Use of a preparation for the manufacture of a medicament for the prevention and / or treatment of cardiac damage associated with myocardial infarction

Abstract

This invention discloses the application of a formulation in the preparation of drugs for the prevention and / or treatment of myocardial infarction-related cardiac injury. The application of an EMP2-overexpressing formulation in the preparation of drugs for the prevention and treatment of myocardial infarction, wherein the formulation is selected from any of the following: a viral expression vector carrying an EMP2 coding sequence; a non-viral nanodelivery system loaded with an EMP2 overexpression plasmid; or extracellular vesicles derived from EMP2-overexpressing vascular endothelial cells. This invention works synergistically at three levels: pre-infarction intervention, timely post-infarction intervention, and long-term microvascular endothelial environment repair, constructing a comprehensive and systematic EMP2-based technical solution for the prevention and treatment of myocardial infarction injury.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cardiovascular disease prevention and treatment, and specifically to the application of a formulation in the preparation of drugs for the prevention and / or treatment of myocardial infarction-related cardiac injury. Specifically, it relates to the application of the AAV9-EMP2 expression vector in the prevention of cardiac injury caused by myocardial infarction, the application of the CPC-EMP2 nucleic acid nanodelivery system in the treatment of cardiac injury after myocardial infarction, and the application of EMP2 overexpression of extracellular vesicles derived from vascular endothelial cells in promoting vascular endothelial cell migration, tube formation, and vascular endothelial repair. Background Technology

[0002] Myocardial infarction (MI) is a serious cardiovascular emergency that severely threatens human life and health. It is essentially caused by a sharp reduction or interruption of coronary blood flow, leading to local myocardial ischemia, hypoxia, and even necrosis. Although current clinical methods (such as percutaneous coronary intervention, thrombolysis, anticoagulation, and drugs to improve cardiac load) can restore blood perfusion and reduce the risk of death in the acute phase to some extent, these methods mainly focus on blood flow reconstruction and cannot fundamentally reverse the already occurring myocardial microvascular damage, myocardial fibrosis, and ventricular remodeling. Therefore, the incidence of sudden cardiac death and ischemic heart failure following MI remains high.

[0003] Following myocardial infarction, the disruption of the microvascular network leads to persistent underperfusion and nutritional deficiencies in local tissues, further exacerbating myocardial damage. Vascular endothelial cells not only maintain vascular barrier function, but their migration and tube-forming capabilities after ischemic injury directly determine the level of angiogenesis. Therefore, promoting endothelial repair and the establishment of new vascular networks, and improving local microcirculation, are key breakthroughs for reversing cardiac damage after myocardial infarction and promoting the recovery of cardiac function.

[0004] Epithelial membrane protein 2 (EMP2) is a transmembrane protein that has been previously studied and is involved in the regulation of cell membrane protein composition, playing a role in biological processes such as cell adhesion, migration, and HIF1A / VEGFA-mediated angiogenesis. However, the exact role of EMP2 in cardiac injury caused by myocardial infarction remains a gap in our understanding. Current techniques have not revealed the effects of intervening in EMP2 expression before and after myocardial infarction on the progression of cardiac injury, nor has it clarified whether EMP2 overexpression can enhance the angiogenesis capacity of vascular endothelial cells and their secreted extracellular vesicles (EVs).

[0005] Furthermore, the application of nucleic acid-based gene therapy in cardiovascular diseases has long been limited by delivery efficiency, tissue targeting, and timing of administration. For early prevention in high-risk groups of myocardial infarction, and for acute or chronic treatment after myocardial infarction, there is an urgent need to develop efficient delivery systems (such as viral vectors or non-viral nanocarriers) adapted to different clinical scenarios.

[0006] In summary, there is currently a lack of effective intervention targets to reverse microvascular damage after myocardial infarction. It is urgent to clarify the role of EMP2 in the prevention and treatment of myocardial infarction-related cardiac injury and vascular endothelial repair, and to develop a new strategy for the prevention and treatment of myocardial infarction centered on EMP2, adaptable to different administration times (prevention / treatment) and different delivery forms (gene vectors / biovesicles), thereby providing a new technical pathway for vascular endothelial repair and improvement of cardiac function after myocardial infarction. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing the application of EMP2-overexpressing formulations in the preparation of drugs for the prevention and treatment of myocardial infarction.

[0008] The second objective of this invention is to provide an EMP2 nucleic acid delivery system for treating cardiac injury following myocardial infarction.

[0009] A third objective of this invention is to provide an extracellular vesicle formulation for promoting vascular endothelial repair and its application.

[0010] A fourth object of the present invention is to provide a composition for the prevention and / or treatment of cardiac injury associated with myocardial infarction.

[0011] To address the aforementioned technical problems, a first aspect of the present invention provides the use of a formulation in the preparation of a medicament for the prevention or treatment of myocardial infarction-related cardiac injury, wherein the formulation is selected from any one of the following:

[0012] (I) Viral expression vector carrying the EMP2 coding sequence;

[0013] (II) Non-viral nanodelivery system loaded with EMP2 overexpression plasmid;

[0014] (III) Extracellular vesicles derived from EMP2-overexpressing vascular endothelial cells.

[0015] The EMP2 coding sequence described in this invention is selected from the coding gene sequence of human EMP2 protein or the coding gene sequence of mouse EMP2 protein.

[0016] In a preferred embodiment, the formulation is an adeno-associated virus type 9 (AAV9) expression vector AAV9-EMP2 carrying the EMP2 coding sequence, and the drug is used to prevent cardiac damage caused by myocardial infarction. The drug is administered to subjects before the onset of myocardial infarction to increase the expression level of EMP2 in cardiac tissue and / or cardiac vascular endothelial cells, thereby enhancing the resistance of cardiac tissue to myocardial infarction damage. Animal experimental results show that, compared with the control group, pre-administration of the AAV9-EMP2 expression vector can reduce mortality after myocardial infarction, improve cardiac systolic function, increase ejection fraction (EF) and / or fractional shortening (FS), reduce myocardial tissue damage, reduce collagen fiber deposition, and inhibit ventricular remodeling after myocardial infarction.

[0017] In another preferred embodiment, the formulation is a non-viral nanodelivery system comprising a cationic nanocarrier modified with a myocardial targeting peptide and an EMP2 overexpression plasmid loaded in the carrier; preferably, the myocardial targeting peptide is a single-targeting peptide or a dual-targeting modified peptide capable of specifically enriching in the cardiac injury region.

[0018] More preferably, the myocardial targeting peptide is CRPPR, the cationic nanocarrier is a CPC carrier containing a cholesterol-terminated polyglycidyl methacrylate backbone, and the non-viral nanodelivery system is a CPC-EMP2 plasmid nanocomposite. The CPC-EMP2 plasmid nanocomposite has a particle size of 190-220 nm and a zeta potential of +30 mV to +50 mV.

[0019] This non-viral nanodelivery system is used to treat or alleviate cardiac damage after myocardial infarction. After administration following myocardial infarction, it can improve cardiac function, increase ejection fraction (EF), increase fractional shortening (FS), reduce myocardial tissue damage, reduce myocardial fibrosis, reduce collagen fiber deposition, inhibit ventricular remodeling, and / or promote vascular repair.

[0020] A second aspect of the present invention provides an EMP2 nucleic acid delivery system for treating cardiac injury after myocardial infarction. The EMP2 nucleic acid delivery system comprises a myocardial-targeting modified cationic nanocarrier and an EMP2 overexpression plasmid loaded in the carrier; preferably, the myocardial-targeting modification is a dual-targeting modification or CRPPR peptide modification with targeting capabilities to the heart and cardiac injury regions, and the cationic nanocarrier comprises a cholesterol-terminated polyglycidyl methacrylate backbone. This delivery system is a non-viral nucleic acid delivery system and can serve as an alternative to the AAV9-EMP2 expression vector for EMP2 overexpression, particularly suitable for therapeutic intervention after myocardial infarction.

[0021] A third aspect of the present invention provides an extracellular vesicle formulation for promoting vascular endothelial repair and its application. The extracellular vesicle formulation is derived from EMP2-overexpressing vascular endothelial cells; preferably, the extracellular vesicles are cup-shaped, spherical, or near-spherical membranous vesicles. More preferably, the EMP2-overexpressing vascular endothelial cells are HUVECs, obtained by treatment with a lentiviral expression vector carrying an EMP2 coding sequence.

[0022] This invention also provides the use of the extracellular vesicle formulation in the preparation of drugs or formulations for promoting vascular endothelial repair. The promotion of vascular endothelial repair includes promoting vascular endothelial cell migration, promoting vascular endothelial cell tubulation, and / or promoting the formation of angiogenesis-like structures.

[0023] A fourth aspect of the present invention provides a composition for the prevention and / or treatment of cardiac injury associated with myocardial infarction. The composition comprises: the aforementioned AAV9-EMP2 expression vector, and the aforementioned EMP2 nucleic acid delivery system (preferably a CPC-EMP2 plasmid nanocomposite); preferably, the composition further comprises the aforementioned extracellular vesicle formulation derived from vascular endothelial cells and overexpressing EMP2. Through this composition, the present invention constructs a comprehensive prevention and treatment technology system from three levels: prevention before myocardial infarction, treatment after myocardial infarction, and vascular endothelial repair.

[0024] Compared with the prior art, the present invention has at least the following beneficial effects:

[0025] First, this invention constructs a multi-dimensional drug intervention system: by providing three different formulations—viral vectors, non-viral nanosystems, and extracellular vesicles—it covers all key clinical aspects, including prevention before myocardial infarction, treatment after myocardial infarction, and microvascular endothelial repair.

[0026] Second, this invention confirms the core role of EMP2 in preventing cardiac injury: in vivo experiments show that increasing EMP2 expression levels before myocardial infarction can reduce mortality, improve cardiac contractile function, reduce myocardial tissue damage and collagen fiber deposition, and effectively inhibit ventricular remodeling.

[0027] Third, this invention develops a novel, highly efficient and targeted non-viral delivery vector: the provided EMP2 nucleic acid delivery system uses a cationic nanocarrier containing a cholesterol-terminated poly(glycidyl methacrylate) backbone, and is combined with single / double targeting peptide (such as CRPPR) modification to achieve precise delivery to specific regions and effectively alleviate cardiac damage after infarction.

[0028] Fourth, this invention provides a novel vesicle formulation with specific morphology and function: This invention extracts EMP2 overexpressing extracellular vesicles with cup-shaped, spherical or near-spherical membranous vesicle structures, and confirms that they can significantly promote the migration and tube formation of receptor vascular endothelial cells, providing a novel biological agent for myocardial infarction-related vascular repair.

[0029] Fifth, this invention proposes a comprehensive prevention and treatment composition scheme: combining advantageous agents at different stages to form a systematic solution that combines the prevention, treatment and vascular endothelial repair of myocardial infarction injury with EMP2 as the core, which has broad prospects for clinical translation. Attached Figure Description

[0030] Figure 1 This is a Western Blot result of the overexpression efficiency of EMP2 protein in mouse heart tissue after AAV9-EMP2 injection in this embodiment of the invention.

[0031] Figure 2 This is a graph showing the effect of AAV9-EMP2 pretreatment on the survival rate and cardiac function indicators of mice with myocardial infarction in this embodiment of the invention.

[0032] Figure A shows the survival rate curves of mice in each group within 28 days after surgery; Figures B to F are statistical graphs of left ventricular fractional shortening (FS), left ventricular ejection fraction (EF), left ventricular end-systolic volume, left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs) obtained by mice in each group on days 0, 7, 14, 21, and 28 after myocardial infarction, respectively.

[0033] Figure 3 The images show the effects of AAV9-EMP2 pretreatment on the pathological morphology and degree of fibrosis of cardiac tissue in mice with myocardial infarction, as described in this embodiment of the invention. The images include HE staining and Masson staining results.

[0034] Figure 4 This is a Western blot result showing the overexpression efficiency of CPC-EMP2 plasmid nanocomposite in the heart tissue of mice with myocardial infarction, as described in this embodiment of the invention.

[0035] Figure 5 This is a graph showing the effect of CPC-EMP2 plasmid nanocomposite on the survival rate and cardiac function indicators of mice with myocardial infarction in the embodiments of the present invention.

[0036] Figure A shows the survival rate curves of mice in each group within 28 days after surgery; Figures B to E are statistical graphs of left ventricular end-systolic diameter (LVIDs), left ventricular ejection fraction (EF), left ventricular end-diastolic diameter (LVIDd), and left ventricular fractional shortening (FS) obtained by mice in each group on days 0, 1, 7, 14, 21, and 28 after myocardial infarction.

[0037] Figure 6 The images show the effects of CPC-EMP2 plasmid nanocomposite on the pathological morphology and degree of fibrosis of cardiac tissue in mice with myocardial infarction, including HE staining and Masson staining results.

[0038] Figure 7 This is a Western Blot result of the efficiency of constructing a lentivirus-mediated EMP2 stably overexpressing HUVECs cell line in an embodiment of the present invention.

[0039] Figure 8 This is a graph showing the detection results of the effect of EMP2 overexpression on extracellular vesicle secretion in an embodiment of the present invention;

[0040] in: Figure 8 The top row shows transmission electron microscopy (TEM) images of control and EMP2 overexpression groups and their secreted extracellular vesicles. Figure 8 The bottom row shows the particle size and concentration distribution of extracellular vesicles secreted by the control group and the EMP2 overexpression group.

[0041] Figure 9 This is a fluorescence image of receptor HUVEC cells taking up PKH26-labeled extracellular vesicles in an embodiment of the present invention.

[0042] The image shows: the left image is a DAPI-stained cell nuclear image; the middle image is a PKH26-labeled extracellular vesicle fluorescence image; and the right image is a merged image of DAPI and PKH26.

[0043] Figure 10 Microscopic images showing the experimental results of the effects of different extracellular vesicles on the migration and tube formation ability of receptor cells in embodiments of the present invention;

[0044] The upper part shows microscopic images of cells from the control group (Control), the group with added control extracellular vesicles (Control-EVs), and the group with added EMP2 overexpressing extracellular vesicles (Over-EMP2-EVs) at 0 hours and 24 hours after scratching. The lower part shows microscopic images of cells from the control group (Control), the group with added control extracellular vesicles (Control-EVs), and the group with added EMP2 overexpressing extracellular vesicles (Over-EMP2-EVs) in the tube formation experiment. Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, conventional improvements, or combinations made based on the disclosure of the present invention should fall within the scope of protection of the present invention. Unless otherwise stated, the experimental materials, reagents, and instruments used in the following embodiments are commercially available, and the experimental methods used are conventional methods in the art.

[0046] Example 1: Validation of the preventive effect of AAV9-EMP2 expression vector in a myocardial infarction model

[0047] This embodiment is used to verify the preventive effect of increasing EMP2 expression before myocardial infarction on cardiac damage caused by myocardial infarction.

[0048] C57BL / 6 mice were randomly divided into three groups: the Sham group (n=10), the MI control group (n=10), and the AAV9-EMP2 pretreatment group (n=15). Mice in the AAV9-EMP2 pretreatment group were injected via tail vein with an AAV9-EMP2 expression vector carrying the EMP2 coding sequence. Mice in the MI control group were injected with an equal volume of the AAV9-Ctrl control vector. Mice in the Sham group underwent sham surgery. The AAV9-EMP2 expression vector was an adeno-associated virus type 9 (AAV9) expression vector carrying the EMP2 coding sequence.

[0049] The specific construction and packaging of the AAV9-EMP2 expression vector are as follows: First, a recombinant AAV shuttle plasmid carrying the mouse EMP2 coding sequence was constructed. GV841 (vector element sequence: TIE promoter-MCS-3flag-T2A-EGFP-WPRE-bGH polyA) was selected as the target vector. This vector is driven by an endothelial-specific TIE promoter to express the target gene and carries a 3xflag tag and an EGFP-enhanced green fluorescent protein reporter gene. To ensure correct in-frame fusion expression of the target gene with the 3xflag tag at the C-terminus of the vector, the natural stop codon of the mouse EMP2 gene (corresponding transcript number NM_007929.2) was removed, and NheI and AgeI restriction sites and homologous arm sequences were designed and introduced at both ends of the sequence, resulting in a 519 bp mouse EMP2 target gene fragment as shown in SEQ ID NO:1. This mouse EMP2 target gene fragment was then directly synthesized using whole-genome synthesis technology. The GV841 vector was double-digested with NheI and AgeI restriction endonucleases, and the linearized vector was purified and recovered by agarose gel electrophoresis. Subsequently, the purified, fully synthesized mouse EMP2 gene fragment was homologously ligated into the linearized GV841 vector using HB infusion™ one-step cloning technology. The ligation product was transformed into DH5α competent cells, and the cells were screened by colony PCR and sequencing. Endotoxin-free plasmids were extracted to obtain the successfully constructed AAV-EMP2 shuttle plasmid. Next, AAV9 adeno-associated virus (AAV9) was packaged and purified. Using a three-plasmid packaging system, the constructed AAV-EMP2 shuttle plasmid, pAAV-RC packaging vector plasmid, and pHelper helper vector plasmid were co-transfected into AAV-293 packaging cells using Lipofiter™ transfection reagent. 72 hours after transfection, cell pellet containing AAV particles was collected. Cells were lysed using a freeze-thaw cycle, and the crude extract was treated with Benzonase to remove cellular genome and residual plasmid DNA. Subsequently, the virus was concentrated and purified using column purification to obtain high-purity AAV9-EMP2 viral fluid. Finally, the purified adeno-associated virus underwent quality testing to ensure it was free from bacterial, fungal, and mycoplasma contamination.

[0050] The AAV9-EMP2 expression vector was administered via a single tail vein injection. Mice were randomly assigned to receive either the AAV9-EMP2 expression vector or the AAV9-Ctrl control vector at a dose of 1 × 10⁻⁶. 11The dosage was vg / mL, and the administration volume was 100 μL per mouse. Administration was performed 9 days prior to myocardial infarction surgery. By administering the AAV9-EMP2 expression vector before establishing the myocardial infarction model, Western blotting experiments successfully detected a significant upregulation of EMP2 protein expression levels in mouse cardiac tissue and / or cardiac vascular endothelial cells (e.g., vg / mL). Figure 1 (As shown in the figure). This result verifies the in vivo overexpression efficiency of the AAV9 vector, laying the foundation for subsequent evaluation of the resistance and preventive effect of EMP2 overexpression on myocardial infarction injury.

[0051] The left anterior descending coronary artery (LAD) was ligated to establish a myocardial infarction model. Mice were first anesthetized, then their limbs were immobilized, and the neck and chest were shaved and disinfected. The skin on the mouse's neck was cut open to expose the trachea, and endotracheal intubation was performed, connecting the mouse to a small animal ventilator. After confirming that the mouse's chest rose and fell rhythmically with the ventilator, the chest skin was cut open, the chest muscles were bluntly dissected, and an opening was made at the fourth intercostal space. A thoracotomy device was used to open the ribs to expose the heart. The LAD was then ligated using 7-0 nylon suture; successful ligation was confirmed by observing whitening at the apex of the heart. After ligation, the intercostal, chest, and neck skins were sutured sequentially, and the mice were kept in a warm environment until awakening. In the Sham group, only the suture was threaded through the neck without ligation.

[0052] The survival of mice in each group was continuously observed after surgery, and mortality was recorded. The survival rate of mice in each group was statistically analyzed within 28 days after surgery. Small animal ultrasound examinations were performed on days 0, 7, 14, 21, and 28 after surgery to evaluate cardiac function indicators, including ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), and left ventricular end-systolic volume.

[0053] Mice were sacrificed at predetermined time points, and cardiac tissue was harvested for histological examination, including HE staining and Masson staining. HE staining was used to observe myocardial tissue structural damage, while Masson staining was used to observe collagen fiber deposition and the degree of myocardial fibrosis. Results showed that, compared with the MI control group, mice pretreated with AAV9-EMP2 had improved survival rates and reduced mortality risk after myocardial infarction (e.g., Figure 2 As shown in A), improvements in cardiac function indicators such as EF and / or FS (e.g. Figure 2 As shown in BF), myocardial tissue structural damage is reduced (e.g. Figure 3 As shown in the HE staining results, collagen fiber deposition is reduced, and the degree of myocardial fibrosis and ventricular remodeling is decreased (e.g., Figure 3 (As shown in the Masson staining results). The above results indicate that increasing EMP2 expression via the AAV9-EMP2 expression vector before myocardial infarction can prevent or reduce cardiac damage caused by myocardial infarction.

[0054] Example 2: Preparation and characterization of CPC-EMP2 plasmid nanocomposites

[0055] This embodiment is used to prepare CPC-EMP2 plasmid nanocomposites and verify their physicochemical properties as a non-viral delivery system for EMP2 overexpression plasmids.

[0056] Preparation of CRPPR-modified cationic nanocarriers CPC:

[0057] The preparation method of the CPC carrier is specifically referred to in the relevant literature (Yihui Shao, et al. OneEndothelium-Targeted Combined Nucleic Acid Delivery System for Myocardial Infarction Therapy. ACS Nano 2024, 18, 8107-8124). The specific preparation steps include: First, 100 mg (approximately 0.0104 mmol) of cholesterol-terminated glycidyl methacrylate (CHO-PGMA) was completely dissolved in 5 mL of dimethyl sulfoxide (DMSO). To prevent oxidation and ensure system stability, nitrogen gas was introduced into the mixing system for degassing for 5 minutes. Next, 1.8 mL of ethanolamine (EA) and 0.2 mL of ethylenediamine (ED) were added to the reaction system. The above reaction mixture was placed at 80 °C and stirred continuously for 2 hours. Through the ring-opening reaction of the epoxy group, CHO-PGMA was converted into CHO-PGEA / ED containing a primary amino group. After the reaction, the resulting crude mixture was transferred to a dialysis membrane with a molecular weight cutoff (MWCO) of 1000 for dialysis purification. The purified solution was lyophilized to obtain the solid CHO-PGEA / ED product. Subsequently, myocardial targeting peptide grafting was performed. 20 mg of the myocardial targeting peptide CRPPR (which is a single-targeting peptide or a dual-targeting modified peptide that can specifically accumulate in the area of ​​cardiac injury) was dissolved in 2 mL of deionized water. To activate the free carboxyl groups on the peptide, 6.0 mg of EDC·HCl and 3.6 mg of NHS were added sequentially as catalysts to the system, and the reaction was stirred at room temperature for 1 hour. After the activation phase was completed, 24 mg of the CHO-PGEA / ED prepared in the previous step was added to the above mixed solution, and the reaction was continued to be stirred at room temperature for 1 day (24 hours) to utilize the primary amino group on CHO-PGEA / ED to undergo an amidation coupling reaction with the myocardial targeting peptide CRPPR. After the coupling reaction was complete, the resulting crude CPC product was subjected to thorough dialysis purification using a dialysis membrane with a molecular weight cutoff (MWCO) of 10,000 to remove unreacted peptides and other small molecule byproducts. The purified dialysate was then lyophilized to finally obtain the cationic nanocarrier CPC containing a cholesterol-terminated poly(glycidyl methacrylate) backbone.

[0058] Construction of EMP2 overexpression plasmid:

[0059] The specific construction method of the EMP2 overexpression plasmid is as follows: Using a 5.4 kb pcDNA3.1(+) cloning vector, the full-length 519 bp mouse EMP2 (EMP2 mus) gene sequence shown in SEQ ID NO:1 was synthesized. Restriction endonucleases HindIII (with a recognition site sequence of 5'-AAGCTT-3') and BamHI (with a recognition site sequence of 5'-GGATCC-3') were introduced at both ends of the sequence. Subsequently, the plasmid was double-digested with HindIII and BamHI, and then directionally cloned into the multiple cloning site (MCS) of the pcDNA3.1(+) vector. In the constructed recombinant expression plasmid, the EMP2 target gene sequence is placed downstream of the cytomegalovirus (CMV) promoter and driven by it to achieve high-level expression in mammalian cells; simultaneously, the gene carries a bovine growth hormone poly(A) signal sequence (bGH poly(A)). In addition, the overexpression plasmid backbone contains an ampicillin resistance gene for amplification screening in prokaryotes (such as Escherichia coli) and a neomycin resistance gene for stable transfection screening in mammalian cells.

[0060] Preparation and characterization of CPC-EMP2 plasmid nanocomposites:

[0061] The CRPPR-modified cationic nanocarrier CPC was mixed with the EMP2 overexpression plasmid constructed above at an N / P ratio of 10. The CPC carrier was then fully compressed and loaded with the EMP2 overexpression plasmid through electrostatic interaction to form an EMP2 nucleic acid delivery system for treating cardiac injury after myocardial infarction, namely the CPC-EMP2 plasmid nanocomposite.

[0062] The physicochemical properties of the CPC-EMP2 plasmid nanocomposite were characterized. Dynamic light scattering was used to detect the nanocomposite particle size, Zeta potential analysis was used to detect its surface potential, and transmission electron microscopy was used to observe the nanoparticle morphology. The results showed that the CPC-EMP2 plasmid nanocomposite had a particle size of approximately 190–220 nm, a Zeta potential of approximately +30 mV to +50 mV, and exhibited spherical or near-spherical nanoparticle morphology under transmission electron microscopy, without significant aggregation. These results indicate that the CRPPR-modified cationic nanocarrier CPC can stably load the EMP2 overexpression plasmid and form a formulation suitable for in vivo delivery.

[0063] Example 3: Validation of the therapeutic effect of CPC-EMP2 plasmid nanocomposite in a myocardial infarction model

[0064] This embodiment is used to verify the therapeutic effect of administering a non-viral nanodelivery system (CPC-EMP2 plasmid nanocomposite) on cardiac injury after myocardial infarction.

[0065] C57BL / 6 mice were randomly divided into three groups: the Sham group (n=7), the MI control group (n=8), and the MI+CPC-EMP2 treatment group (n=7). Except for the Sham group, all other groups underwent left anterior descending coronary artery ligation to establish a myocardial infarction model. After the myocardial infarction model was established, mice in the MI+CPC-EMP2 treatment group were injected with CPC-EMP2 plasmid nanocomposite via the tail vein, while mice in the MI control group were injected with an equal volume of blank CPC plasmid nanocomposite.

[0066] The method for establishing the myocardial infarction model was the same as in Example 1. After model establishment, the CPC-EMP2 plasmid nanocomplex was administered 30 minutes later. The CPC-EMP2 plasmid nanocomplex was administered at a concentration of 25 μg plasmid per mouse, with a total volume of 100 μL. Western blot experiments confirmed that the EMP2 overexpression plasmid was successfully delivered to the tissues and / or vascular endothelial cells of the cardiac injury area, and that effective expression of the EMP2 protein (e.g., ...) was achieved by administering the CPC-EMP2 plasmid nanocomplex after myocardial infarction. Figure 4 (As shown). This result verifies the excellent in vivo delivery efficiency of the nanocomposite of the present invention, thus providing a solid material basis for subsequent evaluation of the therapeutic or alleviating effect of EMP2 overexpression on cardiac damage after myocardial infarction.

[0067] The survival of mice in each group was continuously observed after surgery, and mortality was recorded. Survival rates were statistically analyzed within 28 days post-surgery. Small animal ultrasound examinations were performed on days 0, 1, 7, 14, 21, and 28 post-surgery to evaluate cardiac function parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs). Mice were euthanized after the examinations, and heart tissue was harvested for further analysis.

[0068] HE staining was used to observe myocardial tissue structural damage, and Masson staining was used to observe myocardial collagen fiber deposition and the degree of myocardial fibrosis. Results showed that compared with the MI control group, the survival rate of mice treated with MI+CPC-EMP2 after myocardial infarction was improved (e.g., ...). Figure 5 As shown in A), cardiac function is significantly improved, as evidenced by increased EF and / or FS (e.g., Figure 5 (As shown in BE); In addition, myocardial tissue structural damage was significantly reduced (e.g. Figure 6As shown in the HE staining results, collagen fiber deposition was reduced, the degree of myocardial fibrosis was decreased, and ventricular remodeling was effectively alleviated (e.g., Figure 6 (As shown in the MASSON staining results). The above results indicate that administration of CPC-EMP2 plasmid nanocomplex after myocardial infarction can treat or alleviate cardiac damage that has already occurred after myocardial infarction.

[0069] Example 4: Construction of an EMP2-overexpressing HUVECs cell model

[0070] This embodiment is used to construct a vascular endothelial cell model with stable EMP2 overexpression, providing a cell source for the subsequent preparation of extracellular vesicles derived from EMP2-overexpressing vascular endothelial cells.

[0071] Human umbilical vein endothelial cells (HUVECs) were selected as an in vitro vascular endothelial cell model. HUVECs were infected with a lentiviral expression vector carrying the human EMP2 coding sequence to construct an EMP2-overexpressing HUVECs cell model. An empty vector control group and an EMP2 overexpression group were included in the experiment.

[0072] The specific construction method of the EMP2 overexpression lentiviral expression vector is as follows: Using the coding region sequence of the human EMP2 gene transcript (RefSeq accession number: NM_001424.6) in the NCBI database as a template, upstream and downstream specific PCR primers for constructing the overexpression vector were designed. Upstream primer: 5'-CGCCGGATCCGCCACCATGTTGGTGCTTCTTGCTTTC-3' (SEQ ID NO:3, note: contains the protective base CGCC, the restriction endonuclease BamHI restriction site GGATCC, the Kozak sequence GCCACC, and the 5' start sequence of EMP2). Downstream primer: 5'-CGCCACCGGTTATTTGCGCTTCCTC-3' (SEQ ID NO:4, note: contains the protective base CGCC, the restriction endonuclease AgeI restriction site ACCGGT, and the 3' end sequence of EMP2, and the stop codon has been removed to allow fusion expression with the C-terminal tag on the vector). PCR amplification was performed using a synthesized human EMP2 template (SEQ ID NO:2). The PCR reaction system employed high-fidelity DNA polymerase, with the following reaction program: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, for a total of 30 cycles; and a final extension at 72℃ for 5 min. After identification by 1% agarose gel electrophoresis, the target fragment (approximately 550 bp) was recovered using a DNA gel extraction kit. The double-stranded lentiviral plasmid vector (a lentiviral overexpression vector with a Flag tag) and the recovered PCR product were double-digested with restriction endonucleases BamHI and AgeI. The digested vector backbone was mixed with the EMP2 target fragment at a molar ratio of 1:3, and T4 DNA ligase was added, followed by ligation overnight at 16℃. The ligation product was transformed into E. coli Stbl3 competent cells, and the transformation product was plated on LB agar plates containing 100 μg / mL ampicillin and cultured at 37°C for 16-18 hours. 293T cells in good logarithmic growth phase were seeded into culture dishes. When the cell density reached 70%-80%, the constructed recombinant plasmid pLV-EMP2-OE and lentiviral packaging helper plasmids (psPAX2 and pMD2.G) were diluted according to Opti-MEM and co-transfected into 293T cells using Lipofectamine 3000 or PEI transfection reagent. Cell culture supernatant containing lentiviral particles was collected at 48 and 72 hours post-transfection. The supernatants were combined and centrifuged at 3,000 rpm for 10 min at 4°C to remove cell debris.The supernatant was filtered through a 0.45 μm low-protein-binding filter membrane and then concentrated using the PEG-8000 precipitation method or ultracentrifugation (25,000 rpm, 2 hours). The precipitate was resuspended in sterile PBS, aliquoted, and stored at -80°C. The lentiviral titer was determined using limiting dilution (for fluorescently labeled vectors) or Real-time qPCR, with the final high-concentration lentiviral concentrate having a titer of not less than 1 × 10⁻⁶. 8 TU / mL, used for subsequent in vitro cell transduction experiments; depending on the experimental purpose, after further determination of the route of administration and dosage, it can also be used for corresponding animal experimental studies.

[0073] Human umbilical vein endothelial cells (HUVECs) were seeded in 6-well plates. When cell confluence reached approximately 80%, either purified EMP2-overexpressing lentivirus or an empty vector control lentivirus was added for infection. The multiplicity of infection (MOI) was set to 20, and polybrene was added concurrently at a final concentration of 5 μg / mL to enhance viral transduction efficiency. The medium was replaced with fresh, standard culture medium 24 hours after infection. Forty-eight hours after infection, using the puromycin resistance gene inherent in the vector, a specific concentration of puromycin (1–3 μg / mL) was added to the medium for continuous pressure selection. This selection process continued for 5–7 days until all uninfected blank cells died, thus obtaining a stable population of HUVECs carrying the exogenous EMP2 gene and a corresponding empty vector control cell population. Western blot was used to detect the expression intensity of EMP2 in the cells. The results showed that, compared with the lentiviral control group (SCR), the EMP2 protein band intensity in the EMP2 overexpression group (EMP2 OE) HUVECs cells was significantly higher than that in the empty vector control group, indicating that the EMP2 stably overexpressing HUVECs cell model was successfully constructed, establishing a stable cell line that can be used for subsequent mechanism studies and extraction of extracellular vesicles (e.g., Figure 7 (As shown).

[0074] Example 5: Extraction and identification of extracellular vesicles derived from EMP2-overexpressing HUVECs

[0075] This embodiment is used to extract and identify extracellular vesicles derived from EMP2-overexpressing HUVECs.

[0076] Extracellular vesicles were extracted from the empty vector control HUVECs and EMP2-overexpressing HUVECs obtained in Example 4. After confirming that the cells were free of mycoplasma contamination and in a normal state, the old culture medium was removed, and the cells were washed three times with PBS, followed by the addition of serum-free and exosome-free culture medium. After 48 hours of culture, the cell culture supernatant was collected. The supernatant was transferred to a centrifuge tube and centrifuged at 2000 × g for 10 min at 4 °C; the obtained supernatant was transferred to a new centrifuge tube and centrifuged again at 10000 × g for 30 min at 4 °C; the supernatant was carefully transferred to a new centrifuge tube and ultracentrifuged at 110000 × g for 70 min at 4 °C. The supernatant was then discarded, the precipitate was resuspended in pre-chilled PBS buffer, and washed again by ultracentrifugation at 110000 × g for 70 min at 4 °C. Finally, the supernatant was discarded, and the precipitate was resuspended with an appropriate volume of PBS buffer to obtain extracellular vesicles of the empty vector control group (Control-EVs) and extracellular vesicles of the EMP2 overexpression group (Over-EMP2-EVs), respectively. The prepared samples can be used for subsequent analysis, and the remaining samples are stored at -80℃ for later use.

[0077] The extracted extracellular vesicles were observed using transmission electron microscopy. The results showed that the extracellular vesicles exhibited cup-shaped, spherical, or near-spherical membranous vesicle structures, and particle size distribution was further analyzed using particle size analysis. The results showed that the extracted particles conformed to the morphology and particle size distribution of extracellular vesicles. Compared with empty vector control HUVECs, EMP2 overexpression of HUVECs-derived extracellular vesicles can be used for subsequent receptor vascular endothelial cell uptake, migration, and tube formation experiments (e.g., ...). Figure 8 (As shown).

[0078] Example 6: Extracellular vesicles derived from EMP2-overexpressing HUVECs are taken up by receptor endothelial cells

[0079] This embodiment is used to verify whether extracellular vesicles derived from EMP2-overexpressing HUVECs can be taken up by recipient vascular endothelial cells.

[0080] The Control-EVs and Over-EMP2-EVs obtained in Example 5 were labeled with PKH26 fluorescent dye. After removing the free dye, the labeled extracellular vesicles were added to the receptor HUVECs cell culture system. After co-incubation for 24 hours, the intracellular fluorescence signal of the receptor HUVECs cells was observed using immunofluorescence microscopy or laser confocal microscopy.

[0081] The results showed that the labeled extracellular vesicles could be taken up by the recipient HUVECs cells, indicating that EMP2-overexpressing extracellular vesicles derived from HUVECs can enter the recipient vascular endothelial cells, providing a basis for their regulation of recipient endothelial cell function (e.g., Figure 9 (As shown).

[0082] Example 7: EMP2 overexpression of HUVECs-derived extracellular vesicles promotes receptor endothelial cell migration and tubule formation.

[0083] This embodiment is used to verify the effect of EMP2 overexpression of HUVECs-derived extracellular vesicles on the migration and tube-forming ability of recipient vascular endothelial cells.

[0084] The receptor HUVECs were divided into three groups: Control group, Control-EVs group, and Over-EMP2-EVs group. The Control group consisted of receptor HUVECs without extracellular vesicle treatment; the Control-EVs group was supplemented with extracellular vesicles derived from empty vector control HUVECs; and the Over-EMP2-EVs group was supplemented with extracellular vesicles derived from EMP2-overexpressing HUVECs.

[0085] The migration ability of receptor HUVECs was assessed using a scratch assay. After HUVECs underwent cell fusion, scratch treatment was performed, and corresponding extracellular vesicles were added. Images were taken at 0 and 24 hours, and the scratch healing area, migration distance, or migration rate were calculated. The results showed that the migration ability of HUVECs in the Over-EMP2-EVs group was enhanced compared with that in the Control-EVs group.

[0086] The Matrigel tube formation assay was used to detect the angiogenesis-like structure formation ability of receptor HUVECs. Matrigel was seeded in 96-well plates, HUVECs and corresponding extracellular vesicles were added, and after 4 hours of incubation, images were taken, and parameters such as total tube length, number of branch points, number of network structures, and number of closed loops were recorded. Results showed that, compared with the Control-EVs group, Over-EMP2-EVs treatment promoted the formation of luminal structures (such as...) in HUVECs. Figure 10 (As shown).

[0087] The above results indicate that EMP2 overexpression of extracellular vesicles derived from HUVECs can be taken up by recipient vascular endothelial cells and promote the migration and tube formation of recipient vascular endothelial cells, suggesting that EMP2 overexpression of extracellular vesicles derived from endothelial cells has the effect of promoting vascular endothelial repair.

[0088] Based on the above embodiments, this invention demonstrates that: administering the AAV9-EMP2 expression vector before myocardial infarction can enhance EMP2 expression and prevent cardiac damage; administering the CPC-EMP2 plasmid nanocomposite after myocardial infarction can deliver EMP2 and alleviate existing cardiac damage; and overexpression of EMP2 in extracellular vesicles derived from vascular endothelial cells can promote the migration and tube formation of recipient vascular endothelial cells.

[0089] Based on the superior effects of the above three formulations at different intervention stages and target sites, this invention also provides a composition for the prevention and / or treatment of myocardial infarction-related cardiac injury in some specific embodiments. This composition includes: the prophylactic AAV9-EMP2 expression vector described in Example 1, and the therapeutic EMP2 nucleic acid delivery system (such as CPC-EMP2 plasmid nanocomplex) described in Examples 2 and 3. More preferably, the composition also includes the extracellular vesicle formulation for promoting vascular endothelial repair described in Example 5. By combining the above formulations, this invention synergistically works from three levels: pre-intervention before myocardial infarction, timely intervention after myocardial infarction, and long-term microvascular endothelial environment repair, constructing a comprehensive and systematic technical solution for the prevention and treatment of myocardial infarction injury centered on EMP2.

Claims

1. The use of a formulation in the preparation of a medicament for the prevention and / or treatment of myocardial infarction-related cardiac injury, characterized in that, The formulation is selected from any one of the following: (I) Viral expression vector carrying the EMP2 coding sequence; (II) Non-viral nanodelivery system loaded with EMP2 overexpression plasmid; (III) Extracellular vesicles derived from EMP2-overexpressing vascular endothelial cells.

2. The application according to claim 1, characterized in that, The formulation is an adeno-associated virus type 9 expression vector AAV9-EMP2 carrying the EMP2 coding sequence, and the drug is used to prevent cardiac damage caused by myocardial infarction.

3. The application according to claim 2, characterized in that, The drug is used to increase the expression level of EMP2 in cardiac tissue and / or cardiac vascular endothelial cells, thereby enhancing the resistance of cardiac tissue to myocardial infarction damage.

4. The application according to claim 1, characterized in that, The formulation is a non-viral nanodelivery system comprising a myocardial targeting peptide-modified cationic nanocarrier and an EMP2 overexpression plasmid loaded in the carrier.

5. The application according to claim 4, characterized in that, The myocardial targeting peptide is CRPPR, the cationic nanocarrier is a CPC carrier containing a cholesterol-terminated polyglycidyl methacrylate backbone, and the non-viral nanodelivery system is a CPC-EMP2 plasmid nanocomposite.

6. The application according to claim 5, characterized in that, The CPC-EMP2 plasmid nanocomposite has a particle size of 190-220 nm and a zeta potential of +30 mV to +50 mV.

7. The application according to any one of claims 4-6, characterized in that, The preparation is used to treat or alleviate cardiac damage following myocardial infarction.

8. The application according to claim 7, characterized in that, The treatment or relief of cardiac damage after myocardial infarction includes: improving cardiac function after myocardial infarction, increasing ejection fraction (EF), increasing fractional shortening (FS), reducing myocardial tissue damage, reducing myocardial fibrosis, reducing collagen fiber deposition, inhibiting ventricular remodeling, and / or promoting vascular repair.

9. An EMP2 nucleic acid delivery system for treating cardiac injury after myocardial infarction, characterized in that, The EMP2 nucleic acid delivery system comprises a myocardial-targeted modified cationic nanocarrier and an EMP2 overexpression plasmid loaded in the carrier.

10. The EMP2 nucleic acid delivery system according to claim 9, characterized in that, The myocardial targeting modification is a dual-targeting modification or a CRPPR peptide modification with targeting capabilities to the heart and areas of heart injury. The cationic nanocarrier contains a cholesterol-terminated polyglycidyl methacrylate backbone.

11. An extracellular vesicle formulation for promoting vascular endothelial repair, characterized in that, The extracellular vesicle preparation is derived from EMP2-overexpressing vascular endothelial cells.

12. The extracellular vesicle formulation according to claim 11, characterized in that, The EMP2-overexpressing vascular endothelial cells are HUVECs and are obtained by processing a lentiviral expression vector carrying the EMP2 coding sequence.

13. Use of the extracellular vesicle formulation of any one of claims 11-12 in the preparation of a medicament or formulation for promoting vascular endothelial repair.

14. The application according to claim 13, characterized in that, The promotion of vascular endothelial repair includes promoting vascular endothelial cell migration, promoting vascular endothelial cell tube formation, and / or promoting the formation of angiogenesis-like structures.

15. A composition for the prevention and / or treatment of cardiac injury associated with myocardial infarction, characterized in that, include: The AAV9-EMP2 expression vector as described in claim 2 and the EMP2 nucleic acid delivery system as described in claim 9.

16. The composition according to claim 15, characterized in that, It also includes the extracellular vesicle formulation as described in claim 11.

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