microparticle, which is loaded with exosomes derived from MSCs for the treatment of myocardial infarction (MI). In order to improve its mechanical permeability and precise release in the infarcted tissue, these eggshell microparticles loaded with exosomes are further doped in polylactic-co-glycolic acid (PLGA) to synthesize microneedles. In addition, the bottom of the microneedle patch is designed as an effervescent base to release lipid particles containing VEGF, aiming to reduce the burden on the heart and activate endothelial cells in the infarcted tissue. The experimental results show that the effervescent base of the microneedle patch can be rapidly degraded within a few minutes, while the VEGF lipid particles effectively promote the formation of microvessels outside the MI. The eggshell microparticles use the weak acid environment formed after MI to deliver MSCs-derived exosomes to a deeper level, effectively preventing the subsequent fibrosis of MI. The experiment further showed that the eggshell microparticles improved the acidic environment, thereby saving the myocardial cells in the infarcted area. Therefore, the rapidly degradable base microneedle patch based on the self-propelled eggshell microparticle drug loading system provides a new strategy with great potential for the treatment of MI.

[0057] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific implementation methods. The actual products used in the present invention can be commercially available.

[0058] Example 1

[0059] The self-driving microrobot dual drug delivery system consists of two parts: ① PLGA microneedles and ② effervescent base, where the PLGA microneedles are embedded with a connector of self-driving eggshell microparticles and exosomes derived from mesenchymal cells; the effervescent base is wrapped with VEGF lipid particles. The specific preparation method includes the following steps:

[0060] S1. Construction of azide exosomes

[0061] Human umbilical cord mesenchymal stem cells (HMSC, provided by Jiukang Research Institute) were cultured in Dulbecco's modified Eagle medium / Ham's F12 nutrient medium (DMEM / F12, mixed in a volume ratio of 1:1) containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% CO 2 . In order to generate azide-modified exosomes (N3-exosomes), when HMSC cells were confluent with 80% of the culture plate, N-azidoacetyl-mannosamine tetraacylation (Ac4GlcNAz, abbreviated as GLC) was added at a final concentration of 30 μM, and the cells were treated for 3 days to obtain cells (GLC-HMSC). Exosomes were extracted using Invitrogen's total exosome isolation reagent or ultracentrifugation. The purity of the isolated exosomes was determined using the BCA protein analysis kit.

[0062] The HMSC cells and the obtained GLC-HMSC were stained with 4',6-diamino-2-phenylpyridine (DAPI), dibenzocyclooctyne (DBCO)-fluorescein isothiocyanate (DBCO-FITC), and Merge. At the same time, the exosomes (Exosome) and azido exosomes (N-exosomes) were extracted from HMSC and GLC-HMSC, respectively. 3 -Exosome, hereinafter referred to as N 3 -exosomes) were subjected to CD63-FITC and DBCO-FITC fluorescence staining detection, and the results were as follows Figure 1 As shown, (A) shows that after mesenchymal stem cells (HMSC) metabolize azido monosaccharides (i.e., GLC) for 24 hours, the azido group is fluorescently labeled with DBCO-FITC, and the cell nucleus is labeled with a nuclear dye (DAPI). The results show that mesenchymal stem cells that metabolize azido monosaccharides are successfully labeled with DBCO-FITC, while cells that do not metabolize azido monosaccharides are not labeled; the scale bar in the figure is 50μm. The above results show that mesenchymal stem cells successfully metabolize azido monosaccharides. (B) DBCO-FITC probe and CD63-Cy3 (CD63 is a classic expression protein of exosomes) probe are used to label azido groups and exosomes, respectively. The results show that azido exosomes (N 3-Exosomes) were labeled with both DBCO-FITC and CD63-Cy3 probes, while non-azid exosomes (Exosomes) could only be labeled with CD63-Cy3 probes; the scale bar in the figure is 10μm. The above results show that the azide group was successfully modified on the exosomes. (C) Transmission electron microscopy characterization of exosomes; the scale bar in the figure is 100μm. The results show that the azide-modified exosomes are similar to the unlabeled exosomes in terms of morphology and size. (D) Particle size analysis of exosomes was performed using a particle size analyzer (NanoBrook Omni). The results showed that the particle size of azide exosomes was comparable to that of unmodified exosomes, which was about 100 nanometers. (EH) Azide exosomes and unmodified exosomes were identified using nanoflow cytometry. The results showed that azide exosomes could be highly captured by CD63-FITC and DBCO-FITC probes, respectively, confirming the high-efficiency azidation modification of azide exosomes.

[0063] S2. Construction of functionalized eggshell microparticles

[0064] Construction of functionalized eggshell microparticles: d) The protein membrane surface of the eggshell microparticles is cross-linked with DBCO-NHS. After the eggs are cooked, the eggshells are carefully removed from the eggs, ensuring the integrity of the calcium carbonate part on the outside of the eggshell and the protein membrane on the inside. The eggshells are then cut into pieces with a diameter of less than 5 mm and thoroughly dried in an oven. To produce a uniform fine powder, the dried eggshells are pre-cooled in liquid nitrogen for 10 minutes and then ground in a cryogenic grinder at minus 50°C. The resulting powder is mixed with deionized water to form an emulsion, and the emulsion is filtered through a 5μm and then a 2μm microporous membrane using a vacuum filtration device. The resulting filtrate is freeze-dried (-40°C, frozen overnight), and the dried eggshell powder (ESMP) is packaged and stored in a minus 20°C refrigerator.

[0065] In order to functionalize the eggshell microparticles, 100 μmol of dibenzocyclooctyne-N-hydroxysuccinimide ester (DBCO-NHS) was added to the eggshell microparticle emulsion (0.5 g of dried powder was added to 10 mL of water), and the modified eggshell microparticles (DBCO-ESMP) were obtained by incubation at 37°C for two hours. The NHS group reacts with the primary amine group in the protein or polypeptide to form a stable amide bond. The modified functionalized eggshell microparticles (DBCO-ESMP) were then purified by centrifugation (3000 rpm for 3 min, discarding the supernatant and retaining the precipitate) three times and then resuspended in deionized water.

[0066] S3, N 3 - Connection of exosomes to functionalized eggshell microparticles

[0067] N 3 - Connection of exosomes to functionalized eggshell microparticles: e)N 3-Exosomes were connected to DBCO-ESMP via a click chemistry cycloaddition reaction. 3 - Exosomes are concentrated (by centrifugation) to 60 mg / mL and quantified using a BCA protein assay kit. 3 - Exosomes and DBCO-ESMP were incubated at 37°C for 2 hours at a mass ratio of 1:30. Then, 3 to 5 times the volume of excess deionized water was added and centrifuged three times (3000 rpm for 3 min, the supernatant was discarded, and the precipitate was retained) to elute the free exosomes and obtain the connector of functionalized eggshell microparticles and azidated exosomes.

[0068] The results of functionalized eggshell microparticle identification and connection with azide exosomes are as follows Figure 3 The results of electron microscopy of eggshell powder (ESMP) and functionalized eggshell microparticles and anthocyanin-azide (N 3 -Cy3) fluorescence staining results are shown in Figure 3 As shown in the figure, (A) is a bright field microscope photo of unmodified eggshell microparticles (ESMP) and functionalized eggshell microparticles (DBCO-ESMP), and the scale bar in the figure is 25μm. The above results show that the eggshell microparticles are particles with uniform particle size. (B) The functionalized eggshell microparticles were labeled with N3-Cy3 fluorescent probe, and the scale bar in the figure is 25μm. The above results show that the functionalized eggshell microparticles can be captured by the azide probe. (C) The azide exosomes (N3-exo-FITC) were pre-labeled with DBCO-FITC probe, and the functionalized exosomes (ESMP-Cy3) were pre-labeled with N3-Cy3, and then the two were connected. The scale bar in the figure is 20μm. The above results show that the azide exosomes were successfully connected to the functionalized eggshell microparticles. (D) The particle size of eggshell microparticles (ESMP), functionalized eggshell microparticles (DBCO-ESMP), and the conjugate of functionalized eggshell microparticles and azide exosomes (ESMP-exo) was characterized using a particle size analyzer (NanoBrook Omni). The above results show that the particle size of the three is uniform, about 2 μm.

[0069] S4. Construction of vascular endothelial growth factor lipid particles

[0070] Specifically, the lipid preparation (50 μmol) prepared above was dissolved in chloroform and evaporated under a stream of dry nitrogen. The resulting lipid film was then dissolved in a mixture of 1 ml of dimethyl sulfoxide and ethanol (EtOH), i.e., DMSO: EtOH (7: 3, v / v). The lipid solution was added to 9 ml of 240 mM sodium sulfate in PBS (pH 7.4), while stirring vigorously to reach a final lipid concentration of 5 mM. After 8 freeze-thaw cycles, VEGF (provided by Jiukang Research Institute) was added at a final concentration of 2 μg / ml, and 2 more freeze-thaw cycles were performed. Subsequently, the lipid solution was extruded through a Northern Lipids extruder using a 100 nm polycarbonate nanoporous membrane. The resulting liposomes (DLC-VEGF) were then washed with PBS (pH 7.4) at room temperature using an ultrafiltration tube (Millipore, Germany) with a cutoff value of 30 kDa. The purified DLC-VEGF particles were mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a mass ratio of 1:10 in PBS (pH 7.4) and incubated at room temperature for 6 hours. Unreacted EDC and NHS were removed using an ultrafiltration tube with a cutoff value of 30 kDa.

[0071] VEGF lipid particle identification results Figure 4 As shown in the figure, (A) a bright field microscope photo of VEGF lipid particles, the scale bar in the figure is 20 μm. The above results show that the size of VEGF lipid particles is relatively uniform. (BC) Transmission electron microscopy characterization of VEGF lipid particles, the scale bar in the figure is 200 nm. The above results show that the particle size of VEGF lipid particles is about 100 nanometers. (D) The particle size and potential of lipid particles are characterized using a particle size analyzer (NanoBrook Omni).

[0072] The results showed that the particle sizes of empty shell lipid particles (DLC), lipid particles encapsulating VEGF (DLC-VEGF) and activated VEGF lipid particles (DLC-VEGF / NHS) were uniform, about 100 nm, and the difference in Zeta potential indicated that the lipid particles were successfully loaded with VEGF and activated.

[0073] S5. Construction of a self-driving microrobot dual-drug delivery system

[0074] The microneedle (MN) patch is manufactured using a polydimethylsiloxane (PDMS) microneedle mold: a cone with a diameter of 500 μm and a height of 1000 μm. The needle cavities are arranged in a 20 x 20 array, and the spacing between the bottoms of adjacent cones is 300 μm. The self-driven dual-drug loaded microneedle patch of the present invention consists of two parts: ① PLGA microneedles and ② effervescent base.

[0075] A. Casting of PLGA microneedles. The PLGA microneedle casting solution is 600 mg of polylactic acid-co-glycolic acid (PLGA) dissolved in 1710 mg of diethylene glycol dimethyl ether and 90 mg of deionized water, that is, the solvent is composed of a mixture of diethylene glycol dimethyl ether and deionized water (95% / 5%, w / w), wherein the mass concentration of polylactic acid-co-glycolic acid is 25.3% (w / w). The microneedle mold is centrifuged at a speed of 3000r / min using a spin coater, and the microneedle casting solution is added dropwise to the top of the MN microneedle mold until it is filled. Subsequently, the MN microneedle mold is centrifuged at a speed of 8000r / min for 15 minutes to eliminate bubbles. Afterwards, the mold is placed in a vacuum oven at 60°C for 40 minutes for drying to obtain PLGA microneedles.

[0076] The linker of functionalized eggshell microparticles and azidated exosomes (60 mg / mL) was added to 600 mg of poly(lactic-co-glycolic acid) (PLGA) and 1710 mg of diethanol dimethyl ether, replacing the above volume of deionized water, as the microneedle casting solution, and the above operation was performed to construct microneedles containing self-propelled particles loaded with exosomes.

[0077] B. Casting of the effervescent base. The effervescent base casting liquid solvent is composed of a mixture of 13% (w / v) polyvinyl pyrrolidone (PVP), 4% (w / v) citric acid and 5% (w / v) sodium bicarbonate added to anhydrous ethanol (the above concentration is the final concentration). The specific operation is: 10 ml of anhydrous ethanol, polyvinyl pyrrolidone (0.65 g of molecular weight 55,000; 0.65 g of molecular weight 360,000), 0.4 g of citric acid, and 0.5 g of sodium bicarbonate. DLC-VEGF is added to the effervescent base casting liquid solvent to make the final concentration of 10 mM DLC-VEGF nanoparticles, and mixed to obtain the effervescent base casting liquid.

[0078] After the functionalized particles of the MN microneedle mold loaded with exosomes were dried, 400 μL of the effervescent base casting solution was covered on it. After drying in a 40°C drying oven for three days to ensure complete drying, the mold was placed on ice for one minute, and then the mold was rolled down from both sides to obtain a complete microneedle patch, i.e., a self-driven microrobot dual drug loading system. The microneedle patch and the color-changing silica gel desiccant were placed in a ziplock bag and stored in a -20°C refrigerator.

[0079] Microneedle identification results Figure 5 As shown in the figure, (A) bright field photo of the microneedle patch, the scale bar in the figure is 1 cm. (B) SEM photo of the microneedle patch, the scale bar in the figure is 500μm. (C) 3D reconstruction of the microneedle patch doped with FITC fluorescent dye; this is a 3D reconstruction after fluorescence confocal photography, the scale bar in the figure is 500μm.

[0080] Example 2

[0081] In order to prove that the obtained PLGA microneedle and the effervescent base are connected, the needle tip was doped with Cy3 fluorescent dye (final concentration of 10 μM) and the effervescent base was doped with Cy5 fluorescent dye (final concentration of 10 μM) through preliminary experiments. The bright field photograph of the assembly of the PLGA microneedle and the effervescent base is shown in Figure 2. Figure 6 The picture shows the color of the fluorescent dye itself, and the result proves that the microneedle tip is connected to the base. The red one is the PLGA microneedle, and the blue one is the effervescent base.

[0082] The eggshell microparticles prepared in S2 of Example 1 were subjected to a study on their mobility. Specifically, a custom glass slide was first made: grooves were formed at both ends and connected in the middle by a connecting groove. The entire glass slide was treated to be hydrophilic, and a suspension of eggshell microparticles was added to one of the grooves, and acid was added to another groove. The excess acid overflowed the groove and flowed along the connecting groove into the groove where the eggshell microparticles were located, thereby triggering the movement of the eggshell microparticles. The results are shown in FIG. Figure 7 As shown in the figure, (A) Schematic diagram of eggshell microparticle drive. CaCO 3 Part of the reaction with acid generates bubbles that propel the particles to move. BF uses an ultra-high-speed camera to capture the movement of eggshell microparticles under a microscope. (B) The moment when the trajectory of eggshell microparticles changes significantly from 0 to 12 seconds. Specifically shows the particle propulsion at 0s, 2.4s, 7.2s, and 12s; the scale bar is 10μm. (C) The dotting method is used to describe the trajectory of eggshell microparticles from 0 to 12s. Specifically, CaCO suspended in ultrapure water 3 / The motion trajectory of protein particles driven by citric acid solution with pH = 2. (D) Use three-dimensional coordinates to describe the changes in the spatial position of eggshell microparticles within 0-12 seconds. (E) Extract the displacement changes of eggshell microparticles along the X-axis and make a fitting curve. (F) Extract the displacement changes of eggshell microparticles along the Y-axis and make a fitting curve. The results show that the calcium carbonate surface of eggshell microparticles reacts with acid to produce carbon dioxide, which is the energy source for self-driven particle movement (as shown in Figure A). Figure B depicts in detail the position change trajectory of ESMP at 0.0 seconds, 2.4 seconds, 7.2 seconds and 12.0 seconds. Figure CD further reveals the motion trajectory of ESMP in water, and its motion trajectory unfolds along the X-axis and Y-axis. In-depth analysis of the trajectory of ESMP found that its displacement along the X-axis showed a significant quadratic function characteristic, which conforms to the formula Y1 = -0.03503X 2 +1.10737X-0.33861; while the displacement along the Y axis follows another quadratic function path, given by the formula Y2=0.0115X 2-0.39391X+2.97221 (as shown in Figure EF). By differential processing of the displacement formula, we successfully derived the velocity expression. Specifically, the velocity formula along the X-axis is Y'1=-0.07006X+1.10737, and the velocity formula along the Y-axis is Y'2=0.023X-0.39391. Therefore, once started, the ESMP obtains a speed of about 1.2μm / s in the X-axis direction and a speed of about 0.4μm / s in the Y-axis direction. It can be seen that the eggshell microparticles prepared by the present invention have good motion performance. Since the particle size of the functionalized eggshell microparticles does not change, and the functionalization is a modification of the protein membrane, it will not affect the CaCO 3 The modified molecules do not increase the mass of the particles. Therefore, the unmodified eggshell particles are used to illustrate that the functionalized eggshell microparticles have the same mobility.

[0083] Example 3

[0084] The mechanical properties of the microneedles obtained in Example 1 were tested using a mechanical tester (Biodynamic 5500). The mechanical properties of the microneedle patches were characterized by PLGA cast microneedle patches (PLGA needle / base, i.e., both the needle tip and the base were cast from PLGA solution, and there was no special substance doped in PLGA), microneedle patches assembled from PLGA microneedle tips and effervescent bases (PLGA needle-effervescent base), and microneedle patches assembled from PLGA needle tips doped with eggshell microparticles and effervescent bases (PLGA / ESMP needle-effervescent base). Figure 8 As shown, the results show that the single needle strength of the PLGA needle / base group reaches a maximum value of about 0.7 N, while the PLGA / ESMP needle effervescent base is the weakest, about 0.5 N. The results show that the eggshell-doped microparticles prepared by the present invention have some detrimental effects on the mechanical properties of the microneedle patch, but the mechanical strength of a single needle tip is still sufficient to pierce the heart tissue.

[0085] The degradation test method of the microneedle patch made of PLGA obtained in Example 1 is to immerse the microneedle patch in PBS, put it in a 37°C constant temperature shaker (120 rpm) to simulate the in vivo environment, collect the supernatant every day and supplement it with an equal amount of PBS, and use ultraviolet absorption detection on the supernatant.

[0086] The degradation of the effervescent base is to place the microneedle patch in PBS, the base is rapidly degraded, 200 μL of supernatant is taken every minute, and 200 μL of PBS is added, and the supernatant is detected by ultraviolet absorption. The degradation characteristics of the microneedle are obtained as follows Fig. 9As shown in the figure, (A) the degradation curve of the microneedle patch made of PLGA doped with Cy3 fluorescent dye. (B) The degradation curve of the effervescent base of the microneedle patch doped with Cy5 fluorescent dye in the effervescent base. Figure A shows the in vitro degradation test of the Cy3-labeled MN patch, which degrades by about 24% after 14 days, followed by a rapid degradation stage, and still retains about 4% after 26 days. Figure B shows that the Cy5-labeled effervescent base degrades by 75% within 5 minutes and disappears after 20 minutes. This experiment shows that the effervescent base in this study can be rapidly degraded, thereby releasing VEGF lipid particles for vascular reconstruction of superficial tissues in the early stage of myocardial infarction. In addition, the rapidly degradable microneedle base can reduce the burden on the heart and prevent the base remaining outside the myocardial tissue from bringing out the microneedles inserted into the myocardial tissue during heart beating.

[0087] The microneedle patch obtained in Example 1 was directly inserted into the rat heart tissue. Fig.10 As shown, (A) A bright field photograph of a PLGA microneedle patch (the needle tip and base are all made of PLGA casting) inserted into a rat heart. The scale in the figure is 2 mm. (B) A Cy3-doped PLGA microneedle patch (simply adding 10 μM Cy3 probe dye to the PLGA casting) was inserted into a frozen section of a rat heart. The left picture is a combination of a bright field picture of myocardial tissue and a fluorescent picture of the microneedle, and the right picture is a fluorescent picture of the microneedle patch; the scale in the figure is 200 μm. (C) A microneedle patch with an effervescent base (i.e., a microneedle patch with a PLGA needle tip and an effervescent base) was inserted into the rat heart tissue, and a large number of bubbles were generated and quickly degraded after a small amount of saline was added. The scale in the figure is 2.5 cm. Figure A shows that the MN patch can be inserted into rat heart tissue in a compliant manner, Figure B is a fluorescent image of frozen section heart tissue, and Figure C shows that after adding a small amount of saline, the effervescent base produces a large number of bubbles and rapidly degrades within a few minutes. This is because the effervescent base contains sodium bicarbonate and citric acid, which trigger the reaction of the two after the addition of saline, generating carbon dioxide, so bubbles are generated. The results show that the self-driven micro-robot dual-drug loading system designed by the present invention, i.e., the microneedle patch, can indeed trigger the rapid degradation of the effervescent base after a small amount of saline is added after being inserted into the myocardial tissue.

[0088] Example 4

[0089] The left anterior descending branch of the rats was ligated to prepare the myocardial infarction model. The cardiac function and morphology of the rats were evaluated 28 days after myocardial infarction. Fig.11(AE) Evaluation of rat cardiac function by ultrasound. In the figure, the English meanings are: untreated myocardial infarction group (MI), microneedle patch treatment group with VEGF lipid particles loaded only on the effervescent base (MI+DLC-VEGF), microneedle patch treatment group with exosomes loaded only on the PLGA needle tip (MI+Exosome), microneedle patch treatment group with VEGF lipid particles loaded on the effervescent base and exosomes loaded on the PLGA needle tip (MI+DLC-VEGF / Exo), and microneedle patch treatment group with VEGF lipid particles loaded on the effervescent base and eggshell microparticles and exosome connectors loaded on the PLGA needle tip (MI+DLC-VEGF / ESMP-exo). In the figure, (A) Ultrasound characterization of the heart in the untreated group and the four treatment groups 28 days after myocardial infarction. (BE) Ultrasound parameters left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), ejection fraction (EF), and shortening fraction (FS). (F) HE staining results of rat heart sections 28 days after myocardial infarction. (G) Masson staining results of rat heart sections 28 days after MI. (H) Statistics of cardiac wall thickness in rats 28 days after MI. (I) Statistics of fibrosis area in rat heart tissue 28 days after MI. Relative changes were analyzed based on the LVIDd, LVIDs, EF, and FS values ​​of normal rats (mean values ​​of 5 rats). (FG) Examination of rat cardiac morphology by H&E and Masson staining. Scale bar, 2000 μm. The insets outlined by red and green dashed boxes are shown at higher magnification in the bottom row; the scale bar is 250 μm. The black dashed line indicates the insertion position of the MN patch. (HI) Morphological parameters, including infarct wall thickness and infarct area, were quantified from Masson staining images using ImageJ software. n = 6. All data are expressed as mean ± SD. Comparisons between the two groups were performed using a two-tailed unpaired Student's t test. NS indicates P > 0.1234. *P<0.0332, **P<0.0021. P<0.0002, ****P<0.0001.

[0090] The specific results were that four weeks after myocardial infarction, HE staining showed that the ventricular wall of the rats in the MI group was significantly thinned, resulting in ventricular dilation and deformation ( Fig.11F). The cardiomyocyte layer formed an almost regular ellipsoid with a diameter of approximately 150 to 500 μm, separated from the ventricular wall. In the DLC-VEGF and extracellular vesicle (exosome) groups, the overall ventricular structure remained stable, and no significant changes were observed. However, in the edge and inner regions, the cardiomyocyte nuclei appeared to be arranged disorderly and partially aggregated, suggesting possible myofiber degradation and cardiomyocyte shedding. In the DLC-VEGF / Exo group, the nuclei were neatly arranged in the edge region, and the myofibers showed a regular distribution pattern. Nevertheless, the myofibers in the inner region appeared to be arranged disorderly and had a tendency to disintegrate. In contrast, the cardiomyocytes in the DLC-VEGF / ESMP-exo group showed regular nuclear arrangement and consistent gaps in both the edge and inner regions of the MI. In addition, the myofibers maintained a consistent orientation with no signs of disintegration. Fig.11 G shows the morphology and fibrosis of mouse heart tissue after Masson staining. The measurement results showed that the ventricular wall of the MI group was the thinnest, with a measurement value of 0.443 ± 0.025 mm ( Fig.11 H). In contrast, the ventricular wall thickness increased significantly in the treatment groups. Among these treatment groups, the ventricular wall thickness in the DLC-VEGF, extracellular vesicle, and DLC-VEGF / Exo groups was 1.742±0.055 mm, 1.834±0.097 mm, and 1.782±0.06 mm, respectively. However, the ventricular wall thickness in the DLC-VEGF / ESMP-exo group was significantly thicker, measuring 2.141±0.064 mm. In addition, the DLC-VEGF / ESMP-exo group showed the most significant improvement in infarct size, with an infarct size of only 3.500±1.049% ( Fig.11 I). Although the degree of fibrosis was reduced compared with the other three treatment groups (26.33±1.211%), the presence of fibrosis cannot be ignored. Among these treatment groups, the fibrosis phenomenon was more obvious in the DLC-VEGF group, with a specific value of 13.170±2.401%. There was no significant difference in the degree of fibrosis between the extracellular vesicle group and the DLC-VEGF / Exo group, with specific values ​​of 7.833±1.472% and 6.333±1.366%, respectively.

[0091] It can be seen that the microneedle patch prepared by the present invention, in which the effervescent base is loaded with VEGF lipid particles and the PLGA needle tip is loaded with a connector of self-propelled eggshell microparticles and exosomes derived from mesenchymal cells, can effectively save myocardial cells, rebuild the vascular network, and effectively treat myocardial infarction.

[0092] Example 5

[0093] The self-driving microrobot dual drug delivery system patch prepared by the present invention can deliver the microneedle patch to the heart of rabbits and pigs respectively through thoracoscopy. Fig.12 As shown, the self-driven microrobot dual drug delivery system (MN patch) prepared in Example 1 is minimally invasively delivered to the pig heart with the help of thoracoscopic technology. In the figure, A refers to thoracoscopic entry: the opening in the upper left corner is the thoracoscopic entry port, and the lower right corner is the microneedle delivery port; B refers to microneedle delivery: the red one is the microneedle patch, and the lower right is the negative pressure device for delivery; C refers to the dripping of normal saline; D refers to the generation of bubbles, that is, the dripping of normal saline triggers the degradation of the effervescent base; E refers to the residue of the microneedle patch in the heart tissue ten minutes after the effervescent base has degraded. Fig.13 The self-propelled microrobot dual drug delivery system (MN patch) prepared in Example 1 of the present invention was minimally invasively delivered to the rabbit heart by thoracoscopic technology. In the figure (AB), the MN patch was delivered into the rabbit heart using a negative pressure device. (C) The effervescent base degraded rapidly and produced a large number of bubbles. (D) The remaining MN patch was firmly attached to the surface of the heart.

[0094] The present invention has developed a self-driving microrobot dual drug-carrying system, which uses self-propelled eggshell microparticles, which are loaded with exosomes (MI) derived from MSCs for the treatment of myocardial infarction, and can also be loaded with exosomes for the treatment of other diseases. In order to improve its mechanical permeability and precise release in the infarcted tissue, these functionalized eggshell microparticles loaded with exosomes are further doped in polylactic acid-co-glycolic acid (PLGA) to synthesize microneedles. In addition, the bottom of the microneedle patch is designed as an effervescent base to release lipid particles containing VEGF, aiming to reduce the burden on the heart and activate endothelial cells in the infarcted tissue. The above experimental results show that the effervescent base of the microneedle patch can be rapidly degraded within a few minutes, while the VEGF lipid particles effectively promote the formation of microvessels outside the MI; the eggshell microparticles use the weak acid environment formed after the MI to deliver the exosomes derived from MSCs to a deeper level, effectively preventing the subsequent fibrosis of the MI. The experiment further showed that the eggshell microparticles improved the acidic environment, thereby saving the myocardial cells in the infarcted area. Therefore, the rapidly degradable substrate microneedle patch based on the self-driven eggshell microparticle drug loading system provides a new strategy with great potential for the treatment of MI.

[0095] It can be seen that the self-driven microrobot dual-drug loading system prepared by the present invention can be minimally invasively delivered to the heart through thoracoscopic surgery to treat myocardial infarction, and can also be expanded to the application of treating cerebral infarction and thrombolysis.

[0096] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any person skilled in the art can use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A self-driving micro-robot dual-drug loading system, characterized in that: It consists of a PLGA microneedle and an effervescent base covering the PLGA microneedle, wherein the needle cavity of the PLGA microneedle is inlaid with a connector of self-propelled eggshell microparticles and azidated exosomes, and the effervescent base is wrapped with vascular endothelial growth factor lipid particles.

2. The self-driving micro-robot dual-drug delivery system according to claim 1, characterized in that: The self-propelled eggshell microparticles are eggshell microparticles containing shell membranes modified by dibenzocyclooctyne-N-hydroxysuccinimide ester.

3. The self-driving micro-robot dual-drug delivery system according to claim 1, characterized in that: The eggshell is a chicken eggshell, a bird eggshell, a duck eggshell, a black chicken eggshell, an ostrich eggshell, a pigeon eggshell or a goose eggshell.

4. The self-driving micro-robot dual-drug delivery system according to claim 1, characterized in that: The exosomes are exosomes derived from human umbilical cord mesenchymal stem cells, exosomes derived from bone marrow mesenchymal stem cells, exosomes derived from endothelial cells, or exosomes derived from cardiomyocytes; The vascular endothelial growth factor lipid particles are vascular endothelial growth factor lipid particles activated by carboxylation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide.

5. A method for preparing a self-driving microrobot dual-drug delivery system, characterized in that: It includes the following steps: S1, mesenchymal stem cells were treated with N-azidoacetyl-mannosamine tetraacylation, and then azidated exosomes were extracted; S2, drying and grinding the eggshell containing the shell membrane to obtain eggshell microparticles, and modifying the eggshell microparticles with dibenzocyclooctyne-N-hydroxysuccinimide ester to obtain functionalized eggshell microparticles; S3, co-incubating the azidated exosomes with the functionalized eggshell microparticles to obtain a connector of the functionalized eggshell microparticles and the azidated exosomes; S4, after the vascular endothelial growth factor and the lipid preparation are mixed, the mixture is frozen and thawed, the lipid solution is squeezed out through an extruder, and DLC-VEGF particles are obtained after ultrafiltration, and the DLC-VEGF particles are incubated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to obtain carboxylated vascular endothelial growth factor lipid particles; S5. Add the connector of functionalized eggshell microparticles and azidated exosomes into the microneedle casting liquid and add it into the needle cavity of the microneedle mold. After the first drying, mix the carboxylated vascular endothelial growth factor lipid particles with the effervescent base casting liquid and cover the dried microneedle mold base. After the second drying, take out the microneedle mold to obtain a self-driven dual-drug loaded microneedle patch.

6. The preparation method according to claim 5, characterized in that: The final concentration of the tetraacylated N-azidoacetyl-mannosamine is 15 to 200 μM, and the mesenchymal stem cells are human umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, endothelial cells or cardiomyocytes; the azidated exosomes can be extracted after treating the cells for 1 to 4 days.

7. The preparation method according to claim 5, characterized in that: The particle size of the eggshell microparticles is less than 5 microns. The eggshell microparticles are mixed with water to obtain an eggshell microparticle emulsion. The modification is carried out using dibenzocyclooctyne-N-hydroxysuccinimide ester, DBCO-PEG-NHS or diphenylcyclooctyne polyethylene glycol active ester and the eggshell microparticle emulsion, wherein the final concentration of dibenzocyclooctyne-N-hydroxysuccinimide ester, DBCO-PEG-NHS or diphenylcyclooctyne polyethylene glycol active ester is 1 to 1000 μM, and the modification is incubated at a temperature of 35 to 40° C. for 1 to 3 hours, wherein the molecular weight of PEG can be 400, 600, 1K, 2K, 3.4K, 4K, 5K or 10K.

8. The preparation method according to claim 5, characterized in that: The mass ratio of the azid exosomes to the functionalized eggshell microparticles is 1:10-50; the concentration of the azid exosomes is 30-500 mg / ml, and the incubation conditions are incubation at a temperature of 35-40° C. for 1-3 hours.

9. The preparation method according to claim 5, characterized in that: The microneedle casting liquid is a polylactic acid-glycolic acid copolymer solution with a mass concentration of 15 to 50%, wherein the solvent is diethylene glycol dimethyl ether; The effervescent base casting solution is a mixture of 13% polyvinyl pyrrolidone, 4% citric acid and 5% sodium bicarbonate in anhydrous ethanol at a final concentration by mass volume; The first drying condition is vacuum drying at 50-80°C for 15-60 minutes; The second drying condition is 35-45°C for 1-3 days.

10. Use of the self-propelled microrobot dual-drug delivery system according to any one of claims 1 to 4 or the self-propelled microrobot dual-drug delivery system obtained by the preparation method according to any one of claims 5 to 9 in the preparation of drugs for myocardial infarction, cerebral infarction, and thrombosis.

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