Method for preparing conjugate of engineered exosome and microRNA drug, conjugate and application thereof

Abstract

The invention provides a method for preparing a conjugate of an engineered exosome and a micro RNA (Ribonucleic Acid) drug, the conjugate and application of the conjugate. The method comprises the following steps: (1) purifying by combining continuous ultracentrifugation with size exclusion chromatography to obtain a high-purity milk-derived exosome from cow milk; (2) removing endogenous RNA (Ribonucleic Acid) from the high-purity milk source exosome by adopting an ultrasonication method to obtain an endogenous RNA removed engineered exosome; (3) loading a microRNA drug to the endogenous RNA cleared engineered exosome by an electroporation method to obtain a conjugate of the endogenous RNA cleared engineered exosome and the microRNA drug; and (4) anchoring the ischemic myocardial targeting peptide on the surface of the exosome through cholesterol and PEG2000 to obtain the conjugate of the endogenous RNA cleared myocardial targeting engineered exosome and the microRNA drug. According to the method, endogenous RNA interference is eliminated, the myocardial targeting efficiency is improved, and the production process is standardized.

Description

Technical Field

[0001] This invention relates to the field of myocardial infarction treatment technology, specifically to a method for preparing a conjugate of myocardial-targeted engineered exosomes and microRNA drugs, the conjugate, and its application. Background Technology

[0002] Myocardial infarction (MI) is a necrotizing disease of the myocardium caused by acute and persistent ischemia and hypoxia of the coronary arteries. It is characterized by restricted blood flow in the infarcted area, leading to progressive death of cardiomyocytes and ultimately ventricular remodeling and heart failure. Although existing clinical interventions (such as percutaneous coronary intervention) can partially alleviate acute symptoms, they cannot effectively regulate the imbalanced microenvironment in the infarcted area (including uncontrolled inflammatory response, oxidative stress, and pathological remodeling of the extracellular matrix), resulting in limited long-term efficacy.

[0003] MicroRNAs (miRNAs) are a class of non-coding RNAs, typically about 22 nucleotides in length, that regulate post-transcriptional gene expression by binding to 3' untranslated regions (UTRs). miRNAs have become an emerging strategy for myocardial repair due to their multi-target regulatory capabilities. However, the clinical application of miRNA therapy faces bottlenecks in delivery systems; synthetic carriers (such as liposomes and polymer nanoparticles) suffer from poor biocompatibility, high immunogenicity, and difficulty in penetrating biological barriers. While natural milk-derived extracellular vesicles (mEVs) possess low immunogenicity, long circulating half-life, and barrier-crossing capabilities, their endogenous RNA (especially miRNA) loading exhibits significant batch-to-batch variability, introducing uncontrollable off-target effects and lacking active myocardial targeting, resulting in low drug accumulation efficiency in the infarct area.

[0004] Existing technologies (such as ultracentrifugation-based systems for separating natural mEVs and loading miRNAs) have the following drawbacks: First, insufficient endogenous RNA clearance: conventional methods (extrusion or electroporation) can only remove 30–40% of endogenous RNA, and residual RNA interferes with the function of therapeutic miRNAs; second, lack of targeting ability: unmodified mEVs rely on passive diffusion, resulting in low drug utilization in the myocardium; third, unstable production process: the loading process easily damages the integrity of vesicles, and the drug loading varies greatly between batches.

[0005] Therefore, there is an urgent need to develop a standardized, highly targeted miRNA delivery system that can eliminate endogenous RNA interference in order to achieve long-term functional repair after myocardial infarction. Summary of the Invention

[0006] To address the three major drawbacks of existing natural milk exosome (mEV) delivery systems—low endogenous RNA clearance rate, lack of myocardial targeting, and unstable manufacturing process—this application aims to provide a myocardial-targeted engineered milk exosome conjugate with a miR30d mimic (miR30d-mELVs). IMTP This technology utilizes an innovative process chain of "ultrasound-mediated removal of endogenous RNA-electroporation loading of miR30d-IMTP peptide targeting modification" to construct a high-purity, high-stability delivery system, which continuously improves cardiac function, inhibits fibrosis, and reduces inflammatory response 21 days after myocardial infarction.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] On one hand, the present invention provides a method for preparing a conjugate of engineered exosomes and microRNA drugs, the method comprising the following steps: (1) purifying high-purity milk-derived exosomes from cow's milk by continuous ultracentrifugation combined with size exclusion chromatography; (2) removing endogenous RNA from the high-purity milk-derived exosomes by ultrasonic disruption, thereby obtaining engineered exosomes (mELVs) with endogenous RNA removal; (3) loading the microRNA drug into the engineered exosomes with endogenous RNA removal by electroporation. (3) mELVs), thereby obtaining a conjugate of engineered exosomes for endogenous RNA clearance and a microRNA drug (miR-30d) mimic (miR30d-mELVs); (4) anchoring the ischemic myocardial targeting peptide (IMTP) to the surface of the exosomes in the conjugate of engineered exosomes for endogenous RNA clearance and a microRNA drug using cholesterol and PEG2000, thereby obtaining a conjugate of engineered exosomes for myocardial clearance and a microRNA drug (miR30d-mELVs). IMTP ).

[0009] Optionally, in step (1), the continuous ultracentrifugation includes: centrifuging at 70,000-80,000×g and 4°C for 1-1.5 hours to remove the fat layer; discarding the supernatant, and then centrifuging at 110,000-115,000×g for 1-1.5 hours to remove large particles and cell debris; subsequently centrifuging the resulting supernatant at 110,000-120,000×g for 0.8-1 hours.

[0010] Optionally, in step (2), the ultrasonic conditions used in the ultrasonic fragmentation method include: amplitude 10-15%, 30-40 cycles.

[0011] Optionally, in step (2), RNaseOUT is added after sonication to inhibit the activity of residual RNaseA.

[0012] Optionally, in step (3), the parameters used in the electroporation method are 800-1100 voltage, 8-10ms pulse width, and 2-5 pulse times.

[0013] Optionally, the particle size of the high-purity milk exosomes obtained in step (1) is 130 ± 10 nm.

[0014] On the other hand, the present invention provides a combination of myocardial-targeted engineered exosomes for endogenous RNA clearance and microRNA drugs obtained according to the method described above.

[0015] In another aspect, the present invention provides the use of a combination of myocardial-targeted engineered exosomes for endogenous RNA clearance obtained by the method described above with a microRNA drug in the preparation of a drug for the treatment of myocardial infarction.

[0016] Optionally, the conjugate is used to inhibit the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α.

[0017] Optionally, the combination is used to improve cardiac function parameters: ejection fraction, left ventricular fractional shortening, left ventricular end-diastolic diameter, and left ventricular end-systolic diameter.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] Complete elimination of endogenous RNA interference: The ultrasonic clearance method (amplitude 10%, 30 cycles) increases the endogenous RNA clearance rate from 30-40% in the existing technology to 70%, and combined with RNaseOUT, the stability of exogenous miRNA is >95%, fundamentally solving the off-target effect problem.

[0020] Breakthrough in myocardial targeting efficiency: IMTP peptide modification enables vesicles to actively accumulate in infarcted myocardium, directly manifested as a significant advantage in improving cardiac function parameters and histological features (reduced collagen deposition) at 21 days, overcoming the defect of passive diffusion of unmodified mEVs.

[0021] Standardized production process: The two-step purification of "ultracentrifugation + size exclusion chromatography" increases the purity of vesicles by 5 times. Optimized electroporation parameters (1000V voltage, 10ms pulse width, 2 pulses) achieve an encapsulation rate of 10% while maintaining vesicle integrity (particle size change <5%), and the batch-to-batch fluctuation range is small, which completely solves the problem of batch-to-batch efficacy differences. Attached Figure Description

[0022] Figure 1 The particle size distribution maps of nanoparticle tracking analysis (NTA) in the ultracentrifugation (UC) group and the UC+ size exclusion chromatography (SEC) group are shown.

[0023] Figure 2Transmission electron microscopy (TEM) images of purified mEVs from the UC and UC+SEC groups are shown.

[0024] Figure 3 The results show the positive expression of TSG101, HSP70, and CD9 proteins in the UC group and UC+SEC group, as verified by Western blot.

[0025] Figure 4 A bar chart comparing the particle number / protein ratio between the UC group and the UC+SEC group is shown.

[0026] Figure 5 A bar chart showing the effect of different sonication cycles on endogenous RNA clearance rate as detected by qPCR is presented.

[0027] Figure 6 A schematic diagram of the preparation process of engineered exosomes with endogenous RNA removed in this application is shown.

[0028] Figure 7 The effects of different ultrasonic amplitudes (10%, 20%, 30%) on particle concentration and vesicle integrity (particle size and zeta potential) are shown.

[0029] Figure 8 mELVs and miR30d-mELVs were shown. IMTP NTA particle size distribution map and TEM image.

[0030] Figure 9 A schematic diagram of the timeline of the animal experiment is shown (drug administration 24 hours post-surgery, endpoint analysis at 21 days).

[0031] Figure 10 The Masson trichrome stained section shows the recovery of left ventricular wall thickness.

[0032] Figures 11A-11B A quantitative map of the cross-sectional area of ​​cardiomyocytes stained with wheat germ lectin (WGA) is shown.

[0033] Figure 12 The bar chart shows the ELISA detection of inflammatory factors (IL-1β, IL-6, TNF-α) in cardiac tissue.

[0034] Figure 13 A line graph showing the changes in cardiac function parameters from echocardiography is presented.

[0035] Figure 14 The changes in indicators and key enzyme activities of cardiac ultrasound examinations in mice from different experimental groups are shown.

[0036] Figure 15 The original images of ultrasound examinations of mice in different experimental groups are shown.

[0037] Figure 16 The changes in cardiac ejection fraction and left ventricular function indices in mice from different experimental groups are shown. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments. The described embodiments are only some, not all, of the embodiments of this invention.

[0039] This invention discloses a myocardial-targeted engineered exosome (miR30d-mELVs) IMTP The exosomes, their preparation method, and their application in the treatment of myocardial infarction are described. The exosomes are prepared through the following steps: First, high-purity exosomes (mEVs) with a particle size of 130 nm are obtained by ultracentrifugation (78600 x g, 1 h; 115200 x g, 1.5 h; 115000 x g, 1 h) combined with size exclusion chromatography. Subsequently, 70% of endogenous RNA is removed by ultrasonic disruption (10% amplitude, 30-40 cycles of pulses), and residual enzyme activity is inhibited by RNaseOUT, forming RNA-clearing exosomes (mELVs). The miR30d mimic was further loaded into mELVs via electroporation (parameters: 1000V voltage, 10ms pulse width, 2 pulses) with a loading efficiency of 10%. Simultaneously, an ischemic myocardial targeting peptide (IMTP) was anchored to the exosome surface using a cholesterol-PEG2000 linker, yielding the final product: a conjugate of myocardial-targeting engineered exosomes and the microRNA drug (miR-30d mimic) (miR30d-mELVs). IMTP The particle size was 157 nm. After being injected via the tail vein into a mouse model of myocardial infarction, the conjugate significantly improved key cardiac function parameters, reduced collagen deposition and infarct area, increased left ventricular wall thickness, decreased pro-inflammatory cytokine expression, and alleviated myocardial hypertrophy at 21 days.

[0040] This invention addresses the technical shortcomings of natural milk exosomes, such as interference from endogenous RNA, lack of myocardial targeting, and poor process stability, providing a new strategy for myocardial infarction repair.

[0041] Unless otherwise stated, the raw materials and equipment used in this invention are all commonly used in the art, and the methods used in this invention are all conventional methods in the art.

[0042] Unless otherwise stated, parts are by weight, % is by weight, and temperature is in degrees Celsius.

[0043] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that: in the following embodiments, unless otherwise specified, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer, and the raw materials used in the following embodiments can be obtained from commercially available sources unless otherwise specified.

[0044] The sources and specifications of each raw material are as follows:

[0045] Milk: Commercially available fresh whole milk (fat content ≥ 3.5%).

[0046] miR-30d (or miR30d) analogue: sequence 5'-UGUAAACAUCCCCGACUGGAAG-3' (SEQ ID NO: 1), purchased from Cytiva.

[0047] Ischemic myocardial targeting peptide (IMTP): purity >95%, purchased from Jier Biochemical.

[0048] Cholesterol-PEG2000: CAS No. 474922-22-0, purchased from Laysan Bio, Inc.

[0049] RNaseOUT: Ribonuclease inhibitor, catalog number 10777-019, purchased from Invitrogen.

[0050] The qEV high-purity size exclusion chromatography (SEC) exosome separation column was purchased from Izon.

[0051] Phosphate-buffered saline (PBS): pH 7.4, purchased from Solarbio.

[0052] RNase A: CAS No. 9001-99-4, purchased from Sigma-Aldrich, purity ≥90%.

[0053] 100 kDa MWCO Amicon® ultrafiltration tube, purchased from Merck Biotechnology.

[0054] Male C57BL / 6J mice aged 7-8 weeks were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0055] Example

[0056] Example 1: A conjugate of engineered milk-derived exosomes and miR30d mimics (miR30d-mELVs) IMTP Preparation of )

[0057] 1. Isolation, purification, and characterization of high-purity milk-derived exosomes (mEVs)

[0058] Take 150 mL of commercially available fresh whole milk (fat content ≥3.5%, w / w), mix with 0.05% (w / v) rennet and 0.3% (w / w) CaCl2, and incubate at room temperature for 5-15 minutes. Centrifuge the resulting mixture at 10000 rpm for 30 minutes to obtain whey supernatant. Aliquot the obtained whey supernatant into ultracentrifuge tubes and perform the following continuous ultracentrifugation (UC): centrifuge at 78600×g and 4℃ for 1 hour to remove the fat layer; discard the supernatant, then centrifuge at 115200×g for 1.5 hours to remove large particles and cell debris; subsequently, centrifuge the obtained supernatant at 115000×g for 1 hour, discard the supernatant, and obtain crude exosomes. Resuspend the obtained crude exosomes in phosphate-buffered saline (PBS), centrifuge at 4000×g for 20 minutes using a 100 kDa ultrafiltration tube, concentrate twice, and obtain preliminarily purified mEVs.

[0059] To further purify mEVs, size exclusion chromatography (SEC) was used. The preliminarily purified mEVs were loaded onto a pre-equilibrated 35 nm qEV2 size exclusion column (25 mL column bed volume), eluted with PBS as the mobile phase (flow rate 0.5 mL / min), and the obtained fractions were collected.

[0060] After concentration, the obtained components yielded high-purity mEVs purified by SEC. Nanoparticle tracking analysis (NTA) determined the particle size distribution to be 30–250 nm, with a main peak at 130 ± 10 nm. Figure 1 Transmission electron microscopy (TEM) revealed its distinct, typical cup-shaped morphology. Figure 2 Western blot analysis confirmed that the exosome-specific markers TSG101, HSP70, and CD9 were significantly positive. Figure 3 The key purity indicator—particle count / protein ratio—is 5 × 10⁻⁶. 9 The purity (particles / protein ratio) of exosomes in the UC+SEC group was approximately 5 times that of the UC group. Figure 4 ).

[0061] 2. Endogenous RNA clearance process

[0062] To remove endogenous RNA from mEVs, RNase A was loaded onto the mEVs using sonication. Specifically, RNase A (100 μg / mL) was loaded onto the mEVs with the high-purity mEVs obtained above (1×10⁻⁶). 12Mix (particles / mL); sonicate in an ice bath under the following conditions: amplitude 10%, pulse on / off time 5s / 5s, total cycles 30 (ultrasound machine: Xiaomei XT-100). The Quant-iT™ RiboGreen RNA Quantification Kit (Thermo Fisher Scientific) showed an endogenous RNA clearance rate of approximately 70%. Figure 5 While higher sonication amplitudes (20% and 30%) could further improve the clearance of endogenous RNA, they damaged vesicle integrity, as shown by the reduced particle size, and increased zeta potential. By comparison, an amplitude of 10% and a total of 30 cycles preserved the morphology, size distribution, and surface charge of mEVs.

[0063] To inhibit the degradation activity of residual RNase on subsequently loaded microRNA (miR30d), RNaseOUT (40U per 20μL reaction volume) was added immediately after sonication, and the mixture was incubated at 37°C for 30 minutes to completely inhibit residual RNase activity, thereby maintaining the stability of the exogenously introduced microRNA. The exosomes (vectors) obtained in this step are engineered mEVs for endogenous RNA clearance, hereinafter referred to as mEV-like vesicles (mELVs). Figure 6 Its particle size was 128 nm, and its zeta potential was -18.3 mV, which was not significantly different from that before treatment, confirming that the integrity of the vesicles was preserved. Figure 7 ).

[0064] 3. miR30d-mELVs IMTP Preparation and characterization

[0065] miR30d is a microRNA that plays an important regulatory role in cardiovascular disease and tumorigenesis. It has been shown to simultaneously inhibit cardiomyocyte apoptosis pathways and alleviate cardiac dysfunction. To investigate the role of miR30d in myocardial infarction (MI), RNA-clearing milky exosomes (mELVs) were engineered. An optimized electroporation strategy was used to load miR30d mimics into mELVs. Electroporation conditions were systematically tested to ensure effective loading without compromising vesicle stability.

[0066] Using Neon TM The transfection system (Invitrogen, Carlsbad, CA, USA) was used to prepare miR30d-loaded mELVs (miR30d-mELVs). Specifically, miR30d mimics (50 μM, 1 nmol) were mixed with mELVs (5 × 10⁻⁶ mELVs). 11 Mix (particles); use an electroporator (Neon) TMElectroporation was performed using a Transfection System (parameters: 1000V voltage, 10ms pulse width, 2 pulses) to obtain miR30d-mELVs. The encapsulation efficiency of the fluorescent label was 10% as determined quantitatively.

[0067] IMTP Anchoring: To achieve myocardial targeting, ischemic myocardial targeting peptide (IMTP) was mixed with cholesterol (Chol)-PEG2000 at a molar ratio of 1:1 and reacted at 25°C for 4 hours to synthesize a Chol-PEG-IMTP linker. This Chol-PEG-IMTP linker (final concentration 100 μM) was co-incubated with drug-loaded exosomes (miR30d-mELVs) (25°C, 1 hour), achieving IMTP anchoring through covalent binding of cholesterol into the lipid layer of the vesicle membrane. The final product miR30d-mELVs was obtained. IMTP The average particle size, as determined by NTA, was 157 nm, a slight increase compared to unmodified mELVs (130 nm), likely due to peptide modification. TEM (HITACHI HT7800H, 120 kV, Japan) showed that the vesicles retained their typical goblet structure, and Western blot analysis detected the expression of exosome markers TSG101 and HSP70. Figure 8 Importantly, the binding of miR30d was further verified by quantifying Cy3-labeled miR30d, with an encapsulation efficiency of 10%.

[0068] Encapsulation efficiency (EE%) is calculated using the following formula:

[0069] EE% = Loaded miR30d (pmole) / Total added miR30d (pmole) × 100%

[0070] 4. Measurement of RNA clearance rate

[0071] Total RNA was obtained from 1×10 using the Qiager Life Sciences miRNeasy Micro kit. 11Extracted from individual mEVs or mELVs particles. Total RNA concentration was quantified using a Thermo Fisher Quant-iT RiboGreen RNA assay kit. miR30d encapsulated in mEVs, mELVs, or miR30d-mELVs was quantified using a StepOnePlus™ real-time PCR system via real-time quantitative polymerase chain reaction (RT-qPCR). The relative abundance of miR30d was determined by comparing Ct values ​​with a standard curve of miR30d oligonucleotide synthesis. Results showed that the RNA clearance rate in "2. Endogenous RNA clearance process" was approximately 70%, and in "3. miR30d-mELVs..." IMTP miR30d-mELVs and miR30d-mELVs prepared in the "Preparation and Characterization" section IMTP The encapsulation rate of miR30d is 10%.

[0072] Comparative Example

[0073] Comparative Example 1 (Insufficient Ultrasonic Amplitude)

[0074] The procedure was the same as in Example 1, but the ultrasonic amplitude used for endogenous RNA clearance was reduced to 5%.

[0075] The results showed that the RNA clearance rate was 42.3%, and the residual RNA led to the inhibition of exogenous miR30d function.

[0076] Comparative Example 2 (Incorrect Electroporation Parameters)

[0077] The operation is the same as in Example 1, but when preparing miR30d loaded mELVs (miR30d-mELVs), the electroporation parameters used are changed to 500V voltage, 20ms pulse width, and 5 pulses.

[0078] Results: Encapsulation efficiency was 6.1%, particle size increased to 158 nm, vesicle membrane integrity was insufficient or caused complete rupture.

[0079] Example 2: miR30d-mELVs IMTP Treatment and efficacy verification of myocardial infarction

[0080] 1. Animal models and miR30d-mELVs IMTP Dosage

[0081] All animal experiments were approved by the Animal Experiment Ethics Committee of Shandong First Medical University and Shandong Academy of Medical Sciences (LS2024103). Seven- to eight-week-old male C57BL / 6J mice were used, provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. All animals were housed under specific pathogen-free (SPF) conditions at 22–24°C with a 12-hour light / 12-hour dark cycle. After a one-week acclimatization period, mice were anesthetized with 2% isoflurane followed by maintenance anesthesia with 1.5% isoflurane. The heart was then exposed via a thoracotomy at the fourth intercostal space. The left anterior descending coronary artery (LAD) was then permanently ligated approximately 3 mm from its origin using 8-0 silk sutures. The thoracotomy incision was then sutured layer by layer with 4-0 silk sutures, and air was drained from the pleural cavity to prevent pneumothorax. Postoperatively, mice were transferred to a 37°C heating pad to maintain warmth until recovery. Sham-operated mice underwent the same procedure without LAD ligation.

[0082] To study the therapeutic effect after myocardial infarction, animals were randomly divided into: (1) sham surgery group; (2) myocardial infarction model group (only the myocardial infarction surgery group, which was performed according to the above-mentioned left anterior descending coronary artery ligation procedure, the same below); (3) miR30d-mELVs IMTP Group (mice injected with miR30d-mELVs after surgery) IMTP The injection group received a tail vein injection (1 × 10⁻⁶) after myocardial infarction. 11 Each granule (≥5 mice per group) was administered on days 0, 1, and 2. Figure 9 ).

[0083] 2. Efficacy Verification

[0084] To evaluate miR30d-mELVs IMTP To investigate the sustained therapeutic effect on cardiac function after myocardial infarction, mice were euthanized 21 days after myocardial infarction, and histological and functional analyses were performed. Figure 9 ).

[0085] Histopathology: Three weeks (21 days) after myocardial infarction, Masson trichrome staining showed that, compared with the saline group, the use of miR30d-mELVs... IMTP Treatment resulted in a significant reduction in infarct size and collagen deposition (collagen deposition in the infarct area was 21.9% in the treatment group), and an increase in left ventricular (LV) wall thickness. Figure 10 Consistent with observations made on day 7 after myocardial infarction, miR30d-mELVs IMTP Myocardial hypertrophy was significantly reduced on day 21.

[0086] This effect was further confirmed by wheat germ agglutinin (WGA) staining, which demonstrated a reduction in the cross-sectional area of ​​cardiomyocytes. Figures 11A-11B ).

[0087] Inflammatory regulation: To observe the inflammatory environment in the chronic phase, the levels of representative pro-inflammatory cytokines were assessed. For example... Figure 12 As shown, miR30d-mELVs IMTP Treatment significantly inhibited the expression of IL-1β, IL-6, and TNF-α. ELISA analysis showed that the levels of IL-1β, IL-6, and TNF-α in cardiac tissue were significantly reduced (…). Figure 12 ).

[0088] Cardiac function recovery: Longitudinal assessment of cardiac function was performed by echocardiography on days 7, 14, and 21 post-myocardial infarction. Key parameters were monitored, including ejection fraction (EF), left ventricular fractional shortening (FS), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs). In the myocardial infarction model group, EF and FS steadily decreased from day 7 to day 21, accompanied by a steady increase in LVIDd and LVIDs, indicating progressively worsening left ventricular dilation and continued deterioration of systolic function. Conversely, patients receiving miR30d-mELVs... IMTP The treated mice showed sustained and significant improvements in all four parameters, reflecting a gradual recovery of cardiac structure and function. Specifically, on day 21, the ejection fraction (EF) increased to 45.3%, compared to less than 20% in the myocardial infarction model group, and the left ventricular end-systolic diameter (LVIDs) decreased to 4.1 mm (6.2 mm in the myocardial infarction model group), with continued improvement from day 7 to day 21. Figure 13 In the diagram, the black line (circle) represents the sham surgery group, the orange line (triangle) represents the treatment group, and the blue line (square box) represents the model group. At 21 days, the ranking was: model group > treatment group > sham surgery group.

[0089] These findings highlight miR30d-mELVs IMTP Its therapeutic advantages not only demonstrate acute cardioprotective effects, but also have long-term benefits in reducing pathological remodeling and promoting functional recovery after myocardial infarction.

[0090] Example 3

[0091] To assess the functional specificity of miR30d, miRNA mimics (miRNCs) were used as a negative control group to evaluate various indicators in mice after myocardial infarction. Animals were randomly assigned to: (1) sham surgery group; (2) myocardial infarction model group (MI, myocardial infarction surgery only, performed according to the above-mentioned left anterior descending coronary artery ligation procedure, the same below); (3) miRnc-mELVs group (mice were injected with miRnc-mELVs after surgery); (4) miR30d-mELVs group (mice were injected with miR30d-mELVs after surgery); (5) miR30d-mELVs IMTP Group (mice injected with miR30d-mELVs after surgery) IMTP The injection group received a tail vein injection (1 × 10⁻⁶) after myocardial infarction. 11 Each granule (≥5 mice per group) was administered on days 0, 1, and 2. Figures 14-16 ).

[0092] As expected, serum levels of cardiac injury markers (creatine kinase isoenzyme MB (CK-MB) and cardiac troponin T (cTnT)) were significantly elevated in mice in the myocardial infarction (MI) model group. Notably, only miR30d-mELVs showed elevated levels. IMTP The treatment significantly reduced the levels of these enzymes ( Figure 14 This indicates that myocardial injury has been alleviated. Histological analysis was then performed using Masson's trichrome staining on day 7 post-myocardial infarction. Compared to miRnc-mELVs or miR30d-mELVs, miR30d-mELVs... IMTP The administration significantly reduced myocardial fibrosis and infarct area. Figure 15 In the myocardial infarction model group (MI), myocardial infarction leads to severe cardiac dysfunction, manifested as decreased ejection fraction (EF) and left ventricular fractional shortening (FS), while increasing left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs). Figure 16 It is worth noting that miR30d-mELVs IMTP The treatment significantly improved cardiac function by reducing LVIDd and LVIDs and enhancing EF and FS.

[0093] The above embodiments are merely explanations of this application and are not intended to limit this application. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a conjugate of engineered exosomes and a microRNA drug, wherein the microRNA drug is a miR-30d mimic, characterized in that, The method includes the following steps: (1) High-purity milk-derived exosomes were obtained from cow's milk by continuous ultracentrifugation combined with size exclusion chromatography; (2) Endogenous RNA was removed from the high-purity milk-derived exosomes by ultrasonic disruption, thereby obtaining engineered exosomes with endogenous RNA removal; (3) The microRNA drug is loaded into the engineered exosomes of endogenous RNA clearance by electroporation, thereby obtaining a conjugate of engineered exosomes of endogenous RNA clearance and microRNA drug; (4) Anchoring ischemic myocardial targeting peptides to the surface of the exosomes in the conjugate of the endogenous RNA clearance engineered exosomes and miR-30d mimic by cholesterol and PEG2000, thereby obtaining the conjugate of the endogenous RNA clearance myocardial targeting engineered exosomes and microRNA drugs.

2. The method according to claim 1, characterized in that, In step (1), the continuous ultracentrifugation includes: centrifuging at 70,000-80,000×g for 1-1.5 hours to remove the fat layer; discarding the supernatant, and then centrifuging at 110,000-115,000×g for 1-1.5 hours to remove large particles and cell debris; subsequently centrifuging the obtained supernatant at 110,000-120,000×g for 0.8-1 hours.

3. The method according to claim 1, characterized in that, In step (2), the ultrasonic conditions used in the ultrasonic fragmentation method include: amplitude 10-15%, 30-40 cycles.

4. The method according to claim 1, characterized in that, In step (2), RNaseOUT is added after ultrasound to inhibit the activity of residual RNaseA.

5. The method according to claim 1, characterized in that, In step (3), the parameters used in the electroporation method are 800-1100 voltage, 8-10ms pulse width, and 2-5 pulse times.

6. The method according to any one of claims 1 to 5, characterized in that, The particle size of the high-purity milk exosomes obtained in step (1) is 130±10 nm.

7. A conjugate of myocardial-targeted engineered exosomes for endogenous RNA clearance and microRNA drugs obtained by the method of any one of claims 1 to 6.

8. The use of the combination of myocardial-targeted engineered exosomes for endogenous RNA clearance and microRNA drugs obtained by the method according to any one of claims 1 to 6 in the preparation of drugs for the treatment of myocardial infarction.

9. The application according to claim 8, characterized in that, The conjugate is used to inhibit the expression of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α.

10. The application according to claim 8, characterized in that, The conjugate is used to improve cardiac function parameters: ejection fraction, left ventricular fractional shortening, left ventricular end-diastolic diameter, and left ventricular end-systolic diameter.

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