MOTS-c targeted delivery system based on astragalus-derived exosome and application of MOTS-c targeted delivery system in myocardial ischemia-reperfusion injury

Abstract

The invention relates to an MOTS-c targeted delivery system based on an exosome derived from astragalus membranaceus and application of the MOTS-c targeted delivery system in myocardial ischemia-reperfusion injury. The preparation method comprises the following steps: preparing an exosome AExo from radix astragali from fresh radix astragali as a raw material, modifying a cardiac troponin I targeting peptide LPFFD to the surface of the AExo by using an EDC / NHS chemical cross-linking method to obtain LPFFD-AExo, and loading a mitochondrial derived small peptide MOTS-c into the LPFFD-AExo by using an ultrasonic-assisted penetration method to obtain the LPFFD-AExo-MOTS-c. According to the invention, the accurate delivery efficiency of the damaged myocardial tissue can be improved, and efficient and accurate delivery of MOTS-c to the ischemia reperfusion damaged myocardial tissue is realized, so that the ferroptosis inhibition capability of MOTS-c is obviously enhanced, the myocardial protection effect on MI / R injury is improved, and finally efficient targeted therapy on MI / R injury is realized.

Description

Technical Field

[0001] This invention relates to a functionalized drug delivery system based on plant-derived exosomes, specifically a MOTS-c targeted delivery system that utilizes exosomes derived from Astragalus membranaceus to encapsulate the mitochondrial-derived peptide MOTS-c and modifies it with myocardial targeting peptides for the precise treatment of myocardial ischemia-reperfusion injury, belonging to the field of biomedical technology. Background Technology

[0002] In recent years, myocardial repair strategies using exosomes (Exo) as carriers have gradually become a hot topic in cardiovascular disease research. Among them, extracellular vesicles, represented by mesenchymal stem cell (MSC)-derived exosomes (MSCs-Exo), have shown significant therapeutic potential in inhibiting inflammatory responses, reducing apoptosis, and improving the ischemic microenvironment due to their rich content of various bioactive molecules (such as microRNA, proteins, and lipids). Existing studies have shown that MSCs-Exo have significant cardioprotective effects in experimental models of myocardial infarction and myocardial ischemia-reperfusion (MI / R) injury, and can effectively improve cardiac function.

[0003] Plant-derived exosomes (PExo), as an emerging natural nanocarrier, have shown promising applications in the biomedical field in recent years. Studies have found that exosomes derived from plants such as citrus, ginger, and grape possess good biocompatibility, low immunogenicity, and certain endogenous therapeutic activity, and have achieved significant progress in multiple disease models, including inflammatory diseases and tumor treatment. Astragalus membranaceus, a traditional Chinese medicine, is widely used clinically due to its well-defined antioxidant, anti-inflammatory, and cardiovascular protective effects. In recent years, astragalus membranaceus-derived exosomes (AExo) have gradually attracted attention as a novel nanocarrier for the effective components of astragalus, but there are currently no reports of its direct use in the treatment of MI / R injuries.

[0004] The mitochondrial-derived peptide MOTS-c has recently emerged as a promising area of ​​research in cardiovascular medicine due to its significant biological functions. Studies have shown that MOTS-c can participate in the regulation of cellular energy metabolism, the alleviation of oxidative stress, and the suppression of inflammatory responses by activating the AMPK signaling pathway.

[0005] Although the above studies have shown some promise for clinical translation, there are no reports of combining plant-derived exosomes with MOTS-c and using specific myocardial targeted modification to treat MI / R injury.

[0006] Although existing technologies based on Exo and small molecule active peptides have been extensively studied in the field of myocardial repair, there are still many technical bottlenecks that limit their practical application in the treatment of MI / R injuries.

[0007] First, MSCs-Exo exhibits poor stability and consistency during clinical translation. The bioactivity of MSCs-Exo is easily affected by factors such as cell donor source, culture conditions, and extraction methods, resulting in significant batch-to-batch variations and making it difficult to guarantee efficacy. Furthermore, the preparation process of MSCs-Exo is complex and costly, and its long-term storage stability is poor, hindering large-scale production and clinical application.

[0008] Secondly, although AExo has attracted attention due to its good natural biocompatibility and potential pharmacological activity, it still has limitations as a monotherapy. The natural pharmacological components of AExo are complex, and its therapeutic effects and mechanisms of action in myocardial ischemia-reperfusion models are not yet clear, resulting in significant uncertainty regarding its efficacy. Using AExo alone is unlikely to achieve the desired precise intervention effect.

[0009] Furthermore, there is currently limited research on MOTS-c peptides in cardiovascular diseases, particularly myocardial ischemia-reperfusion injury, with only a few reported cases in diabetic cardiomyopathy. MOTS-c exhibits poor in vivo stability, is easily degraded rapidly, and lacks specific myocardial targeting capabilities, thus limiting its therapeutic efficacy and clinical translation.

[0010] Finally, current exosome delivery systems (including MSCs-Exo and AExo) generally lack specific targeting to myocardial tissue. After intravenous injection, most Exo tends to accumulate in non-target organs such as the liver and spleen, making it difficult to efficiently accumulate in damaged myocardium, thus severely limiting therapeutic efficacy and clinical application prospects.

[0011] In summary, myocardial ischemia-reperfusion injury (MI / R injury), a common and life-threatening cardiovascular pathological process, still faces significant limitations in terms of drug intervention, cell therapy, and drug delivery systems. These limitations prevent effective relief of the pathological damage caused by oxidative stress, inflammation, and apoptosis during MI / R. Exosomes, due to their natural biocompatibility and drug delivery capabilities, are considered to have great potential in cardioprotective therapy; however, further optimization is needed in terms of targeted enrichment efficiency, drug stability, and systemic drug delivery efficacy.

[0012] Therefore, developing a delivery vehicle with precise myocardial targeting capability has become one of the key technical problems that urgently need to be solved in the field of myocardial injury repair. Summary of the Invention

[0013] The main objective of this invention is to overcome the problems existing in the prior art and propose a MOTS-c targeted delivery system based on Astragalus membranaceus exosomes. This system can protect MOTS-c from enzymatic degradation in vivo, prolong its circulating half-life, accurately target and deliver it to myocardial tissue, and achieve local sustained release in the myocardium, thereby increasing the therapeutic concentration and duration of action of the drug while reducing systemic exposure and toxic side effects. The invention also proposes a corresponding preparation method and the application of this system in myocardial ischemia-reperfusion injury.

[0014] The technical solution of this invention to solve its technical problem is as follows:

[0015] A method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes includes the following steps:

[0016] Step 1: Take fresh Astragalus root, add PBS buffer (pH 7.0-7.4), homogenize and filter, collect the filtrate; centrifuge the filtrate multiple times, and collect the precipitate in the last centrifugation to obtain exosomes; purify the exosomes to obtain purified Astragalus-derived exosomes AExo.

[0017] The second step involves adding AExo to MES buffer at pH 5.5–6.5, activating it with EDC and NHS, and then adding a cardiac targeting peptide for coupling reaction to modify the AExo surface. The cardiac targeting peptide is the cardiac troponin I targeting peptide LPFFD, whose amino acid sequence is shown in SEQ ID No. 1. After the reaction, the peptide is dialyzed and centrifuged sequentially to collect the precipitate and obtain the modified LPFFD-AExo.

[0018] The third step involves mixing LPFFD-AExo with the mitochondrial-derived peptide MOTS-c and sonicating the mixture to load MOTS-c into LPFFD-AExo. The amino acid sequence of MOTS-c is shown in SEQ ID No. 2. The mixture is then centrifuged, and the precipitate is collected to obtain LPFFD-AExo@MOTS-c, which is the MOTS-c targeted delivery system based on Astragalus-derived exosomes.

[0019] This method first uses fresh Astragalus root as raw material to prepare Astragalus-derived exosomes AExo. Then, the cardiac troponin I targeting peptide LPFFD is modified onto the surface of AExo using an EDC / NHS chemical cross-linking method to obtain LPFFD-AExo. Finally, the mitochondrial-derived small peptide MOTS-c is loaded into LPFFD-AExo using an ultrasound-assisted permeation method to obtain LPFFD-AExo@MOTS-c. This protects MOTS-c from enzymatic degradation in vivo, prolongs its circulating half-life, precisely targets and delivers it to myocardial tissue, achieves local sustained release in the myocardium, increases the therapeutic concentration and duration of action of the drug, and reduces systemic drug exposure and toxic side effects.

[0020] The technical solution for further improving the preparation method of the present invention is as follows:

[0021] Preferably, in the first step, the mass-to-volume ratio of Astragalus root to PBS buffer is 1g:5mL to 10mL;

[0022] The specific process of centrifuging the filtrate in multiple stages is as follows:

[0023] The filtrate was subjected to three stages of centrifugation: primary centrifugation, secondary centrifugation, and tertiary centrifugation. Primary centrifugation involved centrifuging at 3000–5000 × g for 20–30 min at 1℃–4℃, and collecting the supernatant. Secondary centrifugation involved centrifuging at 10000–12000 × g for 30–40 min at 1℃–4℃, and collecting the supernatant. Tertiary centrifugation involved centrifuging at 100000–120000 × g for 60–120 min at 1℃–4℃, and collecting the precipitate to obtain exosomes.

[0024] The specific process for purifying exosomes is as follows:

[0025] The exosomes were resuspended in PBS buffer at pH 7.0–7.4, and impurities were removed by filtration. Then, the exosome fractions with densities in the range of 1.13–1.19 g / mL were purified by centrifugation using an 8%–60% sucrose density gradient to obtain AExo.

[0026] More preferably, in the first step, the PBS buffer is sterile PBS buffer; a juicer is used to homogenize the PBS for 3 to 5 minutes; after homogenization, the PBS is filtered using medical gauze or medical hemostatic cotton cloth.

[0027] By adopting the above preferred scheme, the specific technical features of the first step can be further optimized, and purified Astragalus-derived exosomes AExo can be obtained better.

[0028] Preferably, in the second step, the mass-to-volume ratio of AExo to MES buffer is 1 mg: 1 ± 0.5 mL; the molar ratio of EDC to NHS is 2:1 to 1:3; activation is performed at room temperature for 20 to 40 min; the final concentration of LPFFD added is 50 to 100 μM; and the coupling reaction is incubated at 1°C to 4°C for 8 to 16 h.

[0029] More preferably, in the second step, dialysis is performed using a dialysis bag with a molecular weight cutoff (MWCO) of 50–150 kDa for at least 24 hours; centrifugation is performed at 100,000–120,000 × g for 1–2 hours.

[0030] By adopting the above preferred scheme, the specific technical features of the second step can be further optimized, and the modified LPFFD-AExo can be obtained better.

[0031] Preferably, in the third step, the concentration of LPFFD-AExo after mixing is 0.5–2 mg / mL, and the concentration of MOTS-c is 5–20 μg / mL; during ultrasonic treatment, the ultrasonic power is 100–200 W, the ultrasonic treatment time is 5–15 min, the ultrasonic working mode is intermittent mode, and the temperature is 0℃–4℃; after ultrasonic treatment, it is left to stand at 0℃–4℃ for at least 1 h to complete the composite reaction.

[0032] More preferably, in the third step, the intermittent mode is to continuously cycle between turning on for 2-5 seconds and turning off for 2-5 seconds; during centrifugation, centrifuge at 100,000-120,000 × g for 1-2 hours.

[0033] By adopting the above preferred scheme, the specific technical features of the third step can be further optimized, and LPFFD-AExo@MOTS-c can be obtained better.

[0034] Preferably, in the first step, the particle size of the obtained AExo is between 50 and 200 nm, the Zeta potential is between -10 and -30 mV, and both exosome marker proteins CD63 and TSG101 are positive; in the second step, the modification efficiency of the obtained LPFFD-AExo is ≥80%; in the third step, the loading efficiency of the obtained LPFFD-AExo@MOTS-c is 70% to 90%.

[0035] By adopting the above preferred scheme, the standards that the products obtained in each step should meet can be further optimized.

[0036] The present invention also proposes:

[0037] The MOTS-c targeted delivery system based on Astragalus-derived exosomes was prepared by the preparation method described above.

[0038] The MOTS-c targeted delivery system based on Astragalus-derived exosomes, as described above, is used to prepare drugs for the treatment of myocardial ischemia-reperfusion injury.

[0039] This invention uses Astragalus-derived exosomes AExo as carriers to load the mitochondrial-derived small peptide MOTS-c, and modifies the exosome surface with the myocardial targeting peptide LPFFD to improve its precise delivery efficiency to damaged myocardial tissue. This achieves highly efficient and precise delivery of MOTS-c to ischemia-reperfusion injury myocardial tissue, thereby significantly enhancing its ferroptosis inhibition capacity and improving the myocardial protection effect against MI / R injury, ultimately achieving highly efficient targeted therapy for MI / R injury. Attached Figure Description

[0040] Figure 1This is a schematic diagram illustrating the main content of Embodiment 1 of the present invention. Figure ① shows the process of encapsulating the mitochondrial-derived peptide MOTS-c into an exosome (AExo) and modifying its surface with the myocardial targeting peptide LPFFD, thereby constructing the targeted delivery system LPFFD-AExo@MOTS-c; Figure ② illustrates the mechanism by which this system, after being injected into rats via the tail vein, is delivered, targeted, enriched, and exerts its therapeutic effect in a myocardial ischemia / reperfusion (MI / R) injury model, involving its effect pathways of improving cardiac function, promoting tissue repair, and protecting mitochondria.

[0041] Figure 2 Figure 1 shows the physicochemical characterization of the three groups in Example 1 of this invention. Figure A is a transmission electron microscope (TEM) image of AExo, showing its typical vesicle-like bilayer membrane structure. Figure B shows the dynamic light scattering (DLS) particle size distribution of AExo, LPFFD-AExo, and LPFFD-AExo@MOTS-c. The particle sizes of the three groups are mainly concentrated between 75 and 130 nm, indicating that the surface modification and drug loading processes did not significantly change their particle size structure. Figure C shows the Western blot results of exosome marker proteins (CD63, TSG101, Alix) and the negative marker (Calnexin), showing that the exosome marker proteins are enriched and have high purity. Figure D shows the Zeta potential test results of the three groups, used to evaluate the electrical changes of surface modification and drug loading. Figure E shows the Fourier transform infrared (FTIR) spectra of each group, further confirming that the myocardial targeting peptide LPFFD has been successfully coupled to the surface of AExo. Figure F shows the ELISA results of the AExo and LPFFD-AExo groups, which are used to evaluate the coupling modification efficiency of the myocardial targeting peptide LPFFD on the surface of exosomes.

[0042] Figure 3 Figure 1 shows the characterization of drug loading and release performance in Example 1 of this invention. Figure A shows the HPLC chromatograms of MOTS-c standard, AExo, AExo@MOTS-c, and LPFFD-AExo@MOTS-c; Figure B shows the MOTS-c standard curve; Figure C shows the detected MOTS-c concentration data; and Figure D shows the in vitro drug release curves of AExo@MOTS-c and LPFFD-AExo@MOTS-c under pH 6.8 and pH 7.4 conditions.

[0043] Figure 4 This diagram illustrates the mechanism by which MOTS-c inhibits ferroptosis in cardiomyocytes in vitro, as demonstrated in Example 1 of this invention. Figures A and B show the fluorescence images and quantitative results of lipid peroxidation detected by the C11-BODIPY probe; Figure C shows the glutathione (GSH) level detection results; and Figure D shows the intracellular Fe... 2+Content detection results; Figures E and F show the Western blot expression of ferroptosis-related proteins (xCT, GPX4, FTH-1) in different treatment groups.

[0044] Figure 5 These are images showing the cardiac function and histopathological evaluation of rats in each group in Example 1 of this invention. Image A shows echocardiography results of left ventricular function parameters, including left ventricular ejection fraction (LVEF) and short-axis shortening (LVFS); Image B shows the results of left ventricular intraventricular diameter (LVDd) and interventricular septal thickness (IVSd); Image C shows the results of HE staining of the heart, observing ventricular structure and myofibrous arrangement; Image D shows the results of Masson staining, assessing the degree of myocardial fibrosis; and Image E shows the quantitative results of myocardial infarction area and fibrosis area.

[0045] Figure 6 Figure 1 shows the biocompatibility evaluation of the exosome delivery system in Example 1 of this invention. Figure A shows the staining images of live / dead cells 24 hours after treatment; Figure B shows the cell viability of cardiomyocytes (H9c2) after 24, 48, and 72 hours of treatment, as detected by the CCK-8 assay, indicating that both the AExo@MOTS-c group and the LPFFD-AExo@MOTS-c group have good cell compatibility. Figure C shows the statistical comparison of fluorescence density of the live cell staining images of each experimental group in Figure A.

[0046] Figure 7 This is a graph evaluating the uptake efficiency of exosomes on cardiomyocytes in Example 1 of the present invention. Graph A shows the fluorescence images of PKH26-labeled AExo@MOTS-c and LPFFD-AExo@MOTS-c uptake in H9c2 cells; Graph B shows the quantitative analysis results of the uptake intensity.

[0047] Figure 8 This diagram illustrates the mechanism by which LPFFD-AExo@MOTS-c inhibits MI / R-induced cardiomyocyte ferroptosis in vivo, as demonstrated in Example 1 of this invention. Figures A and B show C11-BODIPY staining and quantitative fluorescence results in rat myocardial tissue; Figure C shows tissue GSH levels; and Figure D shows Fe... 2+ Content results; Figures E and F show the expression levels of ferroptosis marker proteins (xCT, GPX4, FTH-1) in myocardial tissue of each group.

[0048] Figure 9 This is an in vivo targeting ability evaluation diagram of LPFFD-AExo@MOTS-c in Example 1 of the present invention. Specifically, it is a fluorescence distribution diagram detected by a small animal in vivo imaging system (IVIS) after rats received tail vein injection of different formulations at 2, 12, 24 and 48 hours.

[0049] Figure 10 This is an assessment diagram of cardiac function and tissue repair in each group of rats in Example 1 of the present invention.

[0050] Figures A and B show echocardiograms and their quantitative parameters (LVEF, LVFS, etc.); Figure C shows HE staining results, assessing the recovery of myocardial tissue structure; Figure D shows Masson staining results, assessing the extent of myocardial fibrosis; and Figure E shows the quantitative analysis results of infarct area and fibrosis area.

[0051] Figure 11 The figures show the levels of myocardial inflammation, oxidative stress, and systemic toxicity in each group of rats after treatment in Example 1 of this invention. Figures A and B show the results of blood biochemical indicators (ALT, AST, BUN, Cr) to assess systemic liver and kidney toxicity; Figures C and D show the results of TNF-α, IL-10, MDA, and SOD levels in the myocardial tissue of each group, respectively, to assess anti-inflammatory and anti-lipid peroxidation effects. Detailed Implementation

[0052] The specific research ideas and processes of this invention are as follows.

[0053] I. The core technical problems solved by this invention include:

[0054] (1) Improve the biocompatibility and safety of the system

[0055] Existing mesenchymal stem cell-derived exosomes (MSCs-Exo) suffer from complex preparation processes, high immunogenicity, and limited clinical translation. This invention selects Astragalus-derived exosomes (AExo) as a delivery carrier, which exhibits good biosafety and low immunogenicity, avoiding the immune rejection risks associated with xenogeneic cell exosomes. Simultaneously, AExo can exert the natural pharmacological effects of Astragalus, synergistically enhancing therapeutic efficacy with MOTS-c, making it more suitable for the clinical needs of long-term systemic medication.

[0056] (2) Enhance the stability of the delivery system and the level of production standardization

[0057] Currently, the structural stability and batch-to-batch consistency of exosomes (Exo) are significant bottlenecks hindering clinical translation. This invention optimizes the extraction, purification, and storage methods of AExo, ensuring that it maintains the integrity of its nanocarrier structure and the stability of its biological functions even after encapsulation with MOTS-c and surface-modified cardiac-targeting peptides. Furthermore, the standardized preparation process improves batch-to-batch product consistency, laying a technical foundation for large-scale production and clinical application.

[0058] (3) Enhance the stability and efficacy maintenance of mitochondrial-derived small peptide MOTS-c in vivo.

[0059] MOTS-c, a short peptide derived from mitochondria, exhibits poor stability in vivo, a short half-life, and is easily degraded by enzymes, thus limiting its clinical use. This invention encapsulates MOTS-c with AExo, effectively protecting its biological activity from enzymatic degradation, prolonging its circulating half-life and duration of effective drug action, thereby significantly improving the bioavailability of MOTS-c and enhancing its cardioprotective effect.

[0060] (4) Achieve precise targeted enrichment of MOTS-c on ischemic myocardial tissue

[0061] Existing Exo delivery systems are mostly non-specifically cleared by organs such as the liver and spleen after intravenous injection, resulting in low effective delivery efficiency to myocardial tissue. This invention covalently modifies the surface of AExo with a specific myocardial-targeting peptide, LPFFD, significantly improving the targeted accumulation ability of drug-loaded exosomes in damaged myocardial regions, increasing local drug concentration within myocardial tissue, enhancing therapeutic efficacy, and reducing systemic side effects.

[0062] (5) Optimize the route of administration and improve clinical applicability.

[0063] Conventional administration methods of MOTS-c, such as oral administration and intramuscular injection, suffer from problems such as insufficient tissue targeting, low bioavailability, and lack of innovative drug delivery. This invention, LPFFD-AExo@MOTS-c, allows for non-invasive intravenous injection, delivering directly to damaged myocardial tissue via the bloodstream. This effectively avoids the trauma and operational difficulties associated with local injection, improving drug delivery efficiency, drug targeting, and patient compliance, making it more suitable for clinical translation and application.

[0064] II. The core steps and key parameters of the technical solution of this invention are as follows:

[0065] 1. Extraction and purification of Astragalus-derived exosomes AExo

[0066] Select fresh Astragalus root, wash and cut into sections, add sterile PBS buffer (pH 7.0-7.4) at a mass-to-volume ratio of 1g:5mL-10mL, homogenize using a juicer for 3-5 minutes until there are no obvious lumps of solids, then filter through medical gauze or medical hemostatic cotton cloth and collect the filtrate.

[0067] The filtrate was subjected to three stages of centrifugation: the first stage was centrifuged at 3000–5000×g for 20–30 min at 1℃–4℃ to remove coarse particles (mainly plant fibers) and the supernatant was collected; the second stage was centrifuged at 10000–12000×g for 30–40 min at 1℃–4℃ to remove cell debris and the supernatant was collected; and the third stage was centrifuged at 100000–120000×g for 60–120 min at 1℃–4℃ to enrich exosomes and collect the precipitate.

[0068] The precipitate was resuspended in sterile PBS buffer (pH 7.0–7.4), and impurities were removed by filtration. Then, exosome fractions with densities ranging from 1.13 to 1.19 g / mL were purified by 8%–60% sucrose density gradient centrifugation to obtain AExo. The obtained AExo should have a particle size between 50 and 200 nm, a Zeta potential between -10 and -30 mV, and be positive for exosome marker proteins CD63 and TSG101.

[0069] 2. AExo surface-modified myocardial targeting peptides

[0070] The cardiac troponin I (cTnI) targeting peptide was modified onto the surface of AExo using an EDC / NHS chemical cross-linking method. The amino acid sequence of the cardiac troponin I targeting peptide is SEQ ID No. 1: LPFFD, and it is denoted as cardiac troponin I targeting peptide LPFFD or cardiac targeting peptide LPFFD.

[0071] The cross-linking reaction conditions were as follows: MES buffer at pH 5.5–6.5 was used as the reaction buffer. AExo was added to the MES buffer at a mass-to-volume ratio of 1 mg:1 ± 0.5 mL. Then, appropriate amounts of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added, with a molar ratio of EDC to NHS of 2:1–1:3. AExo was activated at room temperature for 20–40 min. After activation, LPFFD (final concentration 50–100 μM) was added, and the mixture was incubated at 1–4 °C for 8–16 h for the coupling reaction. After the reaction was complete, the mixture was dialyzed and then ultracentrifuged at 100,000–120,000 × g to remove free peptides. The precipitate was collected to obtain the modified LPFFD-AExo. The modification efficiency was detected by ELISA, and the modification efficiency should be ≥80%.

[0072] 3. Loading of mitochondrial-derived small peptide MOTS-c

[0073] MOTS-c was loaded into LPFFD-AExo using an ultrasound-assisted permeation method to obtain LPFFD-AExo@MOTS-c. The amino acid sequence of MOTS-c is SEQ ID No. 2: MRWQEMGYIF YPRKLR.

[0074] The specific conditions are as follows: LPFFD-AExo concentration range is 0.5–2 mg / mL, MOTS-c concentration range is 5–20 μg / mL; ultrasonic power is 100–200 W, total reaction time is 5–15 min, working mode is intermittent (2–5 s on / 2–5 s off), temperature is controlled at 0℃–4℃; after ultrasonication, it is allowed to stand at 0℃–4℃ for at least 1 h to complete the composite reaction; then, it is ultracentrifuged at 100,000–120,000 × g for 1–2 h to remove unloaded MOTS-c, obtaining LPFFD-AExo@MOTS-c. The loading rate is detected by HPLC, and the target loading rate is 70%–90%.

[0075] 4. Verification of the in vitro ferroptosis inhibitory effect of MOTS-c

[0076] An oxygen-glucose deprivation / reoxygenation (OGD / R) model was constructed using H9c2 cardiomyocytes: Cells were incubated in glucose-free, serum-free DMEM under a hypoxic environment (O2 concentration 1%–5%) for 1–4 hours, followed by normal culture for 8 hours. Different concentrations of MOTS-c (0–100 μM) were then added for treatment. The anti-lipid peroxidation level of MOTS-c was detected using C11-BODIPY immunofluorescence; glutathione content was determined using a GSH assay kit; and relative iron content in cell lysates was detected using an iron assay kit. 2+ The expression levels of GPX4, xCT, and FTH-1 proteins were detected by Western blot to comprehensively evaluate the inhibitory effect of ferroptosis.

[0077] 5. MOTS-c in vivo protection of cardiac structure and function

[0078] Four-week-old male SD rats were used to establish a left anterior descending coronary artery (LAD) model (ligation of the left anterior descending coronary artery for 30 minutes, followed by reperfusion for 5 hours). Immediately after model establishment, rats were injected via the tail vein. Experimental groups included the Sham group, MI / R group, PBS group, and MOTS-c group. MOTS-c concentrations ranged from 200 to 800 μM, with an injection dose of 1 mg / kg, administered every two days for 14 days. Cardiac function parameters such as left ventricular ejection fraction (LVEF), left ventricular stenosis (FS), left ventricular diameter (LVDd), and left ventricular diameters (LVDs) were assessed by echocardiography. Heart tissue was collected for HE and Masson staining to assess structural damage, infarct area, and fibrosis.

[0079] 6. Biocompatibility and cellular functional verification of LPFFD-AExo

[0080] H9c2 cardiomyocytes were seeded in 96-well plates and treated with PBS, free MOTS-c, AExo@MOTS-c, and LPFFD-AExo@MOTS-c, respectively. The final concentration of MOTS-c was 100 μM for all groups. Cell viability and survival status were assessed after 24 hours of treatment. DiO fluorescently labeled exosomes were used to treat cells for 30 minutes, and the uptake efficiency of different groups was detected by flow cytometry.

[0081] 7. Evaluation of the in vivo targeted delivery and myocardial repair effects of LPFFD-AExo@MOTS-c

[0082] Four-week-old male SD rats were used to establish a myocardial infarction / reperfusion (MI / R) model (ligation of the left anterior descending coronary artery for 30 minutes, followed by release and reperfusion for 5 hours). Immediately after modeling, rats were injected via tail vein. Experimental groups included PBS, free MOTS-c, AExo@MOTS-c, and LPFFD-AExo@MOTS-c groups. MOTS-c concentrations ranged from 200 to 800 μM, with an injection dose of 1 mg / kg, administered every two days for 14 days. Exosome enrichment in the cardiac region was detected using an IVIS system, with a target heart / liver fluorescence ratio ≥5:1. Cardiac function parameters such as LVEF, FS, LVDd, and LVDs were assessed by echocardiography. Heart tissue was collected for HE and Masso staining to assess structural damage, infarct area, and fibrosis. Fe in infarcted myocardial tissue was detected. 2+ The levels of GSH were measured, and the expression of GPX4, xCT, and FTH-1 proteins were analyzed by Western blot to evaluate the inhibitory effect of each delivery system on ferroptosis.

[0083] III. Advantages of the present invention:

[0084] This invention relates to a precision delivery system based on Astragalus-derived exosomes (AExo) loaded with MOTS-c and surface-modified with myocardial targeting peptides. Compared with existing technologies, this system demonstrates significant advantages in treating myocardial ischemia / reperfusion (MI / R) injury, specifically in the following ways:

[0085] (1) Using AExo as a novel carrier overcomes the shortcomings of existing MSCs-Exo preparation methods, such as complex preparation, low yield, large batch-to-batch variability, and high immunogenicity. AExo has a stable source, simple extraction method, relatively low cost, and good biocompatibility and low immunogenicity. In addition, the endogenous active ingredients of Astragalus membranaceus carried by AExo can also play an auxiliary anti-inflammatory and antioxidant role while delivering MOTS-c, further enhancing the therapeutic effect.

[0086] (2) Improved in vivo stability and therapeutic efficiency of MOTS-c. Although MOTS-c has significant mitochondrial protection and antioxidant effects, it is easily degraded in vivo and has low bioavailability. By encapsulating MOTS-c inside AExo, it is possible to effectively protect MOTS-c from enzymatic degradation in vivo, prolong its blood circulation half-life, and achieve local sustained release in the myocardium, thereby increasing the therapeutic concentration and duration of action of the drug, and reducing systemic exposure and toxic side effects.

[0087] (3) Achieving precise targeted delivery to myocardial tissue. This invention modifies the surface of AExo with a specific myocardial targeting peptide, LPFFD, enabling the delivery system to actively target and recognize cTnI, significantly improving the enrichment efficiency and therapeutic effect of MOTS-c in I / R myocardial tissue. Simultaneously, it reduces the distribution of Exo in non-target organs such as the liver and spleen, lowering potential systemic side effects and improving efficacy and safety.

[0088] (4) It can be administered via non-invasive intravenous injection, which has better clinical feasibility and patient compliance compared to local myocardial injection or interventional drug delivery. With the assistance of targeted peptide modification, even intravenous administration can effectively achieve precise delivery to the lesion area. The operation is simple and convenient, making it suitable for clinical application.

[0089] (5) High safety and good metabolic degradation performance. AExo is derived from the natural plant Astragalus membranaceus, avoiding the potential immune rejection risk of xenogeneic cell-derived Exo. It can be effectively degraded and metabolized in vivo, and long-term use will not produce significant toxic accumulation. At the same time, the precise targeted delivery strategy reduces the distribution of the drug in non-target organs, further ensuring the safety of the treatment process.

[0090] (6) This invention utilizes plant-derived exosomes combined with nanodelivery technology to establish a precision drug delivery system that combines bioactivity and delivery function. This system not only expands the application scope of plant-derived exosomes in the treatment of cardiovascular diseases, but also provides new ideas for the modern delivery application of plant active ingredients. It has positive significance for the application and development of plant-derived delivery carriers and has good clinical application potential and translational value.

[0091] The present invention will be further described in detail below with reference to the embodiments. However, the present invention is not limited to the examples given. Unless specific experimental conditions are specified or only the experimental process is briefly described in the following description, the experimental operations shall be performed according to the conventional conditions of the prior art or the conditions recommended by the manufacturer.

[0092] Example 1

[0093] This embodiment prepares an AExo@MOTS-c delivery system with surface-modified myocardial targeting peptides and verifies its therapeutic effect on MI / R injury. The main contents are as follows: Figure 1 As shown.

[0094] The specific details of this embodiment are as follows:

[0095] (1) Extraction and purification of AExo

[0096] Take 50g of fresh Astragalus root, wash it thoroughly, cut it into sections, place it in a juicer, add 250mL of pre-cooled sterile PBS buffer (pH 7.4), and juice thoroughly until there is no obvious solid residue. Filter the resulting suspension through medical hemostatic cotton gauze and collect the filtrate for later use.

[0097] The filtrate was subjected to three-stage centrifugation: centrifugation at 3000×g for 30 min (4℃) to remove coarse particles (mainly plant fibers), and the supernatant was collected. Then, centrifugation at 10000×g for 30 min (4℃) was used to remove cell debris, and the supernatant was collected. Finally, ultracentrifugation at 100000×g for 2 h (4℃) was performed to collect the precipitate and obtain exosomes.

[0098] The precipitate was resuspended in sterile PBS buffer (pH 7.4) and filtered through a 0.22 μm filter to remove impurities. Then, exosome components with densities in the range of 1.13–1.19 g / mL were separated and purified by centrifugation using an 8%–60% sucrose density gradient to obtain purified AExo.

[0099] The physicochemical properties of AExo were characterized and tested: (i) the vesicle morphology of AExo was observed by transmission electron microscopy (TEM), such as... Figure 2 As shown in Figure A, the results indicate that it has a typical vesicle-like bilayer membrane structure; (ii) the particle size was determined by dynamic light scattering (DLS), as shown in Figure A. Figure 2 The results for AExo in Figure B show that the particle size of AExo is mainly concentrated between 75 and 130 nm. This result serves as the basis for subsequent evaluation of the influence of surface modification and drug loading processes on particle size structure; (iii) Zeta potential was measured by dynamic light scattering (DLS), and the results are shown in Figure B. Figure 2 The results of AExo in Figure D serve as the basis for subsequent evaluation of the effects of surface modification and drug encapsulation on electrical changes; (iv) Western blot analysis of exosome marker protein (CD63, TSG101, Alix) expression was performed, and the expression of intracellular contamination marker proteins (such as Calnexin) was confirmed. Figure 2 The results for AExo in Figure C show that the exosome marker proteins of AExo are enriched and highly pure; (v) Fourier transform infrared spectroscopy (FTIR) detection results are shown in Figure C. Figure 2The results of AExo in the E diagram serve as the basis for subsequent evaluation of whether the myocardial targeting peptide LPFFD was successfully coupled to the surface of AExo.

[0100] (2) AExo surface-modified myocardial targeting peptide

[0101] 1 mg of AExo was resuspended in 1 mL of MES buffer at pH 6.0, and EDC and NHS were added to a final concentration of 10 mM. The mixture was reacted at room temperature for 30 min to activate the surface carboxyl groups. Subsequently, myocardial-specific targeting peptide LPFFD was added to a final concentration of 100 μM, and the mixture was incubated at 4 °C for 12 h to complete the coupling reaction. The reaction system was dialyzed for 24 h through a dialysis bag with a molecular weight cutoff of 100 kDa, and the modified LPFFD-AExo was collected by ultracentrifugation at 100,000 × g for 1 h.

[0102] The physicochemical properties of LPFFD-AExo were characterized and tested: (i) particle size was determined by dynamic light scattering (DLS), such as... Figure 2 The results for LPFFD-AExo in Figure B show that the particle size of LPFFD-AExo is still mainly concentrated between 75 and 130 nm, indicating that surface modification did not significantly change its particle size structure; (ii) the Zeta potential was measured by dynamic light scattering (DLS), and the results are shown in Figure B. Figure 2 The results of LPFFD-AExo in Figure D show that, compared with AExo, the surface electronegativity of LPFFD-AExo is slightly reduced (Zeta potential is upregulated from -17.2mV to -13.6mV), suggesting that the modification of the myocardial targeting peptide LPFFD has a certain shielding effect on the surface charge of exosomes; (iii) Western blot analysis of the expression of exosome marker proteins (CD63, TSG101, Alix) and confirmation of the absence of intracellular contamination marker proteins (such as Calnexin) expression, such as Figure 2 The results of LPFFD-AExo in Figure C are shown, indicating that LPFFD-AExo is enriched and has high purity as an exosomal marker protein; (iv) LPFFD and LPFFD-AExo were detected by Fourier transform infrared spectroscopy (FTIR), and the results are shown in Figure C. Figure 2 The E diagram confirms that LPFFD has been successfully coupled to the AExo surface.

[0103] The coupling modification efficiency of LPFFD peptide on the AExo surface was detected by enzyme-linked immunosorbent assay (ELISA). Equal amounts of AExo protein and LPFFD-AExo were coated onto 96-well plates, and colorimetric analysis was performed using anti-LPFFD antibody. The formula used was: The results are as follows Figure 2As shown in Figure F, compared with AExo, the absorbance of LPFFD-AExo at a wavelength of 450 nm is significantly increased, and the calculated modification efficiency is 87.1%, which meets the design requirements (≥80%). This indicates that LPFFD has been successfully coupled to the surface of exosome AExo and the coupling modification efficiency is high.

[0104] (3) Evaluation of the load and release stability of MOTS-c

[0105] MOTS-c was loaded into LPFFD-AExo using an ultrasonic-assisted permeation method to obtain LPFFD-AExo@MOTS-c. The specific process is as follows:

[0106] 1 mg LPFFD-AExo was resuspended in 1 mL of sterile PBS buffer (pH 7.4), and 10 μg of MOTS-c was added. The mixture was then incubated on ice (0℃~4℃) using a 100W probe-type sonicator (intermittent mode: 5s on / 5s off, 10 min) to promote peptide penetration into the exosome cavity. After sonication, the reaction system was allowed to stand at 4℃ for 1 h to complete the complexation reaction. Unloaded MOTS-c was then removed by centrifugation at 100000×g for 1 h to obtain LPFFD-AExo@MOTS-c.

[0107] Meanwhile, the control group used AExo to replace LPFFD-AExo and repeated the above ultrasound-assisted permeation process to obtain AExo@MOTS-c.

[0108] The physicochemical properties of LPFFD-AExo@MOTS-c were characterized and tested: (i) particle size was determined by dynamic light scattering (DLS), such as... Figure 2 Figure B shows the results for LPFFD-AExo@MOTS-c. The results indicate that the particle size of LPFFD-AExo@MOTS-c is still mainly concentrated between 75 and 130 nm, indicating that the drug encapsulation process did not significantly change its particle size structure; (ii) the Zeta potential was measured by dynamic light scattering (DLS), and the results are shown in Figure B. Figure 2 The results of LPFFD-AExo@MOTS-c in Figure D show that, compared with AExo and LPFFD-AExo, the Zeta potential of LPFFD-AExo@MOTS-c is further upregulated to -11.4 mV, indicating that MOTS-c neutralizes the original negative charge of exosomes. This change in charge helps to improve the fusion ability and biocompatibility of exosomes with cardiomyocyte membranes, laying the foundation for their in vivo delivery.

[0109] High-performance liquid chromatography (HPLC) was used to determine the loading efficiency. Specifically, exosomes were first lysed, then the concentration of MOTS-c was determined by HPLC, and finally the loading rate of MOTS-c in the exosomes was calculated. The chromatographic conditions were briefly described below: C18 reversed-phase column, acetonitrile-water gradient elution mobile phase, flow rate 1.0 mL / min, sample loading volume 20 μL, and detection wavelength 230 nm. After detection, the actual content was calculated by comparing with the MOTS-c standard curve. The loading rate was calculated using the following formula: The results are as follows Figure 3 Figures A through C are shown. Figure A presents the HPLC chromatograms of MOTS-c standard, AExo, AExo@MOTS-c, and LPFFD-AExo@MOTS-c. Each sample exhibits a distinct characteristic peak at the MOTS-c retention time (approximately 12.5 min) at 230 nm. Comparative analysis reveals differences in the absorption peak intensity at the MOTS-c retention time (approximately 12.5 min) among different samples. Figure B presents the MOTS-c standard curve, plotted based on MOTS-c standards of different concentrations, showing good correlation (R0). 2 =0.9976), used to calculate the MOTS-c concentration in each sample; Figure C shows the detected MOTS-c concentration data. The results show that the MOTS-c loading of the LPFFD-AExo@MOTS-c group is higher than that of the AExo@MOTS-c group. After conversion based on the standard curve, the loading rate of LPFFD-AExo@MOTS-c is approximately 81.25%. The above results indicate that the MOTS-c loading rate of LPFFD-AExo@MOTS-c is within the target range (70%–90%), the results are reliable, and it has a good loading effect.

[0110] In vitro release experiments were conducted in pH 7.4 PBS buffer and pH 6.8 PBS simulating a MI / R microenvironment, respectively. Dialysis bags with a molecular weight cutoff (MWCO) of 8–14 kDa were used, and the bags were incubated at 37°C with shaking. Samples were taken from the ambient liquid outside the dialysis bags at regular intervals within 0–72 h. The release amount was determined using the HPLC method described above, and release curves were plotted. The results are as follows: Figure 3 As shown in Figure D, the results indicate that LPFFD-AExo@MOTS-c possesses excellent sustained-release properties.

[0111] (4) Verification of the in vitro ferroptosis inhibition effect of MOTS-c

[0112] To clarify the cell protection mechanism of MOTS-c, an OGD / R model was established without the addition of exosomes: H9c2 cardiomyocytes were seeded in 6-well plates and incubated in glucose-free, serum-free DMEM under a hypoxic environment (O2 concentration 1%–5%) for 2 h followed by 8 h. Then, they were treated with 0, 25, 50, and 100 μM MOTS-c, respectively.

[0113] The level of MOTS-c anti-lipid peroxidation was detected by C11-BODIPY immunofluorescence, and the results are as follows: Figure 4 Figures A and B are shown; intracellular glutathione levels were detected using a GSH assay kit, and the results are as follows. Figure 4 Figure C shows the results; the relative Fe in cell lysate was detected using an iron detection kit. 2+ Horizontal, the result is as follows Figure 4 The results are shown in Figure D; Western blot analysis of the expression and quantitative changes of ferroptosis-related proteins GPX4, xCT, and FTH1 are shown in Figure D. Figure 4 The E and F plots are shown to verify the inhibitory effect of MOTS-c on hypoxia-induced ferroptosis.

[0114] The above results indicate that MOTS-c delivered via AExo can significantly inhibit OGD-induced cardiomyocyte ferroptosis, specifically by reducing intracellular iron content. 2+ The levels of glutathione (GSH) decreased, while the levels of ferroptosis-related proteins (such as GPX4, xCT, and FTH-1) increased.

[0115] (5) Validation of MOTS-c in vivo protection of cardiac structure and function

[0116] Four-week-old male SD rats were selected to establish a MI / R model by ligating the left anterior descending coronary artery (LAD) for 30 minutes, releasing it, and then reperfusing for 5 hours. Immediately after model establishment, tail vein injection was performed. The experimental groups included the Sham group, MI / R group, PBS group, and MOTS-c group. The MOTS-c concentration was 500 μM in all groups, and the injection dose was 1 mg / kg, once every 2 days for 14 days.

[0117] Cardiac function indicators such as LVEF, FS, LVDd, and LVDs were assessed by echocardiography. The results are as follows: Figure 5 Figures A and B are shown in the diagram. Heart tissue was collected for HE and Masson staining to assess structural damage, infarct area, and fibrosis. The results are as follows: Figure 5 Figures C through E are shown. These results demonstrate that MOTS-c significantly improves ventricular remodeling.

[0118] Although the above (4) and (5) indicate that MOTS-c has a cardioprotective effect, MOTS-c has poor in vivo stability, is easily degraded rapidly, and lacks specific myocardial targeting ability. These are key factors restricting the therapeutic effect and clinical translation of MOTS-c. In this embodiment, the stability of MOTS-c and the enrichment efficiency in myocardial tissue are improved by preparing the LPFFD-AExo@MOTS-c system, as follows.

[0119] (6) Biocompatibility and cellular functional verification of LPFFD-AExo

[0120] Biocompatibility and cytotoxicity evaluation: H9c2 cardiomyocytes were seeded in 96-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were added to different treatment groups: PBS control group, free MOTS-c group, AExo@MOTS-c group and LPFFD-AExo@MOTS-c group, with a final MOTS-c concentration of 100 μM for all groups.

[0121] After 24 hours of treatment, cell viability was measured using a CCK-8 assay kit; Live / Dead fluorescence staining was performed simultaneously to observe cell survival status; cytotoxicity data were used to assess biocompatibility by combining absorbance values ​​and fluorescence imaging. Results are as follows: Figure 6 As shown, Figure 6 Figures A and C show that there was no significant cytotoxicity in any group; Figure 6 Figure B shows that both the AExo@MOTS-c group and the LPFFD-AExo@MOTS-c group have good cell compatibility.

[0122] Cellular uptake capacity assessment: Exosomes were labeled with DiO fluorescence and treated with H9c2 cells for 30 minutes. The uptake efficiency of each exosome group was detected by flow cytometry to assess the effect of LPFFD modification on the targeted uptake capacity of cardiomyocytes (e.g., ...). Figure 7 (As shown in the figure). The results indicate that LPFFD modification significantly enhances the uptake efficiency of exosomes by cardiomyocytes.

[0123] (7) Evaluation of the in vivo targeted delivery and myocardial repair effects of LPFFD-AExo@MOTS-c

[0124] Four-week-old male SD rats were selected, and a MI / R model was established using the same method described above. Immediately after successful model establishment, rats were treated with tail vein injection (1 mg / kg, MOTS-c concentration 500 μM, both containing Dir fluorescent labeling), once every two days for 14 days. Experimental groups included: saline group, free MOTS-c group, AExo@MOTS-c group, and LPFFD-AExo@MOTS-c group.

[0125] The relative Fe content in cell lysate was detected using an iron assay kit. 2+ The levels of GSH were determined using a GSH kit, and the expression of GPX4, xCT, and FTH-1 proteins was detected by Western blot and grayscale analysis was performed to comprehensively evaluate the inhibitory effects of each delivery system on ferroptosis. Figure 8 As shown, the results indicate that LPFFD-AExo@MOTS-c can effectively reverse MI / R-induced ferroptosis-related abnormalities.

[0126] In vivo fluorescence distribution at different time points was obtained using an IVIS imaging system, and the targeting efficiency was assessed by calculating the heart / liver fluorescence ratio. Figure 9 As shown, the results indicate that LPFFD modification can significantly enhance the enrichment ability of LPFFD-AExo@MOTS-c in the cardiac region, demonstrating good targeting.

[0127] Echocardiography was performed on day 14 to measure LVEF, FS, LVDd, and LVDs to assess cardiac function recovery. Results are as follows: Figure 10 Figures A and B are shown; paraffin sections of cardiac tissue were stained with Masson stain to assess the area of ​​fibrosis, and H&E staining was used for myocardial structural observation to assess structural damage, infarct area, and fibrosis status. The results are as follows. Figure 10 Figures C through E are shown. These results demonstrate that LPFFD-AExo@MOTS-c is superior to other treatment groups in promoting tissue repair.

[0128] Systemic toxicity was further analyzed using blood biochemical indicators (ALT, AST, BUN, Cr), and the results were as follows: Figure 11 Figures A and B are shown; the levels of TNF-α, IL-6, MDA, and SOD in myocardial tissue were detected to assess the anti-inflammatory and anti-lipid peroxidation effects. The results are as follows. Figure 11 Figures C and D are shown. These results indicate that the LPFFD-AExo@MOTS-c treatment group has significant advantages in reducing inflammatory responses, inhibiting oxidative damage, and improving systemic toxicity.

[0129] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. A method for preparing a MOTS-c targeted delivery system based on Astragalus membranaceus-derived exosomes, characterized in that, Includes the following steps: Step 1: Take fresh Astragalus root, add PBS buffer (pH 7.0-7.4), grind and homogenize, filter, and collect the filtrate; centrifuge the filtrate multiple times, and collect the precipitate in the last centrifugation to obtain exosomes; After purification of the exosomes, purified Astragalus-derived exosomes AExo were obtained; The second step involves adding AExo to MES buffer at pH 5.5–6.5, activating it with EDC and NHS, and then adding a cardiac targeting peptide for coupling reaction to modify the AExo surface. The cardiac targeting peptide is the cardiac troponin I targeting peptide LPFFD, whose amino acid sequence is shown in SEQ ID No.

1. After the reaction, the peptide is dialyzed and centrifuged sequentially to collect the precipitate and obtain the modified LPFFD-AExo. The third step involves mixing LPFFD-AExo with the mitochondrial-derived peptide MOTS-c and sonicating the mixture to load MOTS-c into LPFFD-AExo. The amino acid sequence of MOTS-c is shown in SEQ ID No.

2. The mixture is then centrifuged, and the precipitate is collected to obtain LPFFD-AExo@MOTS-c, which is the MOTS-c targeted delivery system based on Astragalus-derived exosomes.

2. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 1, characterized in that, in the first step, the mass-to-volume ratio of Astragalus root to PBS buffer is 1g:5mL~10mL; The specific process of centrifuging the filtrate in multiple stages is as follows: The filtrate was subjected to three stages of centrifugation: primary centrifugation, secondary centrifugation, and tertiary centrifugation. Primary centrifugation involved centrifuging at 3000–5000 × g for 20–30 min at 1℃–4℃, and collecting the supernatant. Secondary centrifugation involved centrifuging at 10000–12000 × g for 30–40 min at 1℃–4℃, and collecting the supernatant. Tertiary centrifugation involved centrifuging at 100000–120000 × g for 60–120 min at 1℃–4℃, and collecting the precipitate to obtain exosomes. The specific process for purifying exosomes is as follows: The exosomes were resuspended in PBS buffer at pH 7.0–7.4, and impurities were removed by filtration. Then, the exosome fractions with densities in the range of 1.13–1.19 g / mL were purified by centrifugation using an 8%–60% sucrose density gradient to obtain AExo.

3. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 2, characterized in that, in the first step, the PBS buffer is sterile PBS buffer; a juicer is used to homogenize the mixture for 3-5 minutes; after homogenization, the mixture is filtered with medical gauze or medical hemostatic cotton cloth.

4. A method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to any one of claims 1 to 3, characterized in that, In the second step, the mass-to-volume ratio of AExo to MES buffer is 1 mg: 1 ± 0.5 mL; the molar ratio of EDC to NHS is 2:1 to 1:3; activation is performed at room temperature for 20 to 40 min; the final concentration of LPFFD added is 50 to 100 μM; and the coupling reaction is incubated at 1℃ to 4℃ for 8 to 16 h.

5. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 4, characterized in that, In the second step, dialysis is performed using a dialysis bag with a molecular weight cutoff (MWCO) of 50–150 kDa for at least 24 hours; centrifugation is performed at 100,000–120,000 × g for 1–2 hours.

6. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 4, characterized in that, In the third step, the concentration of LPFFD-AExo after mixing is 0.5–2 mg / mL, and the concentration of MOTS-c is 5–20 μg / mL; during ultrasonic treatment, the ultrasonic power is 100–200 W, the ultrasonic treatment time is 5–15 min, the ultrasonic working mode is intermittent, and the temperature is 0℃–4℃; after ultrasonic treatment, it is left to stand at 0℃–4℃ for at least 1 h to complete the composite reaction.

7. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 6, characterized in that, In the third step, the intermittent mode is to continuously cycle between turning on for 2-5 seconds and turning off for 2-5 seconds; during centrifugation, centrifuge at 100,000-120,000 × g for 1-2 hours.

8. The method for preparing a MOTS-c targeted delivery system based on Astragalus-derived exosomes according to claim 6, characterized in that, in the first step, the particle size of the obtained AExo is between 50 and 200 nm, the Zeta potential is between -10 and -30 mV, and both exosome marker proteins CD63 and TSG101 are positive; in the second step, the modification efficiency of the obtained LPFFD-AExo is ≥80%; in the third step, the loading efficiency of the obtained LPFFD-AExo@MOTS-c is 70% to 90%.

9. The MOTS-c targeted delivery system based on Astragalus-derived exosomes prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the MOTS-c targeted delivery system based on Astragalus-derived exosomes as described in claim 9 for preparing a medicament for treating myocardial ischemia-reperfusion injury.

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