Engineered vesicle as well as preparation method and application thereof

Abstract

The invention provides an engineered vesicle as well as a preparation method and application thereof. The engineered vesicle provided by the invention can be used for effectively targeting the heart and remarkably promoting polarization of M2 type macrophages, and has the functions of increasing the heart retention rate, inhibiting cardiac fibrosis, improving the heart function and improving the injured myocarditis level.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an engineered vesicle and a preparation method and application thereof. Background Art

[0002] Myocardial infarction (MI) is a common and widespread disease with high morbidity and mortality worldwide. Myocardial infarction leads to myocardial remodeling and subsequent heart failure. Although timely reperfusion and concomitant reoxygenation can largely reduce the infarct size, myocardial infarction survivors still face a poor prognosis due to the deterioration of cardiac function and the progression of heart failure.

[0003] Currently, stem cell transplantation is expected to become an alternative therapy for repairing damaged hearts. Although animal experimental results show that stem cell transplantation therapy can effectively improve the cardiac function of MI animals, the results of clinical trials have not shown significant efficacy and long-term prognosis benefits. This shows that the current cell therapy strategy still needs to be optimized. Summary of the invention

[0004] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0005] The first aspect of the present invention provides an engineered vesicle, wherein the engineered vesicle contains a vesicle, miR-181a, and a platelet membrane.

[0006] In the present invention, "vesicle" refers to the membrane vesicles released after the fusion of intracellular multivesicular bodies with the cell membrane. Vesicles include exosomes, microvesicles, and apoptotic bodies.

[0007] Furthermore, the vesicles are nanovesicles.

[0008] In the present invention, the nanovesicle is a nanoscale vesicle structure. The present invention obtains nanovesicles with a diameter of 100-200 nm by filtering with filter membranes of different filter diameters.

[0009] Furthermore, the vesicles are stem cell vesicles.

[0010] In the present invention, the stem cells include but are not limited to hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

[0011] Furthermore, the mesenchymal stem cells include but are not limited to bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0012] Furthermore, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells.

[0013] In the present invention, miR-181a is a non-coding RNA molecule belonging to the miRNA family. The two arms of the miR-181a precursor each produce a functional mature miRNA, named "-5p" and "-3p", such as miR-181a-5p and miR-181a-3p, indicating that they are processed from the 5' end arm and 3' end arm of the miR-181a precursor, respectively.

[0014] Furthermore, the miR-181a is selected from miR-181a-5p.

[0015] The second aspect of the present invention provides a pharmaceutical composition, which comprises the engineered vesicles described in the first aspect of the present invention.

[0016] In the present invention, the pharmaceutical composition is administered in a dosage form for enteral administration or parenteral administration.

[0017] Furthermore, the gastrointestinal dosage forms include but are not limited to solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pills, suppositories, aerosols, sprays, powder mists, patches, ointments or creams.

[0018] Furthermore, the non-gastrointestinal dosage forms include, but are not limited to, injection dosage forms, respiratory tract dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0019] Furthermore, the injectable dosage forms include but are not limited to intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the respiratory tract administration dosage forms include but are not limited to sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops, pills, etc., which are used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0020] In the present invention, the pharmaceutical composition and at least one additional therapeutic agent or therapy may be administered sequentially, simultaneously and / or alternately, wherein the at least one additional therapeutic agent or therapy includes but is not limited to percutaneous coronary intervention, coronary artery bypass grafting, thrombolytic therapy, antiplatelet therapy, heparin, warfarin, fibrinolytic agents, oxygen therapy, vasodilators, analgesics, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, glycoprotein antagonists, statins, aldosterone antagonists, implantable cardiac defibrillators or any combination thereof.

[0021] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

[0022] In the present invention, "pharmaceutically acceptable carriers and / or excipients" include, but are not limited to, diluents, binders, surfactants, humectants, adsorption carriers, lubricants, disintegrants, emulsifiers, bioavailability enhancers, suspending agents, sweeteners, flavoring agents, colorants, excipients, preservatives, solubilizers, dispersants and / or wetting agents. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; adhesives include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and hexadecanol; humectants include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium stearate, magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0023] The third aspect of the present invention provides a method for preparing the engineered vesicles according to the first aspect of the present invention, the method comprising combining a vesicle containing miR-181a and a platelet membrane.

[0024] In the present invention, the vesicles containing miR-181a may be cell-derived vesicles naturally carrying miR-181a, cell-derived vesicles transfected with miR-181a, or vesicles obtained by fusing miR-181a with cell-derived vesicles.

[0025] In the present invention, the term "transfection" refers to a process in which a nucleic acid molecule is introduced into a cell using a non-viral or virus-based method. The nucleic acid molecule can be a sequence encoding an entire protein or a functional portion thereof. Non-viral transfection methods include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce nucleic acid molecules into cells. Exemplary non-viral transfection methods include, but are not limited to, calcium phosphate transfection, liposome transfection, nuclear transfection, sonoporation, heat shock transfection, magnetofection, and electroporation. For viral-based methods, any useful viral vector known in the art can be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, and adeno-associated viral vectors. In some aspects, the nucleic acid molecule is introduced into a cell using a retroviral vector according to standard procedures known in the art.

[0026] In the present invention, miR-181a can be fused with cell-derived vesicles using methods including but not limited to electroporation, chemical reagents, viral vectors, microinjection, ultrasound, freeze-thaw, and membrane fusion agents.

[0027] Furthermore, non-viral transfection methods are used to introduce nucleic acid molecules into cells.

[0028] Furthermore, the non-viral transfection method is selected from liposome transfection.

[0029] In the present invention, liposomes include but are not limited to positively charged liposomes, neutral liposomes, and negatively charged liposomes.

[0030] In the present invention, the positively charged lipids in the positively charged liposomes are the core components in liposome transfection. They bind to negatively charged nucleic acids (such as DNA or RNA) through their positive charges to form lipid complexes. At physiological pH, positively charged lipids are usually uncharged, which reduces their interaction with the anionic membranes of non-target cells, thereby improving the specificity and efficiency of transfection. In the endosomes, protonation destroys the stability of the endosomal membrane and promotes the release of nucleic acids. The neutral lipids in the neutral liposomes mainly play a structural support role in the transfection process in the liposomes, maintaining the stability of the liposomes and the fluidity of the membrane. They are usually not directly involved in the transfection of nucleic acids, but can play an indirect role by affecting the stability and transfection efficiency of the overall liposomes. The transfection efficiency of negatively charged liposomes is usually lower than that of positively charged liposomes. In addition, polyethylene glycol (PEG) can be used to modify liposomes. PEG can prolong the circulation time, provide steric hindrance, reduce the size of liposomes and prevent their aggregation, which helps to increase the circulation time of liposomes in the body and improve their biocompatibility and safety; in addition, PEGylation can also protect liposomes from recognition and clearance by the immune system.

[0031] Furthermore, the liposome is selected from positively charged liposomes.

[0032] Furthermore, the positively charged liposomes include Lipo3000 or Lipo2000.

[0033] Furthermore, the positively charged liposome is selected from Lipo3000.

[0034] Furthermore, the vesicles are nanovesicles.

[0035] Furthermore, the diameter of the nanovesicles is 100-200 nm.

[0036] Furthermore, the method also includes a method for preparing nanovesicles, and the preparation method includes filtering cells using filter membranes with different filter diameters.

[0037] Furthermore, the filter diameter ranges from 0.1 μm to 10 μm.

[0038] Furthermore, the order of using the filter membranes with different filter diameters is to first use the filter membrane with a large filter diameter and then use the filter membrane with a small filter diameter.

[0039] Furthermore, the solution is repeatedly passed through the filter membrane of each filter diameter.

[0040] Further, repeat three times.

[0041] Furthermore, the method comprises filtering the cells using 5 μm, 1 μm, and 0.4 μm filter membranes in sequence, repeatedly passing through filter membranes of each filter size, and then filtering using a 0.22 μm filter membrane.

[0042] Furthermore, the mass ratio of the miR-181a-containing vesicles to the platelet membrane is 1:1-1:20.

[0043] Furthermore, the mass ratio of the miR-181a-containing vesicles to the platelet membrane is 1:2.

[0044] Furthermore, the method comprises mixing bone marrow mesenchymal stem cell nanovesicles containing miR-181a-5p with platelet membranes in a mass ratio of 1:2, and filtering the mixture using filter membranes with different filter diameters.

[0045] Furthermore, the filter diameter ranges from 0.1 μm to 10 μm.

[0046] Furthermore, the order of using the filter membranes with different filter diameters is to first use the filter membrane with a large filter diameter and then use the filter membrane with a small filter diameter.

[0047] Furthermore, the method comprises filtering the mixture using 5 μm, 1 μm, and 0.4 μm filter membranes in sequence.

[0048] Furthermore, the method also includes transfecting miR-181a into stem cells.

[0049] A fourth aspect of the present invention provides any of the following applications: (1) Use of the engineered vesicles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in the preparation of a drug for treating heart disease; (2) Application of miR-181a and platelet membrane in the preparation of drugs for the treatment of heart diseases; (3) Application of miR-181a and platelet membrane in the preparation of drugs to enhance the efficacy of vesicles in treating heart diseases.

[0050] Furthermore, the heart disease includes but is not limited to myocardial infarction, myocardial ischemia-reperfusion injury, pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, ventricular dysfunction, heart failure, coronary artery disease, end-stage heart disease, angina pectoris, rheumatic heart disease or cardiovascular disease.

[0051] Furthermore, the heart disease is selected from myocardial infarction.

[0052] In the present invention, the drug is administered at a therapeutically effective dose. The term "therapeutically effective dose" refers to a level or amount of the target agent that does not produce significant negative or adverse side effects: (1) delay or prevent the onset of heart disease; (2) slow or prevent the progression, aggravation or worsening of one or more symptoms of heart disease; (3) improve the symptoms of heart disease; (4) reduce the severity or incidence of heart disease; or (5) cure heart disease. The therapeutically effective dose can be administered before the onset of heart disease for a preventive effect; or, the therapeutically effective dose can be administered after the onset of heart disease for a therapeutic effect or to maintain a therapeutic effect.

[0053] Furthermore, the effects of the treatment include but are not limited to promoting myocardial repair, increasing cardiac retention rate, inhibiting cardiac fibrosis, improving cardiac function, reducing inflammation levels, promoting M2 macrophage polarization, and reducing myocardial infarction area.

[0054] Furthermore, the miR-181a and platelet membrane play a synergistic role in enhancing the efficacy of vesicles in treating heart diseases.

[0055] A fifth aspect of the present invention provides any of the following applications: (1) Use of the engineered vesicles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in regulating macrophage polarization; Further, the engineered vesicle or pharmaceutical composition promotes polarization of M2 macrophages; (2) Use of the engineered vesicles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in promoting the proliferation of anti-inflammatory macrophages.

[0056] A sixth aspect of the present invention provides any of the following methods: (1) A method for regulating macrophage polarization in vitro, the method comprising regulating macrophage polarization by the engineered vesicle described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention; Further, the engineered vesicle or pharmaceutical composition promotes polarization of M2 macrophages; (2) A method for promoting the proliferation of anti-inflammatory macrophages in vitro, the method comprising administering the engineered vesicles described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention to promote the proliferation of anti-inflammatory macrophages.

[0057] The present invention has the following advantages and beneficial effects: The present invention provides an engineered vesicle and a preparation method and application thereof. The engineered vesicle provided by the present invention can effectively target the heart, significantly promote the polarization of M2 macrophages, and has the functions of increasing the heart retention rate, inhibiting cardiac fibrosis, improving cardiac function, and improving the level of inflammation in damaged myocardium. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Figure 5 is an analysis of the morphology and biological characteristics of P-181-NV. Figure A is a schematic diagram of the preparation process of P-181-NV nanovesicles; Figure B is the expression results of miR-181a-5p in stem cells; Figure C is the expression results of miR-181a-5p in vesicles; Figure D is an electron microscope photo of PM, NV, and P-NV; Figures E, F, and G are the expression level results of Alix, P-selectin, and β-actin in PM, NV, P-NV, 181-NV, and P-181-NV; Figures H and I are the NTA results of NV, P-NV, 181-NV, and P-181-NV; Figure J is the ZETA results of NV, P-NV, 181-NV, and P-181-NV.

[0059] Figure 2 These are the results of P-181-NV reducing cardiac fibrosis and improving cardiac function (bar=50μm). A and B are the cardiac IVIS in vivo imaging data; C and D are the Masson staining results and quantitative graphs; E is the echocardiography result; F is the EF result; G is the CardiacOutput result; H is the FS result; I is the Stroke Volume result; J is the LVID;s result; K is the LVID;d result.

[0060] Figure 3Figure 3 is the result of P-181-NV promoting the differentiation of CX3CR1-positive anti-inflammatory macrophages on the 3rd and 7th days after MI (bar=50μm). Figures A-C are the fluorescence images of anti-inflammatory macrophages and pro-inflammatory macrophages on the 3rd day after MI; Figures D-F are the fluorescence images of anti-inflammatory macrophages and pro-inflammatory macrophages on the 7th day after MI.

[0061] Figure 4 These are the results of P-181-NV promoting the differentiation of M2 macrophages 3, 7, and 14 days after MI (bar=50μm). Panel A and Panel B are the results of immunofluorescence detection of fluorescence intensity of CD206-positive M2 macrophages 3 days after MI; Panel C and Panel D are the results of immunofluorescence detection of fluorescence intensity of CD206-positive M2 macrophages 7 days after MI; Panel E and Panel F are the results of immunofluorescence detection of fluorescence intensity of CD206-positive M2 macrophages 14 days after MI.

[0062] Figure 5 Figures A and B are the results of P-181-NV promoting macrophage polarization in vitro experiments. Figures C to F are flow cytometry results of P-181a-NV phagocytosis. Figures G to I are Western blot results of P-181a-NV polarization on macrophages.

[0063] PM: platelet membrane; NV: nanovesicle; 181-NV: nanovesicle transfected with miR-181a-5p; P-NV: nanovesicle wrapped with platelet membrane; P-181-NV: nanovesicle transfected with miR-181a-5p was wrapped with platelet membrane. TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis. *P<0.05, **P<0.01, ***P<0.001. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0065] Example

[0066] 1. Experimental Methods 1. Cell Culture Mouse bone marrow mesenchymal stem cells were purchased from Prosai (CP-M131) and the culture medium was Prosai mouse bone marrow mesenchymal stem cell-specific culture medium (CM-M131-100). THP-1 cells were purchased from Starfish (TCH-C361) and cultured in 1640 medium (Gibco) containing 10% FBS (Gibco) at 37°C and 5% CO2. T293 cells were purchased from Starfish (TCH-C101) and cultured in high-glucose DMEM (Gibco) containing 10% FBS (Gibco) at 37°C and 5% CO2.

[0067] 2. Transfection When the density of mouse bone marrow mesenchymal stem cells reached 60%, miR-181a-5p mimics were transfected into the cells according to Lipo3000 (Invitrogen), and after being cultured in an incubator for 24 h, the cells were collected for PCR verification and preparation of vesicles loaded with miR-181a-5p.

[0068] 3. Preparation of Platelet Membranes Take 10 ml of mouse blood, centrifuge and take the upper layer into a blood collection tube. Take 2 ml of the supernatant, mix it with PBS buffer containing EDTA and PGE1, gently turn it upside down, and centrifuge it at room temperature for minutes. Resuspend the lower layer in PBS containing protease inhibitors and phosphatase inhibitors, and freeze and thaw it repeatedly at -80℃ for 3 times. Centrifuge the platelet extract after it is completely thawed at room temperature, and repeat 5 times. Discard the supernatant, resuspend and wash 3 times with PBS containing protease inhibitors and phosphatase inhibitors, and then resuspend it with PBS, and prepare a mixture of platelet membrane fragments by ultrasound. Detect the platelet membrane morphology by electron microscopy.

[0069] 4. Preparation of Nanovesicles When the cell fusion degree of bone marrow mesenchymal stem cells (BM-MSCs) reached 80%, the cells were collected by trypsin digestion, washed twice with PBS, and then collected for the preparation of nanovesicles. The cells were squeezed through 5μm, 1μm, and 0.4μm filter membranes (Whatman) using LiposoFastLF-50 (Avestin, York, UK) in turn. Each pore size was squeezed repeatedly three times, and finally filtered through a 0.22μm filter membrane to obtain nanovesicles with a diameter of 100-200nm, which were packaged and stored at -80°C. Subsequently, the protein concentration of the nanovesicles was determined by the BCA protein concentration assay kit. After mixing evenly at a mass ratio of platelets: vesicles = 2:1, they were squeezed through three pore size filters again to finally prepare platelet-coated nanovesicles. See the preparation flow chart. Figure 1 Figure A in .

[0070] 5. Identification of Vesicles Transmission electron microscopy, nanoparticle tracking technology and zeta potential analyzer were used to measure the vesicle morphology, size, size distribution and membrane potential. Western blot was used to detect the expression levels of exosome protein marker Alix (Proteintech, 20597-1-AP), platelet membrane marker Pselect (Proteintech, 60322-1-Ig) and intracellular reference protein β-actin (Abbkine, A23910, A23720).

[0071] 6. Flow Cytometry When the cell density reached 80%, cells were collected by trypsin digestion. Washed with sterile PBS 2-3 times. Resuspended cells in 100μl PBS / group, and flow cytometry antibodies CD206 (Proteintech, 2344972), CD163 (Proteintech, 333606) and CD68 (Proteintech, 333814) were added according to the instructions. After incubation at room temperature for 20 minutes, washed twice with PBS, resuspended with 500μl PBS, and detected by flow cytometry within 30 minutes.

[0072] 7. PCR detection Total RNA was extracted using the Trizol method, and the sample RNA concentration, 260 / 230 value, and 260 / 280 value were determined using a nucleic acid analyzer (NanoDrop, ND1000). Reverse transcription and fluorescence quantitative amplification were performed using the reverse transcription system (R323) and qRTPCR system (Q711) of Novagen, respectively. The relative expression of the target gene was calculated using the 2^-ΔΔCt method.

[0073] 8. Western blot Total protein from cells or vesicles was extracted, and the protein content was determined using a BCA protein concentration assay kit (Vazyme, E112). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using a Bio-Rad electrophoresis instrument, and proteins were transferred from SDS-PAGE to a PVDF membrane using a semi-dry transfer system. Afterwards, the results were obtained by blocking (5% skim milk), incubating with primary antibodies, and incubating with secondary antibodies, respectively. The primary antibodies we used were Alix (Proteintech, 12422-1-AP, 1:5000), P-select (60322-1-Ig, 1:2000), CD206 (18704-1-AP, 1:1000), CD163 (16646-1-AP, 1:1000), and β-actin (Affinity, T0022). Anti-mouse fluorescent secondary antibody (Abbkine, A23910) and anti-rabbit fluorescent secondary antibody (Abbkine, A23720) were diluted 1:500 and incubated at room temperature in the dark for 1.5 hours, then washed three times with TBST and tested on a LI-COR Odyssey CLX-0664. The grayscale value of the bands was determined using the ImageJ imaging system and statistical analysis was performed.

[0074] 9. Preparation of Myocardial Infarction Animal Model SPF grade C57 mice (6-8 weeks old) were purchased from Beijing Huafukang Company. Mice were anesthetized with isoflurane gas. After the body position was fixed, the skin was cut transversely from 5 mm to the left of the sternum and the fourth intercostal space on the left side, the subcutaneous tissue was separated, and the pectoralis major and pectoralis minor muscles were separated to expose the intercostal muscles. The intercostal space was opened and the heart was squeezed with the left hand until the heart bulged. The left atrial appendage was quickly ligated 5 mm below the lower edge with 8-0 fine thread, and the myocardium was observed to turn white. The chest was quickly closed and the skin was sutured with 3-0 thread.

[0075] 10. In vivo tracing and echocardiography in small animals Nanovesicles were labeled with DIR (D1220A) and injected into the rat tail vein. In vivo imaging was performed using an instrument (PerkinElmer) on days 1, 3, 7, and 14. Echocardiography (VisualSonics) was performed on day 28 to detect EF (Ejection Fraction), FS (Fractional Shortening), CO (Cardiac Output), SV (Stroke Volume), LVID;s, and LVID;d.

[0076] 11.Masson Mouse heart tissues were obtained at 0, 3, 7, 14, and 28 days after vesicle drug injection, fixed with 4% paraformaldehyde, transferred to ethanol for immersion and paraffin embedding. Paraffin sections were prepared for Masson trichrome staining, and the size of fibrosis area was determined using ImageJ.

[0077] 12. Tissue Fluorescence Staining After obtaining the heart tissue, fix the heart tissue with 4% paraformaldehyde, dehydrate with 15%-30% gradient sucrose solution, embed in OCT, and store at -80°C. Heart slices were incubated with primary antibodies at 4°C overnight. The primary antibodies were SarcomericAlphaActinin, CX3CR1, CCR2, and CD206. Then fluorescent secondary antibodies (Servicebio, GB22403, GB21303) and TSA (Servicebio, G1222, G1223) were added in sequence and antigen repair was performed. Finally, DAPI was used to seal the slices. Observe under a microscope (NIKON Eclipse ci).

[0078] 13. Statistical Analysis This study used prism statistical software for data analysis and drawing. The results of quantitative data were expressed as mean ± standard deviation (Mean ± SD). The t-test was used for comparison between the two groups, and one-way ANOVA was used for comparison between multiple groups. The test level was α = 0.05, and P < 0.05 was considered statistically significant.

[0079] 2. Experimental Results 1. Construction and identification of P-181-NV nanovesicles The experimental results are as follows Figure 1 As shown. PCR results showed that compared with the untransfected group, the miR-181a-5p level in the stem cells transfected with miR-181a-5p mimics group was significantly increased (P<0.05, Figure B), and the miR-181a-5p level in the vesicles derived from them was also significantly increased (P<0.05, Figure C), proving that the miR-181a-5p-modified mesenchymal stem cell-derived nanovesicles (181-NV) were successfully prepared. Platelet-modified NV-miR181a-5p (P-181-NV) was further prepared by platelet membrane coating. Transmission electron microscopy results showed that the engineered nanovesicle particles were cup-shaped or round in shape, and the platelet membrane-coated NVs were a double-layer membrane structure (Figure D). Western blot showed that the vesicles all expressed Alix, and both P-181-NV and P-NV significantly expressed the platelet-specific marker p-selectin, indicating that the platelet membrane coating was successful (P<0.05, Figure 1 EG). NTA results show that the diameter is about 100-200nm, ( Figure 1HI). The ZETA potential results showed that there was no significant difference in the potential of the four types of vesicles (Figure J). The above results proved that the P-181-NV nanovesicles were successfully prepared.

[0080] 2. P-181-NV nanovesicles have high targeting and can improve cardiac function after MI To verify the high targeted migration ability of the engineered nanovesicle P-181-NV to the injured myocardium, we injected DIR-labeled nanovesicles into mice through the tail vein. The mice were randomly divided into three groups (Sham, NV, P-181-NV), and NV and P-181-NV were injected 24 hours after myocardial infarction, and the ability of the vesicles to home to the injured myocardium was tested on the 1st, 3rd, 7th, and 14th days. The results are shown in Figure 2 As shown, the heart homing rate of the P-181-NV group was significantly increased on days 1, 3, and 7, and the vesicle fluorescence signal in the heart almost disappeared on day 14 ( Figure 2 AB). It can be seen that P-181-NV nanovesicles can significantly improve the cardiac retention rate (P<0.05, Figure 2 AB), laying an important foundation for the next step of exploring its functional characteristics of repairing damaged myocardium. Next, we divided the mice into 6 groups (Sham group, MI group, NV group, P-NV group, 181-NV group, and P-181-NV group) and gave them different treatments, and performed Masson staining after 28 days.

[0081] Masson staining results showed that P-181-NV significantly reduced the area of ​​cardiac fibrosis (P<0.05, Figure 2 CD), the cardiac fibrosis of the 181-NV group was reduced by 51.07% compared with the NV group, the cardiac fibrosis of the P-NV group was reduced by 16.61% compared with the NV group, and the cardiac fibrosis rate of the P-181-NV group was 73.39% lower than that of the NV group. According to the reduction rate of cardiac fibrosis, the synergistic q value was calculated using the King's formula to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in reducing the area of ​​cardiac fibrosis. The q=E was calculated. A+B / (E A +E B -E A *E B )=0.7339 / (0.5107+0.1661-0.5107*0.1661)=1.2398, that is, q=1.2398, q>1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in reducing the area of ​​cardiac fibrosis.

[0082] The results of echocardiography showed that the P-181-NV group had significantly improved EF, FS and CO compared with other groups (P<0.05, Figure 2 EK).

[0083] Compared with the NV group, the EF of the 181-NV group increased by 11.46%, the EF of the P-NV group increased by 2.68%, and the EF of the P-181-NV group increased by 25.78%. According to the EF increase rate, the synergistic q value was calculated using the King's formula to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in improving EF. The result of q=EA+B / (EA+EB-EA*EB)=0.2578 / (0.1146+0.0268-0.1146*0.0268)=1.8636 was obtained, that is, q=1.8636, q>1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in improving EF.

[0084] The FS of the 181-NV group increased by 11.21% compared with the NV group, the FS of the P-NV group increased by 1.75% compared with the NV group, and the FS of the P-181-NV group increased by 30.38% compared with the NV group. According to the FS improvement rate, the synergistic q value was calculated using the King's formula to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in improving FS. The result of the calculation was q=EA+B / (EA+EB-EA*EB)=0.3038 / (0.1121+0.0175-0.1121*0.0175)=2.3801, that is, q=2.3801, q>1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in improving FS.

[0085] The SV of the 181-NV group increased by 11.85% compared with the NV group, the SV of the P-NV group increased by -7.68% compared with the NV group, and the SV of the P-181-NV group increased by 27.13% compared with the NV group. According to the SV increase rate, the synergistic q value was calculated using the King's formula to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in increasing SV. The result of the calculation was q=EA+B / (EA+EB-EA*EB)=0.2713 / (0.1185-0.0768+0.1185*0.0768)=5.3402, that is, q=5.3402, q>1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in increasing SV.

[0086] The LVID;s of the 181-NV group decreased by 2.93% compared with the NV group, the LVID;s of the P-NV group decreased by 2.92% compared with the NV group, and the LVID;s of the P-181-NV group decreased by 32.47% compared with the NV group. According to the LVID;s reduction rate, the synergistic q value was calculated using the King's formula to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in reducing LVID;s. The result of the calculation was q=EA+B / (EA+EB-EA*EB)=0.3247 / (0.0293+0.0292-0.0293*0.0292)=5.6326, that is, q=5.6326, q>1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in reducing LVID;s.

[0087] The above results indicate that P-181-NV can significantly increase cardiac retention rate, inhibit cardiac fibrosis and improve cardiac function in ischemic heart disease, and that miR-181a-5p and platelet membrane have a synergistic effect.

[0088] 3. P-181-NV nanovesicles promote the increase of anti-inflammatory macrophage levels after myocardial infarction To investigate whether P-181-NV nanovesicles can repair damaged myocardium after myocardial infarction by regulating macrophage polarization, four groups (Sham, MI, P-NV, P-181-NV) of nanovesicles were injected into mice with myocardial infarction, CX3CR1 and CD206 marked anti-inflammatory macrophage phenotypes, and CCR2 marked pro-inflammatory macrophage phenotypes. The results showed that on the 3rd and 7th days, P-181-NV nanovesicles had a significantly higher level of CX3CR1 (P<0.05, Figure 3 AF) and CD206 positive cell rates were significantly increased (P<0.05, Figure 4 AD), CD206 level also increased significantly on day 14 (P<0.05, Figure 4 EF). These results suggest that engineered nanovesicles P-181-NV can significantly improve the inflammatory level of damaged myocardium after myocardial infarction.

[0089] 4. P-181-NV nanovesicles regulate macrophage polarization This study further explored the role of P-181-NV in regulating the polarization of anti-inflammatory macrophages. First, Did-labeled NVs were co-cultured with macrophages for different periods of time, and the phagocytic efficiency of macrophages was detected by flow cytometry. It was found that after 2 hours of co-incubation, almost all vesicles were phagocytosed by macrophages, proving that they can be well taken up by macrophages ( Figure 5AB). RAW246.7 was then used to verify the ability of the vesicles to promote macrophage polarization. RAW246.7 macrophages were divided into three groups (Control, P-NV, and P-181-NV) and treated differently. Flow cytometry was performed 48 hours after treatment. The results showed that the proportion of M2 macrophages in the P-181-NV group was significantly increased (P < 0.05, Figure 5 CF), Western blot results showed that the expression levels of M2 macrophage markers CD163 and CD206 in the P-181-NV group were significantly increased (P < 0.05, Figure 5 GI). These results suggest that P-181-NV nanovesicles can significantly promote the polarization of M2 anti-inflammatory macrophages. The above experimental results show that P-181-NV nanovesicles can significantly promote the polarization of M2 macrophages.

[0090] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An engineered vesicle, characterized in that: The engineered vesicle contains vesicle, miR-181a, and platelet membrane; Preferably, the vesicles are nanovesicles; Preferably, the diameter of the nanovesicles is 100-200 nm.

2. The engineered vesicle according to claim 1, characterized in that The vesicle is a stem cell vesicle; Preferably, the stem cells include hematopoietic stem cells, neural stem cells, and mesenchymal stem cells; Preferably, the mesenchymal stem cells include bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, and dental pulp mesenchymal stem cells; Preferably, the mesenchymal stem cells are selected from bone marrow mesenchymal stem cells.

3. The engineered vesicle according to claim 1, characterized in that The miR-181a includes miR-181a-5p and miR-181a-3p; Preferably, the miR-181a is selected from miR-181a-5p.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the engineered vesicle according to any one of claims 1 to 3; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

5. A method for preparing the engineered vesicle according to any one of claims 1 to 3, characterized in that: The method comprises combining a vesicle containing miR-181a and a platelet membrane; Preferably, the miR-181a-containing vesicles include cell-derived vesicles naturally carrying miR-181a, cell-derived vesicles transfected with miR-181a, and vesicles obtained by fusing miR-181a with cell-derived vesicles; Preferably, the vesicles are nanovesicles; Preferably, the diameter of the nanovesicles is 100-200 nm; Preferably, the method further comprises a method for preparing nanovesicles, the preparation method comprising filtering cells using filter membranes of different filter diameters; Preferably, the filter diameter ranges from 0.1 μm to 10 μm; Preferably, the filter membranes with different filter diameters are used in the order of first using the filter membrane with a large filter diameter and then using the filter membrane with a small filter diameter; Preferably, the filter is passed through the filter membrane of each filter size repeatedly; Preferably, repeated three times; Preferably, the method comprises filtering the cells using 5 μm, 1 μm, and 0.4 μm filter membranes in sequence, repeatedly passing through the filter membranes of each filter size three times, and then filtering using a 0.22 μm filter membrane.

6. The method according to claim 5, characterized in that The mass ratio of the miR-181a-containing vesicle to the platelet membrane is 1:1-1:20; Preferably, the mass ratio of the miR-181a-containing vesicles to the platelet membrane is 1:

2.

7. The method according to claim 6, characterized in that The method comprises mixing bone marrow mesenchymal stem cell nanovesicles containing miR-181a-5p with platelet membranes in a mass ratio of 1:2, and filtering the mixture using filter membranes with different filter diameters; Preferably, the filter diameter ranges from 0.1 μm to 10 μm; Preferably, the filter membranes with different filter diameters are used in the order of first using the filter membrane with a large filter diameter and then using the filter membrane with a small filter diameter; Preferably, the method comprises filtering the mixture using 5 μm, 1 μm and 0.4 μm filter membranes in sequence.

8. Any of the following applications: (1) Use of the engineered vesicle according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of a drug for treating heart disease; (2) Application of miR-181a and platelet membrane in the preparation of drugs for the treatment of heart diseases; (3) Application of miR-181a and platelet membrane in the preparation of drugs to enhance the efficacy of vesicles in treating heart diseases; Preferably, the heart disease comprises myocardial infarction, myocardial ischemia-reperfusion injury, pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, ventricular dysfunction, heart failure, coronary artery disease, end-stage heart disease, angina pectoris, rheumatic heart disease or cardiovascular disease; Preferably, the heart disease is selected from myocardial infarction; Preferably, the effects of the treatment include promoting myocardial repair, increasing cardiac retention rate, inhibiting cardiac fibrosis, improving cardiac function, reducing inflammation levels, promoting M2 macrophage polarization, and reducing myocardial infarction area.

9. Any of the following applications: (1) Use of the engineered vesicle according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in regulating macrophage polarization; Preferably, the engineered vesicle or pharmaceutical composition promotes M2 macrophage polarization; (2) Use of the engineered vesicle according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in promoting the proliferation of anti-inflammatory macrophages.

10. Any of the following methods: (1) A method for regulating macrophage polarization in vitro, characterized in that: The method comprises regulating macrophage polarization by the engineered vesicle of any one of claims 1 to 3 or the pharmaceutical composition of claim 4; Preferably, the engineered vesicle or pharmaceutical composition promotes M2 macrophage polarization; (2) A method for promoting the proliferation of anti-inflammatory macrophages in vitro, characterized in that the method comprises administering the engineered vesicles described in any one of claims 1 to 3 or the pharmaceutical composition described in claim 4 to promote the proliferation of anti-inflammatory macrophages.

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