An engineered vesicle and methods of making and using the same

Abstract

The application provides an engineered vesicle and a preparation method and application thereof. The engineered vesicle provided by the application can effectively target the heart, significantly promote M2 type macrophage polarization, and has the functions of improving heart retention rate, inhibiting cardiac fibrosis, improving heart function, and improving the inflammation level of damaged myocardium.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an engineered vesicle and a preparation method and application thereof. BACKGROUND

[0002] Myocardial infarct (MI) has a high incidence and mortality worldwide. Myocardial infarction leads to myocardial remodeling and subsequent heart failure, which is a common and universal disease. Although timely reperfusion and accompanying reoxygenation can greatly reduce the infarct size, MI survivors still face poor prognosis due to worsening cardiac function and progression of heart failure.

[0003] At present, stem cell transplantation is expected to become a replacement therapy for repairing damaged hearts. Although animal experiment results show that stem cell transplantation therapy can effectively improve the cardiac function of MI animals, the results of clinical trials have not shown obvious efficacy and long-term prognosis benefits. This shows that the current cell therapy strategy still needs to be optimized. SUMMARY

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

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

[0006] In the present application, "vesicle" refers to a membrane small vesicle released after the fusion of intracellular multivesicular bodies and cell membranes. The vesicle includes exosomes, microvesicles and apoptotic bodies.

[0007] Further, the vesicle is a nanovesicle.

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

[0009] Further, the vesicle is a stem cell vesicle.

[0010] In the present application, the stem cell includes but is not limited to hematopoietic stem cells, neural stem cells and mesenchymal stem cells.

[0011] Further, the mesenchymal stem cell includes but is not limited to bone marrow mesenchymal stem cells, adipose tissue mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells and dental pulp mesenchymal stem cells.

[0012] Further, the mesenchymal stem cell is selected from bone marrow mesenchymal stem cells.

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

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

[0015] The second aspect of the present application provides a pharmaceutical composition comprising the engineered vesicle of the first aspect of the present application.

[0016] In the present application, the pharmaceutical composition is administered in a gastrointestinal administration dosage form or a non-gastrointestinal administration dosage form.

[0017] Further, the gastrointestinal administration dosage form includes but is not limited to solution, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, dripping pills, suppositories, aerosols, sprays, powder sprays, patches, ointments or creams.

[0018] Further, the non-gastrointestinal administration dosage form includes but is not limited to injection administration dosage form, respiratory administration dosage form, cavity administration dosage form, mucosal administration dosage form, skin administration dosage form.

[0019] Further, the injection administration dosage form includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection and intracavity injection, etc.; the respiratory administration dosage form includes but is not limited to spray, aerosol, powder spray, etc.; the cavity administration dosage form includes but is not limited to suppository, aerosol, effervescent tablet, drop, dripping pill, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage form includes but is not limited to eye drops, nose drops, eye ointment, gargle, sublingual tablet, sticking tablet, patch film, etc.; the skin administration dosage form includes but is not limited to external solution, lotion, liniment, ointment, plaster, paste, patch, etc.

[0020] In the present application, the pharmaceutical composition and at least one additional therapeutic agent or therapy can 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, anti-platelet therapy, heparin, warfarin, fibrinolytic agent, oxygen therapy, vasodilator, analgesic, beta-blocker, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, glycoprotein antagonist, statin, aldosterone antagonist, implantable cardioverter-defibrillator or any combination thereof.

[0021] Further, the pharmaceutical composition includes pharmaceutically acceptable carriers and / or excipients thereof.

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

[0023] The third aspect of the present application provides a method for preparing the engineered vesicle of the first aspect of the present application, which comprises combining a miR-181a-containing vesicle and a platelet membrane.

[0024] In the present application, the miR-181a-containing vesicle can be a cell-derived natural vesicle with miR-181a, a cell-derived vesicle transfected with miR-181a, or a vesicle obtained by fusing miR-181a with a cell-derived vesicle.

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

[0026] In the present application, 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, membrane fusion agents.

[0027] Further, the nucleic acid molecule is introduced into the cell using a non-viral transfection method.

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

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

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

[0031] Further, the liposomes are selected from positively charged liposomes.

[0032] Further, the positively charged liposome comprises Lipo3000 or Lipo2000.

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

[0034] Further, the vesicle is a nanovesicle.

[0035] Further, the nanovesicle has a diameter of 100-200 nm.

[0036] Further, the method further comprises a method for preparing the nanovesicle, wherein the method comprises filtering the cells using filters with different pore sizes.

[0037] Further, the pore size ranges from 0.1 μm to 10 μm.

[0038] Further, the filters with different pore sizes are used in the order of using a filter with a large pore size first and then using a filter with a small pore size.

[0039] Further, the filter with each pore size is used repeatedly.

[0040] Further, the filter with each pore size is used repeatedly three times.

[0041] Further, the method comprises using filters with pore sizes of 5 μm, 1 μm, and 0.4 μm in sequence to filter the cells, repeatedly using the filter with each pore size, and then using a filter with a pore size of 0.22 μm to filter.

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

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

[0044] Further, the method comprises mixing the bone marrow mesenchymal stem cell nanovesicle containing miR-181a-5p with the platelet membrane at a mass ratio of 1:2 and filtering the mixture using filters with different pore sizes.

[0045] Further, the pore size ranges from 0.1 μm to 10 μm.

[0046] Further, the filters with different pore sizes are used in the order of using a filter with a large pore size first and then using a filter with a small pore size.

[0047] Further, the method comprises using filters with pore sizes of 5 μm, 1 μm, and 0.4 μm in sequence to filter the mixture.

[0048] Further, the method further comprises transfecting miR-181a into the stem cells.

[0049] The fourth aspect of the present application provides any one of the following uses:

[0050] (1) the use of the engineered vesicle of the first aspect of the present application or the pharmaceutical composition of the second aspect of the present application in the preparation of a medicament for treating a cardiac disease;

[0051] (2) the use of miR-181a and platelet membrane in the preparation of a medicament for treating a cardiac disease;

[0052] (3) the use of miR-181a and platelet membrane in the preparation of a medicament for enhancing the efficacy of vesicles in treating a cardiac disease.

[0053] Further, the cardiac 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 cardiomyopathy, ventricular dysfunction, heart failure, coronary artery disease, end-stage heart disease, angina pectoris, rheumatic heart disease, or cardiovascular disease.

[0054] Further, the cardiac disease is selected from myocardial infarction.

[0055] In the present application, the drug is administered in a therapeutically effective amount. The term "therapeutically effective amount" refers to the level or amount of the target agent that does not produce significant negative or adverse side effects: (1) delays or prevents the onset of a cardiac disease; (2) slows down or stops the progression, aggravation or worsening of one or more symptoms of a cardiac disease; (3) improves the symptoms of a cardiac disease; (4) reduces the severity or incidence of a cardiac disease; or (5) cures a cardiac disease. The therapeutically effective amount can be administered before the onset of a cardiac disease for a prophylactic effect; or the therapeutically effective amount can be administered after the onset of a cardiac disease for a therapeutic effect or maintenance of the therapeutic effect.

[0056] Further, the effect of the treatment includes, but is not limited to, promoting myocardial repair, increasing cardiac retention rate, inhibiting cardiac fibrosis, improving cardiac function, reducing inflammation level, promoting M2 macrophage polarization, and reducing infarct size.

[0057] Further, the miR-181a and platelet membrane have a synergistic effect in enhancing the efficacy of vesicles in treating a cardiac disease.

[0058] The fifth aspect of the present application provides any one of the following uses:

[0059] (1) the use of the engineered vesicle of the first aspect of the present application or the pharmaceutical composition of the second aspect of the present application in regulating macrophage polarization;

[0060] Further, the engineered vesicle or the pharmaceutical composition promotes M2 type macrophage polarization;

[0061] (2) Use of the engineered vesicle according to the first aspect of the present application or the pharmaceutical composition according to the second aspect of the present application in promoting proliferation of anti-inflammatory macrophages.

[0062] The sixth aspect of the present application provides any one of the following methods:

[0063] (1) A method for regulating macrophage polarization in vitro, the method comprising regulating macrophage polarization by the engineered vesicle according to the first aspect of the present application or the pharmaceutical composition according to the second aspect of the present application;

[0064] Further, the engineered vesicle or the pharmaceutical composition promotes M2 type macrophage polarization;

[0065] (2) A method for promoting proliferation of anti-inflammatory macrophages in vitro, the method comprising administering the engineered vesicle according to the first aspect of the present application or the pharmaceutical composition according to the second aspect of the present application to promote proliferation of anti-inflammatory macrophages.

[0066] The present application has the advantages and beneficial effects:

[0067] The present application provides an engineered vesicle and a preparation method and application thereof. The engineered vesicle provided by the present application can effectively target the heart and significantly promote M2 type macrophage polarization, and has the functions of improving heart retention rate, inhibiting cardiac fibrosis, improving heart function and improving the level of inflammation in damaged myocardium. BRIEF DESCRIPTION OF DRAWINGS

[0068] Figure 1 is a morphological and biological property analysis diagram of P-181-NV, A is a schematic diagram of the preparation process of P-181-NV nanovesicle; B is an expression result diagram of miR-181a-5p in stem cells; C is an expression result diagram of miR-181a-5p in vesicles; D is an electron microscope photo of PM, NV, P-NV; E, F, G are expression level result diagrams of Alix, P-selectin and β-actin in PM, NV, P-NV, 181-NV, P-181-NV; H, I are NTA result diagrams of NV, P-NV, 181-NV, P-181-NV; J is a ZETA result diagram of NV, P-NV, 181-NV, P-181-NV.

[0069] Figure 2Figure is the result of P-181-NV reducing cardiac fibrosis and improving cardiac function (bar = 50 pm), A, B are the IVIS live imaging data of heart; C, D are the Masson staining result and quantification; E is the echocardiogram result; F is the EF result; G is the Cardiac Output result; H is the FS result; I is the Stroke Volume result; J is the LVID;s result; K is the LVID;d result.

[0070] Figure 3 Figure is the result of P-181-NV promoting the differentiation of CX3CR1 positive anti-inflammatory macrophages at 3 and 7 days after MI (bar = 50 pm), A-C are fluorescence images of anti-inflammatory macrophages and pro-inflammatory macrophages at 3 days after MI; D-F are fluorescence images of anti-inflammatory macrophages and pro-inflammatory macrophages at 7 days after MI.

[0071] Figure 4 Figure is the result of P-181-NV promoting the differentiation of M2 macrophages at 3, 7, 14 days after MI (bar = 50 pm), A, B are the results of fluorescence intensity of CD206 positive M2 macrophages detected by immunofluorescence at 3 days after MI; C, D are the results of fluorescence intensity of CD206 positive M2 macrophages detected by immunofluorescence at 7 days after MI; E, F are the results of fluorescence intensity of CD206 positive M2 macrophages detected by immunofluorescence at 14 days after MI.

[0072] Figure 5 Figure is the result of P-181-NV promoting macrophage polarization in in vitro experiments, A, B are the results of flow cytometry detecting the phagocytosis of P-181a-NV; C-F are the results of flow cytometry detecting the polarization of macrophages by P-181a-NV and their statistical graphs; G-I are the results of Western blot detecting the polarization of macrophages by P-181a-NV and their statistical graphs.

[0073] 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 wrapped with platelet membrane. TEM: transmission electron microscopy; NTA: nanoparticle tracking analysis. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0074] The application will be further described below in connection with the embodiments. The following description is only the preferred embodiments of the application and is not intended to limit the application in other forms. Any skilled person in the art can modify the above-mentioned technical content into equivalent embodiments with equivalent changes. Any simple modification or equivalent change to the following embodiments without departing from the technical essence of the application falls within the protection scope of the application.

[0075] Embodiments

[0076] I. Experimental methods

[0077] 1. Cell culture

[0078] Mouse bone marrow mesenchymal stem cells were purchased from Plnomer (CP-M131), and the culture medium was Plnomer mouse bone marrow mesenchymal stem cell special culture medium (CM-M131-100). THP-1 cells were purchased from Haixing Biology (TCH-C361), and were cultured in 1640 culture medium (Gibco) containing 10% FBS (Gibco) at 37°C and 5% CO2. T293 cells were purchased from Haixing Biology (TCH-C101), and were cultured in high-glucose DMEM (Gibco) containing 10% FBS (Gibco) at 37°C and 5% CO2.

[0079] 2. Transfection

[0080] 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 then the cells were placed in an incubator for 24 hours. The cells were collected for PCR verification and preparation of miR-181a-5p-loaded vesicles.

[0081] 3. Preparation of platelet membrane

[0082] Take 10 ml of mouse blood, centrifuge and take the upper layer to the blood collection tube. Take 2 ml of supernatant and mix with PBS buffer containing EDTA and PGE1, gently invert up and down, and centrifuge at room temperature for minutes. Resuspend the lower layer in PBS containing protease inhibitors and phosphatase inhibitors, and freeze-thaw repeatedly at-80°C for 3 times. After the platelet extract is completely thawed at room temperature, centrifuge and repeat 5 times. Discard the supernatant, resuspend and wash 3 times with PBS containing protease inhibitors and phosphatase inhibitors, then resuspend with PBS, and ultrasonically prepare platelet membrane fragment mixture. Electron microscope is used to detect the morphology of platelet membrane.

[0083] 4. Preparation of nanovesicles

[0084] Bone marrow mesenchymal stem cells (BM-MSCs) were trypsinized when the confluence reached 80%. After washing twice with PBS, the cells were collected for the preparation of nanovesicles. The cells were extruded through 5 μm, 1 μm, and 0.4 μm filters (Whatman) using a LiposoFast LF-50 (Avestin, York, UK) three times for each pore size. Finally, the nanovesicles with a diameter of 100-200 nm were obtained by filtration through a 0.22 μm filter and stored at -80°C. The protein concentration of the nanovesicles was determined using a BCA protein concentration determination kit. The platelets and nanovesicles were mixed at a mass ratio of 2:1, and then extruded through the three filters. The platelet-coated nanovesicles were finally prepared. The preparation process is shown in FIG. A of Figure 1 .

[0085] 5. Identification of nanovesicles

[0086] The morphology, size, particle size distribution, and membrane potential of the nanovesicles were determined using transmission electron microscopy, nanoparticle tracking technology, and a zeta potential analyzer. Western blot was used to detect the expression levels of the exosome protein marker Alix (Proteintech, 20597-1-AP), the platelet membrane marker Pselectin (Proteintech, 60322-1- Ig), and the intracellular reference protein β-actin (Abbkine, A23910, A23720).

[0087] 6. Flow cytometry

[0088] When the cell density reached 80%, the cells were collected by trypsinization. The cells were washed with sterile PBS for 2-3 times. The cells were resuspended in 100 μl PBS per group, and the flow detection 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, the cells were washed with PBS for 2 times, resuspended in 500 μl PBS, and then detected by flow cytometry within 30 minutes.

[0089] 7. PCR detection

[0090] Total RNA was extracted using the Trizol method, and the RNA concentration, 260 / 230 value, and 260 / 280 value of the sample were determined using a nucleic acid detector (NanoDrop, ND1000). Reverse transcription and fluorescent quantitative amplification detection were performed using the reverse transcription system (R323) and qRTPCR system (Q711) of Nuoyuan Company, respectively. The relative expression level of the target gene was calculated using the 2^-ΔΔCt method.

[0091] 8. Western blot

[0092] Total protein of cells or vesicles was extracted, and 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 apparatus, and proteins were transferred from SDS-PAGE to PVDF membranes using a semi-dry transfer system. After blocking (5% skim milk), primary antibody incubation, and secondary antibody incubation, respectively, the results were obtained using an instrument (LICOR Odyssey CLX-0664). The primary antibodies we used were Alix (Proteintech, 12422-1-AP, 1:5000), P-selectin (60322-1-Ig, 1:2000), CD206 (18704-1-AP, 1:1000), CD163 (16646-1-AP, 1:1000), and β-actin (Affinity, T0022). After the anti-mouse fluorescent secondary antibody (Abbkine, A23910) and the anti-rabbit fluorescent secondary antibody (Abbkine, A23720) were diluted 1:500 and incubated at room temperature for 1.5 hours in the dark, they were washed with TBST three times, and the results were detected using an LI-COR Odyssey CLX-0664 instrument. The band gray value was determined using an ImageJ imaging system, and statistical analysis was performed.

[0093] 9. Preparation of animal model of myocardial infarction

[0094] SPF C57 mice (6-8 weeks old) were purchased from Beijing Huafukang Company. The mice were anesthetized with isoflurane gas, and after body position fixation, the skin was cut transversely 5 mm from the left side of the sternum, the subcutaneous tissue was separated, and the pectoralis major and pectoralis minor muscles were suddenly separated, and the intercostal muscle was exposed. Open the intercostal space and squeeze the heart with the left hand until the heart is swollen. Ligation was performed with 8-0 fine thread at 5 mm below the left auricle, and the myocardium was observed to be white. Quickly close the chest and suture the skin with a 3-0 thread.

[0095] 10. Small animal live tracking and echocardiography detection

[0096] DIR (D1220A) labeled nanovesicles were injected into the tail vein of mice, and live imaging was performed using a PerkinElmer instrument at 1, 3, 7, and 14 days, respectively. At 28 days, EF (Ejection Fraction), FS (Fractional Shortening), CO (Cardiac Output), SV (Stroke Volume), and LVID;s and LVID;d were detected by echocardiography (VisualSonics).

[0097] 11. Masson

[0098] Heart tissues were collected at 0, 3, 7, 14, and 28 days after vesicular drug injection, respectively. After fixation with 4% paraformaldehyde, the tissues were transferred to ethanol for paraffin embedding. Paraffin sections were prepared for Masson trichrome staining. The fibrosis area size was determined using ImageJ.

[0099] 12. Tissue fluorescence staining

[0100] After the heart tissues were collected, the heart tissues were fixed with 4% paraformaldehyde, dehydrated with 15%-30% gradient sucrose solution, and then embedded with OCT for storage at -80°C. The heart sections were incubated with primary antibodies overnight at 4°C. The primary antibodies included Sarcomeric Alpha Actinin, 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 for mounting. Microscopy (NIKON Eclipse ci) was used for observation.

[0101] 13. Statistical analysis

[0102] In this study, prism statistical software was used for data analysis and plotting. The results of the measurement data were expressed as mean ± standard deviation (Mean ± SD). The t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. The test level was α = 0.05, and P < 0.05 was considered statistically significant.

[0103] II. Experimental results

[0104] 1. Construction and identification of P-181-NV nanovesicles

[0105] The experimental results are shown in Table 1. Figure 1The results of PCR showed that the level of miR-181a-5p in stem cells of the group transfected with miR-181a-5p mimics was significantly increased (P<0.05, FIG. B) compared with the untransfected group, and the level of miR-181a-5p in the exosomes derived therefrom was also significantly increased (P<0.05, FIG. C), proving that the mesenchymal stem cell-derived nanovesicles modified with miR-181a-5p (181-NV) were successfully prepared. Further, the NV-miR181a-5p was prepared into platelet-modified NV-miR181a-5p (P-181-NV) by using platelet membrane coating. The results of transmission electron microscopy showed that the engineered nanovesicle particles were in the form of cups or circles, and the NVs coated with platelet membrane were in the form of double-membrane structure (FIG. D). Western blot proved that the vesicles all expressed Alix, and P-181-NV and P-NV both significantly expressed the platelet-specific marker p-selectin, indicating that the platelet membrane coating was successful (P<0.05, Figure 1 E-G). The results of NTA showed that the diameters were about 100-200 nm, Figure 1 H-I). The results of ZETA potential showed that there was no significant difference in the potentials of the four types of vesicles (FIG. J), and the above results proved that the P-181-NV nanovesicles were successfully prepared.

[0106] 2. The P-181-NV nanovesicles have high targeting and can improve the cardiac function after MI

[0107] To verify the high targeting migration ability of the engineered nanovesicles P-181-NV to the injured myocardium, the DIR-labeled nanovesicles were injected into the body of 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 respectively 24 hours after myocardial infarction, and the homing ability of the vesicles to the injured myocardium was detected on day 1, 3, 7 and 14. The results are shown in Figure 2 As shown, the heart homing rate of the P-181-NV group was significantly increased on day 1, 3 and 7, and the fluorescence signal of the vesicles in the heart almost disappeared on day 14 (FIG. A-B). Figure 2 As can be seen, the P-181-NV nanovesicles can significantly improve the retention rate in the heart (P<0.05, Figure 2 A-B), which laid an important foundation for exploring its functional characteristics of repairing the injured myocardium. Next, we divided the mice into 6 groups (Sham group, MI group, NV group, P-NV group, 181-NV group, P-181-NV group) and gave different treatments, and performed Masson staining after 28 days.

[0108] The results of Masson staining showed that P-181-NV significantly reduced the area of cardiac fibrosis (P<0.05, Figure 2(CD), the P-181-NV group showed a 51.07% reduction in cardiac fibrosis compared to the NV group, the P-NV group a 16.61% reduction, and the P-181-NV group a 73.39% lower rate of cardiac fibrosis compared to the NV group. Based on 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. Substituting the values ​​into the calculation, we obtain q=E. 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.

[0109] Echocardiographic results showed that the P-181-NV group significantly improved EF, FS, and CO compared to other groups (P<0.05). Figure 2 EK).

[0110] The EF of the 181-NV group was increased by 11.46% compared to the NV group, the P-NV group by 2.68%, and the P-181-NV group by 25.78%. Based on the EF improvement rate, the synergistic q-value was calculated using the King's Law to evaluate whether miR-181a-5p and platelet membrane have a synergistic effect in improving EF. Substituting the values ​​into the calculation, we get q = EA + B / (EA + EB - EA * EB) = 0.2578 / (0.1146 + 0.0268 - 0.1146 * 0.0268) = 1.8636, i.e., q = 1.8636, q > 1.15, indicating that miR-181a-5p and platelet membrane have a synergistic effect in improving EF.

[0111] The FS in the 181-NV group was 11.21% higher than that in the NV group, the FS in the P-NV group was 1.75% higher than that in the NV group, and the FS in the P-181-NV group was 30.38% higher than that in the NV group. Based on 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. Substituting the values ​​into the calculation, we get 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.

[0112] 181-NV group relative to the NV group SV increased by 11.85%, P-NV group relative to the NV group SV increased by -7.68%, P-181-NV group relative to the NV group SV increased by 27.13%. According to the SV increase rate, the synergistic q value is calculated by using the Jin's formula, to evaluate whether miR-181a-5p and platelet membrane have synergistic effect in improving SV, and the calculation is brought in 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 synergistic effect in improving SV.

[0113] 181-NV group relative to the NV group LVID;s decreased by 2.93%, P-NV group relative to the NV group LVID;s decreased by 2.92%, P-181-NV group relative to the NV group LVID;s decreased by 32.47%. According to the LVID;s decrease rate, the synergistic q value is calculated by using the Jin's formula, to evaluate whether miR-181a-5p and platelet membrane have synergistic effect in reducing LVID;s, and the calculation is brought in 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 synergistic effect in reducing LVID;s.

[0114] The above results show that P-181-NV can significantly improve the heart retention rate, inhibit cardiac fibrosis and improve the cardiac function of ischemic heart disease, and miR-181a-5p and platelet membrane have synergistic effect.

[0115] 3. P-181-NV nanovesicles promote the increase of anti-inflammatory macrophage levels after myocardial infarction

[0116] To explore whether P-181-NV nanovesicles play a role in repairing the damaged myocardium after myocardial infarction by regulating macrophage polarization, 4 groups (Sham, MI, P-NV, P-181-NV) of nanovesicles were injected into the body of the myocardial infarction mice, CX3CR1 and CD206 labeled anti-inflammatory macrophage phenotype, and CCR2 labeled pro-inflammatory macrophage phenotype. The results showed that on the 3rd and 7th days, the CX3CR1 (P<0.05, Figure 3 A-F) and CD206 positive cell rates of P-181-NV nanovesicles were significantly increased (P<0.05, Figure 4A-D), and CD206 levels were also significantly increased at 14 days (P<0.05, Figure 4 E-F). These results suggest that engineered nanovesicles P-181-NV can significantly improve the inflammatory levels of damaged myocardium after myocardial infarction.

[0117] 4. P-181-NV nanovesicles regulate macrophage polarization

[0118] This study further explored the polarization effect of P-181-NV on anti-inflammatory macrophages. First, we co-cultured Did-labeled NVs with macrophages for different times, and flow cytometry was used to detect the phagocytosis efficiency of macrophages. We found that almost all vesicles were phagocytosed by macrophages 2 hours after co-incubation, which proved that they could be well taken up by macrophages (Fig. 4A-B). Figure 5 A-B). Subsequently, we used RAW246.7 to verify the ability of vesicles to promote macrophage polarization. RAW246.7 macrophages were divided into three groups (Control, P-NV, P-181-NV) and treated differently. Flow cytometry was used to detect the proportion of M2 macrophages 48 hours after treatment, and the results showed that the proportion of M2 macrophages in the P-181-NV group was significantly increased (P<0.05, Figure 5 C-F), and 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 G-I). 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.

[0119] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. An engineered vesicle, characterized in that, The engineered vesicle contains a vesicle, miR-181a, and a platelet membrane; The vesicle is a stem cell-derived vesicle; The miR-181a is miR-181a-5p; The mass ratio of the stem cell-derived vesicle + miR-181a-5p to the platelet membrane is 1:

2.

2. The engineered vesicle of claim 1, wherein, The vesicle is a nanovesicle.

3. The engineered vesicle of claim 2, wherein, The nanovesicle has a diameter of 100-200 nm.

4. The engineered vesicle of any one of claims 1-3, wherein, The stem cell includes a hematopoietic stem cell, a neural stem cell, and a mesenchymal stem cell.

5. The engineered vesicle of claim 4, wherein, The mesenchymal stem cell includes a bone marrow mesenchymal stem cell, an adipose tissue-derived mesenchymal stem cell, an umbilical cord-derived mesenchymal stem cell, a placenta-derived mesenchymal stem cell, and a dental pulp-derived mesenchymal stem cell.

6. The engineered vesicle of claim 5, wherein, The mesenchymal stem cell is selected from a bone marrow mesenchymal stem cell.

7. A pharmaceutical composition, characterized by, The pharmaceutical composition includes the engineered vesicle of any one of claims 1-6.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient thereof.

9. A method of making the engineered vesicle of any one of claims 1-6, wherein, The method includes combining a vesicle containing miR-181a and a platelet membrane, and the mass ratio of the vesicle containing miR-181a to the platelet membrane is 1:

2.

10. The method of claim 9, wherein, The vesicle containing miR-181a is a vesicle derived from a stem cell after transfection of miR-181a into the stem cell.

11. The method of claim 9, wherein, The vesicle is a nanovesicle.

12. The method of claim 11, wherein, The nanovesicle has a diameter of 100-200 nm.

13. The method of claim 11, wherein, The method further includes a preparation method of the nanovesicle, and the preparation method includes filtering cells using filters with different filter diameters.

14. The method of claim 13, wherein, The filter diameter ranges from 0.1 μm to 10 μm.

15. The method of claim 13, wherein, The sequence of using filters with different filter diameters is to use a filter with a large filter diameter first and then use a filter with a small filter diameter.

16. The method of claim 15, wherein, The cells are repeatedly filtered through filters with each filter diameter.

17. The method of claim 16, wherein, The repetition is three times.

18. The method of claim 13, wherein, The method includes sequentially using filters with filter diameters of 5 μm, 1 μm, and 0.4 μm to filter the cells, repeatedly filtering the cells through filters with each filter diameter three times, and then using a filter with a filter diameter of 0.22 μm to filter.

19. The method of claim 9, wherein, The method includes mixing bone marrow mesenchymal stem cell nanovesicles containing miR-181a-5p and a platelet membrane at a mass ratio of 1:2, and filtering the mixture using filters with different filter diameters.

20. The method of claim 19, wherein, The filter diameter ranges from 0.1 μm to 10 μm.

21. The method of claim 19, wherein, The sequence of using filters with different filter diameters is to use a filter with a large filter diameter first and then use a filter with a small filter diameter.

22. The method of claim 21, wherein, The method includes sequentially using filters with filter diameters of 5 μm, 1 μm, and 0.4 μm to filter the mixture.

23. Use of the engineered vesicle of any one of claims 1-6 or the pharmaceutical composition of any one of claims 7-8 in the preparation of a medicament for treating a heart disease, and the heart disease is myocardial infarction.

24. A method of modulating macrophage polarization in vitro, comprising contacting macrophages with a compound of any one of claims 1-23. 25 The method includes regulating macrophage polarization by the engineered vesicle of any one of claims 1-6 or the pharmaceutical composition of any one of claims 7-8.

25. The method of claim 24, wherein, The engineered vesicle or the pharmaceutical composition promotes M2-type macrophage polarization.

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