Myocardial targeting engineered extracellular vesicle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a myocardial targeted engineered extracellular vesicle as well as a preparation method and application thereof. The invention provides an engineered extracellular vesicle. The engineered extracellular vesicle is an engineered extracellular vesicle subjected to overexpression modification of gap junction protein alpha 1; the amino acid sequence of the gap connecting protein alpha 1 is shown as SEQ ID NO. 1. The engineered extracellular vesicles are modified by expression gap connecting protein alpha1, and compared with a control, the engineered extracellular vesicles modified by the gap connecting protein alpha1 are more ingested by myocardial cells, have higher heart targeting performance and can provide targeting drug carriers for patients with heart diseases. The invention provides a new drug research and development approach and means for clinical treatment of heart failure or myocardial injury, also provides a heart targeted modification tool for industrial research and development and basic scientific research users, and has very important medicinal value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a myocardial-targeted engineered extracellular vesicle, its preparation method, and its application. Background Technology

[0002] Cardiovascular diseases (CVD) are a serious threat to human health, mainly including ischemic heart disease, hypertensive heart disease, and cardiomyopathy, often leading to heart failure in the end stage. However, there are currently no targeted therapeutic drugs that target cardiomyocytes in clinical practice. Therefore, exploring effective drug carriers is of great significance for improving the prognosis of patients with myocardial damage and heart failure.

[0003] Extracellular vesicles (EVs) are lipid vesicles with a double-membrane structure secreted by cells. They contain various substances such as proteins, lipids, and nucleic acids and play an important role in cell communication. Due to their low immunogenicity and circulatory stability, they can be used as drug delivery carriers to carry therapeutic molecules in clinical practice. Increasing the targeting specificity of EVs is of great significance for enhancing their medical value.

[0004] Currently, the targeting ability of extracellular vesicles can be enhanced by overexpressing artificial peptides. However, these artificial peptides may trigger an immune response, leading to rapid clearance of EVs, and may also alter membrane protein conformation, interfering with the inherent homing, uptake, and signaling functions of EVs. Therefore, how to screen natural proteins with highly efficient targeting capabilities for the functional modification of extracellular vesicles has become a key problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a myocardial-targeting engineered extracellular vesicle, its preparation method, and its application. This invention identifies a natural cellular protein gap junction protein α1 with myocardial targeting capability. The engineered extracellular vesicle, after being modified by expressing gap junction protein α1, exhibits enhanced cardiac targeting and can be used as a drug carrier. It also provides a cardiac-targeting modification tool for industrial R&D and basic scientific research users.

[0006] This invention provides a myocardial-targeted engineered extracellular vesicle, wherein the engineered extracellular vesicle is an engineered extracellular vesicle modified by overexpression of gap junction protein α1; the amino acid sequence of the gap junction protein α1 is shown in SEQ ID NO. 1.

[0007] The present invention also provides a method for preparing the engineered extracellular vesicles described above, wherein a vector overexpressing gap junction protein α1 is mixed with human kidney embryo 293T cells for culture, the cell supernatant is collected, and the engineered extracellular vesicles are obtained by centrifugation.

[0008] As a preferred embodiment, the vector overexpressing gap junction protein α1 comprises a lentiviral vector overexpressing gap junction protein α1.

[0009] As a preferred embodiment, the base vector of the lentiviral vector includes pLVX-puro.

[0010] As a preferred embodiment, the gap junction protein α1 is inserted into the pLVX-puro BamH I and Age Between Ⅰ enzyme cleavage sites.

[0011] As a preferred embodiment, the vector for overexpressing gap junction protein α1 includes a vector for overexpressing humanized gap junction protein α1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0012] The present invention also provides the application of the engineered extracellular vesicles described above as drug delivery carriers.

[0013] The present invention also provides the use of the engineered extracellular vesicles described above in the preparation of medicaments for the prevention and / or treatment of heart disease.

[0014] As a preferred embodiment, the cardiac disease includes myocardial injury and / or heart failure.

[0015] The present invention also provides a medicament for the prevention and / or treatment of heart disease according to the above-described scheme, the medicament comprising the engineered extracellular vesicles described above and an active ingredient loaded on the engineered extracellular vesicles.

[0016] This invention provides a myocardial-targeting engineered extracellular vesicle, wherein the engineered extracellular vesicle is modified by overexpression of gap connexin α1; the amino acid sequence of gap connexin α1 is shown in SEQ ID NO. 1. The engineered extracellular vesicles of this invention, modified by expression of gap connexin α1, exhibit greater uptake by cardiomyocytes and higher cardiac targeting compared to the control, providing a targeted drug carrier for patients with heart disease. This invention provides a new drug development pathway and method for the clinical treatment of heart failure or myocardial injury, and also provides cardiac-targeting modification tools for industrial R&D and basic research users, possessing significant pharmaceutical value. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The spectrum of the hsa-pLVX-GJA1-OE-puro vector in Example 1; Figure 2 The images show the results of RT-qPCR and Western blot analysis to verify the overexpression efficiency of humanized gap junction protein α1 at the cellular level in Example 1; where A represents the GJA1 gene expression level and B represents the GJA1 protein expression level. Figure 3 The following are the detection results in Test Example 2; where A is the Western blot result of the efficiency of human kidney embryo 293T cell line stably expressing gap connexin α1; B~C are the relevant characterization results of engineered extracellular vesicles modified with humanized gap connexin α1; and D is the Western blot result of the expression efficiency of gap connexin α1 in engineered extracellular vesicles. Figure 4 The figure shows the results of the experiment on the uptake of engineered extracellular vesicles modified with humanized gap junction protein α1 by cardiomyocytes in Example 3; where A is primary neonatal rat cardiomyocytes NRCM; and B is the human cardiomyocyte cell line AC16. Figure 5 Figure 4 shows the results of an experiment detecting the uptake of engineered extracellular vesicles modified with humanized gap junction protein α1 by mouse heart tissue in test example 4. Figure 6 The figure shows the experimental results of the uptake of engineered extracellular vesicles modified with humanized gap junction protein α1 and extracellular vesicles connected to cardiac targeting peptides by cardiac tissue in mice after acute myocardial ischemia-reperfusion injury surgery in Example 5. Detailed Implementation

[0019] This invention provides a myocardial-targeted engineered extracellular vesicle, wherein the engineered extracellular vesicle is an engineered extracellular vesicle modified by overexpression of gap junction protein α1; the amino acid sequence of the gap junction protein α1 is shown in SEQ. IDNO.1 shows: MGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPT LLYLAHVFYVMRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTC KRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSMSPPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRASSRASSRPRPDDLEI. The gap junction protein α1 has a Gene ID of 2697 in NCBI.

[0020] Gap junction protein α1 (GJA1) is an intercellular communication linker protein that is highly specific and abundantly expressed in the heart, enriched in ventricular cardiomyocytes, and expressed at very low levels or not at all in non-cardiomyocyte tissues. This makes GJA1 a specific molecular marker for cardiomyocytes and provides a clear target recognition tag for extracellular vesicles (EVs). The engineered extracellular vesicles of this invention, after being modified with GJA1 overexpression, can interact homologously with GJA1 on the cardiomyocyte membrane, triggering the opening of GJA1 hemichannels on the cardiomyocyte membrane, or inducing the formation of a "gap junction-like contact area" between EVs and the cell membrane, thereby promoting the efficient uptake of EVs by cardiomyocytes through endocytosis. Furthermore, the GJA1-modified engineered EVs have a surface GJA1 that acts as a "self-recognition signal," reducing phagocytosis by macrophages and dendritic cells, prolonging the circulation time of EVs in the blood, and enhancing their cardiac targeting.

[0021] The present invention also provides a method for preparing the engineered extracellular vesicles described above, wherein a vector overexpressing gap junction protein α1 is mixed with human kidney embryo 293T cells for culture, the cell supernatant is collected, and the engineered extracellular vesicles are obtained by centrifugation.

[0022] In one embodiment, the vector overexpressing gap junction protein α1 includes a lentiviral vector overexpressing gap junction protein α1. The present invention does not impose any particular limitation on the packaging process of the lentivirus; conventional lentiviral packaging methods in the art can be used.

[0023] In one embodiment, the base vector of the lentiviral vector includes pLVX-puro. In another embodiment, the gap junction protein α1 is inserted into the pLVX-puro. BamH I and Age Between Ⅰ enzyme cleavage sites. BamH I and Age I. The restriction enzyme sites are adjacent to the multiple cloning site of pLVX-puro and are free from interference by other redundant restriction enzyme sites. After restriction enzyme digestion, it can ensure that the target gene is inserted into the correct region downstream of the CMV promoter and expressed stably; and BamH I and Age All enzymes are commercially available and mature restriction enzymes, with short recognition sequences, high specificity, high cleavage efficiency, and high recombination efficiency. BamH I and Age The restriction enzyme recognition sequence is not located in the LTR, ψ packaging signal, or puro gene region, so restriction enzyme digestion will not affect the vector's packaging efficiency and screening function, ensuring the efficiency of stable transgenic line construction; at the same time, BamH I and Age I. The choice of restriction enzyme sites does not affect the subsequent secretion efficiency and function of extracellular vesicles, and is suitable for subsequent experimental requirements.

[0024] This invention involves co-culturing a vector overexpressing gap junction protein α1 with human kidney embryo 293T cells. As one implementation method, human kidney embryo 293T cells are cultured to the logarithmic growth phase. When the cell confluence reaches 70%–80%, the old cell culture medium is removed, and transfection reagent is added for transfection. Puromycin is added to remove cells that have not been transfected with lentivirus. This invention utilizes human kidney embryo 293T cells to prepare engineered extracellular vesicles, offering several advantages: the 293T cell line grows rapidly and secretes extracellular vesicles efficiently; the 293T cell line can grow stably in serum-free medium, and collecting the serum-free supernatant avoids interference from bovine extracellular vesicles in serum; the 293T cell line is non-tumorigenic and does not secrete toxins or pathogenic factors, resulting in higher safety of the secreted extracellular vesicles in subsequent animal experiments; the 293T cell line has a clear genetic background, reducing non-specific interference, and the stable transfected strain constructed from the 293T cell line can maintain target gene expression for a long period, resulting in higher stability of the secreted extracellular vesicle load.

[0025] After mixed culture, the cell supernatant is collected and centrifuged to obtain the engineered extracellular vesicles. This invention does not specifically limit the centrifugation method for the engineered extracellular vesicles; conventional centrifugation methods in the art are acceptable. As one embodiment, the centrifugation process further includes filtering to obtain the supernatant, ultracentrifuging, and discarding the supernatant to obtain the engineered extracellular vesicles. In a specific embodiment of this invention, the specific operations after collecting the cell supernatant include: centrifugation at 500g for 5 min; centrifugation at 3000g for 10 min; centrifugation at 12000g for 45 min; filtering the supernatant using a 0.22 μm filter; and centrifuging the filtered supernatant at 100000g for 70 min. All centrifugations are performed at 4℃ to obtain engineered extracellular vesicles modified with gap junction protein α1 overexpression. As one embodiment, the concentration of the engineered extracellular vesicles modified with gap junction protein α1 in this invention is 40 μg / mL or 10 μg / mL. 10 / mL; the concentration of the engineered extracellular vesicles modified with gap junction protein α1 is the concentration in phosphate buffer solution.

[0026]

[0027] This invention also provides the application of the engineered extracellular vesicles described above as drug delivery carriers. The engineered extracellular vesicles of this invention, modified by overexpression of gap junction protein α1, can promote the efficient uptake of EVs by cardiomyocytes via endocytosis, exhibiting higher cardiac targeting and providing targeted drug carriers for patients with heart disease.

[0028] This invention also provides the application of the engineered extracellular vesicles described above in the preparation of drugs for the prevention and / or treatment of heart diseases. As one embodiment, the heart diseases include myocardial injury and / or heart failure. The engineered extracellular vesicles of this invention have higher cardiac targeting specificity, providing new drug development pathways and methods for the clinical treatment of heart failure or myocardial injury, while also providing cardiac-targeting modification tools for industrial R&D and basic research users.

[0029] This invention also provides a medicament for the prevention and / or treatment of heart disease, the medicament comprising the engineered extracellular vesicles described above and an active ingredient loaded on the engineered extracellular vesicles. As one embodiment, the medicament further comprises pharmaceutically acceptable excipients. This invention does not specifically limit the type of excipients; they can be selected according to conventional drug dosage forms. For example, the excipients may include one or more of buffers, encapsulating agents, fillers, binders, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorbents. As another embodiment, this invention selects the corresponding excipients according to the drug dosage form. As one embodiment, the dosage form of the medicament of this invention includes tablets, powders, granules, capsules, decoctions, oral liquids, injections, or suppositories, more preferably granules, capsules, decoctions, oral liquids, or injections, and more preferably injections.

[0030] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1 1. Construction of humanized gap junction protein α1 overexpression plasmid, the steps are as follows: (1) Initial vector digestion The pLVX-puro vector (Addgene plasmid #125839) was mixed according to the system described in Table 1, gently aspirated and mixed, and incubated in a 37°C water bath for 1 hour. After enzyme digestion, agarose gel electrophoresis was performed to recover the target fragment. The pLVX-puro vector was as follows: Figure 1 As shown.

[0032] Table 1. Vector Enzyme Digestion System

[0033] (2) Obtaining humanized gap junction protein α1 fragment The nucleotide sequence of the positive-strand primer hsa-GJA1-F for preparing humanized gap junction protein α1 is 5'-CGCGGATCCATGGGTGACTGGAGCGCCTTA-3' (SEQ ID NO.3); the nucleotide sequence of the reverse-strand primer hsa-GJA1-R is 5'-TATAACCGGTCTAGATCTCCAGGTCATCAGGCCG-3' (SEQ ID NO.4). The sequences obtained by amplification using the above primers include the restriction enzyme sites linked to the pLVX-puro vector and the hsa-GJA1 sequence; the target fragment is subjected to agarose gel electrophoresis; the recovered target fragment is mixed according to the system described in Table 2, gently aspirated and mixed, and incubated in a 37℃ water bath for 1 h; after enzyme digestion, the target fragment is cleaned and recovered.

[0034] Table 2. Enzyme digestion system for the target fragment

[0035] (3) Connection of the target fragment to the carrier The pLVX-puro vector digested in step (1) and the target fragment digested in step (2) were mixed according to the system described in Table 3 and ligated overnight at 16°C.

[0036] Table 3 Connection System

[0037] The ligation product obtained in step (3) was then transformed into *E. coli*. After transformation, the target strain was screened using antibiotic-free LB medium and sent to Qingke Company for sequencing. Plasmid was extracted using a nucleic acid extraction kit to obtain a humanized gap connexin α1 overexpression plasmid (hereinafter referred to as hsa-GJA1-OE plasmid). Sequencing results showed that the sequencing results were consistent with the target sequence, indicating that the humanized hsa-GJA1-OE plasmid was successfully constructed.

[0038] Example 2 Plasmid Transfection 293T cells were randomly divided into two groups: a control group and an experimental group, denoted as the Ctr, hsa-GJA1-OE group. The specific procedures are as follows: (1) 293T cells were passaged into 12-well plates and cultured to a cell density of 70%–80%; (2) Preparation of cell transfection reagent A: Add 2 μg of hsa-GJA1-OE plasmid to 500 μL of serum-free culture medium; add an equal amount of pLVX-puro plasmid to the Ctr group; mix gently and let stand for 5 min; (3) Prepare cell transfection reagent B: Add 4 μL of Lipo2000 transfection reagent to 500 μL of serum-free culture medium, mix gently, and let stand for 5 min; (4) Gently mix cell transfection solution A with solution B and let stand at room temperature for 15-20 minutes; (5) Remove the old cell culture medium from the cell plate and add 1 mL of the mixed plasmid transfection reagent to each well; (6) Replace with normal culture medium after 6 to 8 hours.

[0039] Test Example 1 1. Using 293T cells transfected with the control pLVX-puro plasmid as the control group, real-time quantitative PCR was used to detect the 293T cells transfected with the hsa-GJA1-OE plasmid in Example 2. The internal reference gene was 18s. The steps are as follows: (1) The fluorescence quantitative PCR reaction system was prepared according to the reaction system in Table 4. Each sample was replicated twice for fluorescence quantitative PCR detection. The primers were purchased from Beijing Qingke Biotechnology Co., Ltd., and the sequence information is shown in Table 5.

[0040] Table 4. PCR detection system

[0041] Table 5 Primer sequences for real-time quantitative PCR

[0042] (2) After the configuration is completed, the real-time fluorescence quantitative PCR reaction can be performed according to the reaction procedure in Table 6.

[0043] Table 6 Real-time quantitative PCR reaction procedure

[0044] (3) The experimental analysis was conducted using a relative quantitative method, which reflects the relative expression level of the target gene in each experimental group relative to the control group. After taking the average value of parallel replicates, the results were analyzed using 2... -ΔΔCt The calculation is performed, where ΔCt = target gene Ct value - internal reference Ct value, and ΔΔCt = ΔCt values ​​of all groups including the control group - average ΔCt value of the control group. The calculation results are as follows: Figure 2 As shown in A and Table 7, where Indicates significance of difference P <0.001.

[0045] Table 7 Test Results

[0046] Depend on Figure 2From A, we can conclude that the hsa-GJA1-OE plasmid was successfully constructed and stably overexpressed humanized gap junction protein α1 at the RNA level.

[0047] 2. Western blot analysis of the expression efficiency of hsa-GJA1-OE plasmid in Example 2 After the treatment in Example 2 was completed, the cell model was subjected to Western blot detection according to the following steps: (1) Remove the cell culture medium, add 100 μL of protein lysis buffer to the cell culture plate, scrape the cells off with a cell scraper, transfer them to a 1.5 mL centrifuge tube, and incubate on ice for 20 min for lysis. (2) Centrifuge at 12000 rpm for 20 min at 4℃, and transfer the supernatant to a new 1.5 mL centrifuge tube; (3) Add 5× protein loading buffer and incubate at 100℃ for 5 min to allow the protein to denature fully; (4) Add samples to the gel wells. After the samples are loaded, use 80V constant voltage for concentration electrophoresis to compress the samples to the same level and the protein markers are clearly separated. Then adjust to 120V constant voltage for separation electrophoresis. (5) After electrophoresis, the membrane was transferred under a constant current of 300mA; (6) After the transfer is completed, the PVDF membrane is placed in 5% skim milk and sealed on a slow shaker at room temperature for 2 hours. (7) Wash the membrane 3 times with PBST, 5 min each time; (8) Dilute the required antibody to a specific concentration with 5% BSA, incubate the antibody strips overnight on a slow shaker at 4°C; (9) Primary antibody recovery, PBST washing of the membrane 3 times, 5 min / time, 5% skim milk powder to prepare the corresponding secondary antibody, incubation at room temperature for 2 h; (10) Wash the membrane 3 times with PBST, 5 min each time; (11) Develop using a Tanon exposure machine; (12) The grayscale values ​​of the strips were statistically analyzed using ImageJ software.

[0048] The results are as follows Figure 2 As shown in B and Table 7, Indicates significance of difference P <0.001. (By...) Figure 2 From B, we can conclude that the hsa-GJA1-OE plasmid was successfully constructed and stably overexpressed humanized gap junction protein α1 at the protein level.

[0049] The above examples demonstrate that the overexpression of humanized gap junction protein particles was successfully constructed and can stably overexpress humanized gap junction protein α1.

[0050] Example 3 1. Preparation of hsa-GJA1-OE lentivirus: The construction steps are as follows.

[0051] (1) Host cells 293T were passaged into 10cm dishes and cultured to a cell density of 60%~70% before transfection; (2) Preparation of cell transfection reagent: Add 1.25 μg of pMD2-G plasmid, 3.75 μg of psPAX.2 plasmid, 5 μg of hsa-GJA1-OE plasmid, and 40 μL of PEI to 500 μL of serum-free culture medium, mix gently, and let stand for 20 min. (3) After the settling is complete, add it evenly to the petri dish and place it in the incubator for incubation; (4) Replace with normal culture medium after 12 hours and continue culturing; (5) After culturing for 48-72 hours, collect the culture medium containing lentivirus according to the culture status, filter the culture medium containing lentivirus with a 0.45μm filter, dispense it into aliquots and store it in a -80℃ freezer for later use.

[0052] 2. Preparation of engineered extracellular vesicles modified by overexpression of gap junction protein α1, the construction steps are as follows: (1) Host cells 293T were passaged into 12-well plates and cultured to a cell density of 70%–80%; (2) Preparation of cell transfection reagent: Add 500 μL of serum-free medium containing hsa-GJA1-OE lentivirus from step 1 and 1 μL of polybrene to 500 μL of serum-free medium, mix gently, and let stand for 5 min. (3) Remove the old cell culture medium from the cell plate and add 1 mL of mixed transfection reagent to each well; (4) Replace with normal culture medium after 24 hours; (5) After 48 hours, puromycin (1 μg / mL) was added to remove cells that had not been transfected with lentivirus, and the cell status was observed after 24 hours. (6) Maintain a suitable Puro concentration until no floating dead cells are found after adding Puro and the cells grow normally, and obtain a stable 293T transgenic cell that overexpresses humanized gap junction protein α1, denoted as 293T-hsa-GJA1; (7) Expand the cells in the 12-well plate to a culture dish and continue to culture them in normal culture medium containing Puro.

[0053] (8) Collect the supernatant of the 293T stable transgenic cell line that stably overexpresses humanized hsa-GJA1 obtained in step (7) and perform ultracentrifugation. The specific steps include: centrifugation at 500g for 5 min; centrifugation at 3000g for 10 min; centrifugation at 12000g for 45 min; filter the supernatant using a 0.22 μm filter, and centrifuge the filtered supernatant at 100000g for 70 min. All centrifugations are performed at 4℃ to obtain engineered extracellular vesicles modified by overexpression of gap junction protein α1, denoted as EVs-hsa-GJA1.

[0054] Test Example 2 1. Western blot analysis of the expression efficiency of the stable 293T cell line overexpressing humanized hsa-GJA1 in Example 3. The control was the ordinary 293T cell line (293T). The Western blot analysis method was the same as in Example 1. The results are as follows: Figure 3 As shown in A and Table 8, where Indicates significance of difference P <0.001.

[0055] Depend on Figure 3 From A, we can conclude that the 293T stable transgenic strain of humanized hsa-GJA1 was successfully constructed and stably overexpressed the humanized gap junction protein α1.

[0056] 2. To confirm whether the extracted product was an extracellular vesicle, Western blotting was used to detect the protein markers CD63 (Abclonal, A5271), Calnexin (CST, #2679), CD9 (CST, #98327), Tsg101 (Abclonal, A2216), and Alix (Abclonal, A2215) in the supernatant of the stable transgenic 293T cell line that stably overexpressed humanized hsa-GJA1 in Example 3. The Western blotting method was the same as in Test Example 1. The detection results are as follows. Figure 3 As shown in B.

[0057] Depend on Figure 3 From B, we can conclude that this embodiment successfully obtained engineered extracellular vesicles modified by overexpression of gap junction protein α1.

[0058] 3. The size and concentration of extracellular vesicles in the supernatant of the stable 293T transgenic cell line overexpressing humanized hsa-GJA1 were detected using nanoparticle tracking analysis technology. The procedure was performed according to [Wang, Hongyun, et al. "Percutaneous Intracoronary Delivery of Plasma Extracellular Vesicles Protects the Myocardium Against Ischemia–Reperfusion Injury in Canis." Hypertension, vol. 78, no. 5, Nov. 2021, pp. 1541–54. doi:10.1161 / HYPERTENSIONAHA.121.17574.]. The results are as follows. Figure 3 As shown in C.

[0059] Depend on Figure 3 From C, we can conclude that: This embodiment successfully obtained engineered extracellular vesicles modified by overexpression of gap junction protein α1, with a particle size of 147.7 nm and a concentration of 5.6E+11 particles / mL.

[0060] 4. Western blot analysis of the expression level of gap junction protein α1 in the extracellular vesicles of the 293T stable transgenic cell line stably overexpressing humanized hsa-GJA1 in Example 3. The Western blot analysis method was the same as in Test Example 1. The results are as follows: Figure 3 As shown in D and Table 8.

[0061] Depend on Figure 3 From D, it can be concluded that the extracellular vesicles isolated from the stable transgenic strain successfully enriched humanized gap junction protein α1.

[0062] Table 8 Test Results

[0063] The above examples demonstrate that the engineered extracellular vesicles modified by overexpression of gap junction protein α1 were successfully constructed and can stably overexpress humanized gap junction protein α1.

[0064] Example 4 1. Uptake of EVs-hsa-GJA1 by primary cardiomyocytes of newborn rats Newborn rat primary cardiomyocytes (NRCMs) were randomly divided into three groups: control extracellular vesicle treatment group (EVs-Ctr group), engineered extracellular vesicle treatment group modified with gap junction protein α1 overexpression (EVs-hsa-GJA1 group), and phosphate buffered saline (PBS) group. (1) The extracted EVs-Ctr and EVs-hsa-GJA1 were diluted to 1 mL with 1×PBS, and 10 μL of DiD dye was added. The staining was carried out in the dark for 30 min. An equal amount of PBS was added to the control group. (2) Centrifuge each group of EVs and DiD dye mixtures at 100,000g for 70min to remove excess dye; (3) Mix DiD-labeled EVs with culture medium (concentration 40 μg / mL) and add to cell plates, then incubate in the dark for 2 h.

[0065] 2. Human cardiomyocytes take up EVs-hsa-GJA1 Human cardiomyocytes (AC16) were randomly divided into three groups: control extracellular vesicle treatment group (EVs-Ctr), engineered extracellular vesicle treatment group modified with gap junction protein α1 overexpression (EVs-hsa-GJA1), and phosphate-buffered saline (PBS) group. (1) The extracted EVs-Ctr and EVs-hsa-GJA1 were diluted to 1 mL with 1×PBS, and 10 μL of DiD dye was added. The staining was carried out in the dark for 30 min. An equal amount of PBS was added to the control group. (2) Centrifuge each group of EVs and DiD dye mixtures at 100,000g for 70min to remove excess dye; (3) Mix DiD-labeled EVs with culture medium (concentration 40 μg / mL) and add to cell plates, then incubate in the dark for 2 h.

[0066] Test Example 3 1. After the treatment in Example 4 was completed, immunofluorescence staining was used to detect the uptake of EVs-hsa-GJA1 by NRCM and AC16 cells. The steps are as follows: (1) After the cell culture medium was removed, the cells were washed with 1×PBS; (2) Fix with 4% paraformaldehyde at room temperature for 30 min, wash with 1×PBS 3 times, 5 min each time; (3) Incubation of nuclear dye: Prepare 1:2000 (v:v) Hoechst with 5% BSA and incubate at room temperature in the dark for 20 min; (4) Observe and photograph under a confocal microscope.

[0067] The results are as follows Figure 4 As shown in Table 9, Indicates significance of difference P <0.001. (By...) Figure 4 From A, we can conclude that, compared to the control, engineered extracellular vesicles modified with overexpression of gap junction protein α1 showed increased uptake by neonatal rat primary cardiomyocytes. Figure 4 From B, we can conclude that, compared with the control, the engineered extracellular vesicles modified by overexpression of gap junction protein α1 were taken up more by human cardiomyocytes.

[0068] Table 9 Results of extracellular vesicle uptake detection

[0069] From the above examples, it can be concluded that, compared with the control, engineered extracellular vesicles modified by overexpression of gap junction protein α1 are taken up more by neonatal rat primary cardiomyocytes / human cardiomyocytes, and engineered extracellular vesicles modified by overexpression of gap junction protein α1 can serve as targeted drug carriers for the treatment of cardiac injury and / or heart failure.

[0070] Example 5 1. Uptake of EVs-hsa-GJA1 in mouse heart tissue Wild-type C57BL / 6J mice were randomly and equally divided into three groups: control extracellular vesicle myocardial injection group (EVs-Ctr group), engineered extracellular vesicle myocardial injection group modified with gap junction protein α1 overexpression (EVs-hsa-GJA1 group), and phosphate-buffered saline (PBS) myocardial injection group. (1) The extracted EVs-Ctr and EVs-hsa-GJA1 were diluted to 1 mL with 1×PBS, and 10 μL of DiD dye was added. The staining was carried out in the dark for 30 minutes. An equal amount of PBS was added to the control group. (2) Centrifuge the mixture of EVs and DiD dye at 100,000g for 70 minutes to remove excess dye; (3) DiD-labeled EVs (dose of 40 μg / mouse) were injected into the myocardium of mice. The injection was performed at three points, and heart tissue was collected 24 hours after the injection.

[0071] Test Example 4 1. After the treatment in Example 5 was completed, immunofluorescence staining was used to detect the uptake of EVs-hsa-GJA1 in mouse heart tissue. The steps are as follows: (1) Frozen sections of mouse heart tissue were placed in a humidified chamber and warmed at room temperature for 20 min, then washed three times with 1×PBS for 5 min each time; (2) Fix with 4% paraformaldehyde at room temperature for 30 min, wash with 1×PBS 3 times, 5 min each time; (3) Disrupt the membrane with 0.5% Triton X-100 for 20 min, wash 3 times with 1×PBS, 5 min each time; (4) Block with 5% BSA at room temperature for 1 hour; (5) Incubation of primary antibody: 1:200 (v:v) α-actinin was prepared with 5% BSA and incubated overnight at 4°C; (6) The next day, wash with 1×PBS 3 times, 5 min each time; (7) Incubation of secondary antibody: Prepare 1:200 (v:v) 488 mouse secondary antibody with 5% BSA and incubate at room temperature in the dark for 2 hours; (8) Sections should be protected from light and washed three times with 1×PBS, 5 min each time; (9) Incubation of nuclear dye: Prepare 1:2000 (v:v) Hoechst with 5% BSA and incubate at room temperature in the dark for 20 min; (10) Observe and photograph under a confocal microscope.

[0072] The results are as follows Figure 5 As shown in Table 10, where Indicates significance of difference P <0.001. (By...) Figure 5 The results show that, compared with the control, engineered extracellular vesicles modified by overexpression of gap junction protein α1 were taken up by cardiac tissue with increased uptake.

[0073] Table 10 Results of extracellular vesicle uptake detection

[0074] The above examples demonstrate that, compared to the control, engineered extracellular vesicles modified with overexpression of gap junction protein α1 exhibit increased uptake by cardiac tissue and thus cardiac targeting. Engineered extracellular vesicles modified with overexpression of gap junction protein α1 can serve as targeted drug carriers for the treatment of cardiac injury and / or heart failure.

[0075] Example 6 1. Uptake of EVs-hsa-GJA1 in cardiac tissue after acute myocardial ischemia-reperfusion injury in mice Wild-type C57BL / 6J mice were randomly and equally divided into four groups: control extracellular vesicle myocardial injection group (EVs-Scr group), target peptide extracellular vesicle myocardial injection group (EVs-CHP group), engineered extracellular vesicle myocardial injection group modified with gap junction protein α1 overexpression (EVs-hsa-GJA1 group), and phosphate buffered saline (PBS) myocardial injection group. (1) The extracted EVs-Ctr were linked to the cardiac targeting peptide (CHP, SEQ ID NO.9: CSTSMLKAC) and the control peptide (Scr, SEQ ID NO.10: CSKTALSMC) via DOPE-NHS, respectively. The procedure was performed according to [Vandergriff, Adam, et al. "Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide." Theranostics, vol. 8, no. 7, 2018, pp. 1869-1878.DOI: 10.7150 / thno.20524.]; (2) Dilute EVs-Scr, EVs-CHP and EVs-hsa-GJA1 to 1 mL with 1×PBS, add 10 μL of DiD dye and stain in the dark for 30 minutes; add an equal amount of PBS to the control group; (3) Centrifuge the mixture of EVs and DiD dye at 100,000g for 70 minutes to remove excess dye; (4) An acute myocardial ischemia-reperfusion injury model in mice was constructed by ligating the left anterior descending coronary artery and reperfusion blood flow. The operation method was as follows: [Bei, Yihua, et al. "Cathelicidin-related antimicrobial peptide protects against myocardial ischemia / reperfusion injury." BMC Medicine, vol. 17, no. 1, 2019, p. 42. DOI: 10.1186 / s12916-019-1268-y.] (5) After the ligation line was loosened, mice were injected with DiD-labeled EVs (dose of 40 μg / mouse) in three points. Heart tissue was collected 24 hours after injection.

[0076] Test Example 5 1. After the treatment in Example 6 was completed, immunofluorescence staining was used to detect the uptake of EVs-hsa-GJA1 in mouse heart tissue. The procedure was performed according to Test Example 4, and the results were as follows. Figure 6 As shown in Table 11, where Indicates significance of difference P <0.001, Indicates significance of difference P <0.01. (By...) Figure 6It can be concluded that, compared with extracellular vesicles that are linked to cardiac-targeting peptides, engineered extracellular vesicles modified by overexpression of gap junction protein α1 are taken up more by cardiac tissue.

[0077] Table 11 Results of extracellular vesicle uptake detection

[0078] The above examples demonstrate that, compared to extracellular vesicles linked to cardiac-targeting peptides, engineered extracellular vesicles modified with overexpression of gap connexin α1 exhibit increased uptake by cardiac tissue and thus cardiac targeting. Engineered extracellular vesicles modified with overexpression of gap connexin α1 can serve as targeted drug carriers for the treatment of cardiac injury and / or heart failure. This provides a new drug development pathway and method for the clinical treatment of heart failure or myocardial injury, while also offering cardiac-targeting modification tools for industrial R&D and basic research users.

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A myocardial-targeted engineered extracellular vesicle, characterized in that, The engineered extracellular vesicles are engineered extracellular vesicles modified by overexpression of gap junction protein α1; the amino acid sequence of the gap junction protein α1 is shown in SEQ ID NO.

1.

2. The method for preparing engineered extracellular vesicles according to claim 1, characterized in that, The vector overexpressing gap junction protein α1 was co-cultured with human kidney embryo 293T cells, the cell supernatant was collected, and the engineered extracellular vesicles were obtained by centrifugation.

3. The preparation method according to claim 2, characterized in that, The vector overexpressing gap junction protein α1 includes a lentiviral vector overexpressing gap junction protein α1.

4. The preparation method according to claim 3, characterized in that, The base vector of the lentiviral vector includes pLVX-puro.

5. The preparation method according to claim 4, characterized in that, The gap junction protein α1 is inserted into the pLVX-puro BamH I and Age Between Ⅰ enzyme cleavage sites.

6. The preparation method according to claim 2, characterized in that, The vector overexpressing gap connexin α1 includes a vector overexpressing humanized gap connexin α1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

7. The application of the engineered extracellular vesicles as described in claim 1 as a drug delivery carrier.

8. The use of the engineered extracellular vesicles of claim 1 in the preparation of medicaments for the prevention and / or treatment of heart disease.

9. The application according to claim 8, characterized in that, The heart disease includes myocardial damage and / or heart failure.

10. A medicament for the prevention and / or treatment of heart disease, characterized in that, The drug comprises the engineered extracellular vesicles of claim 1 and an active ingredient loaded on the engineered extracellular vesicles of claim 1.

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