Mitochondrial-targeting carbon dot-engineered exosome and preparation method and application thereof

Abstract

The present application relates to a kind of mitochondria targeted carbon dot engineering exosome and its preparation method and application;With natural panax notoginseng saponin as carbon source preparation carbon dot, and covalent grafting triphenylphosphine (TPP) constructs mitochondria targeted carbon dot module, subsequently using mild ultrasonic and electrostatic self-assembly, it is compounded with M2 macrophage exosome (M2-EVs) to form mitochondria targeted carbon dot engineering exosome.Mitochondria targeted carbon dot engineering exosome forms unique inside and outside double load configuration, carbon dot is as the antioxidant shield of exosome surface to resist ischemic microenvironment degradation, also mediate it to be accurately targeted to damaged mitochondria after entering cell;Carbon dot engineering exosome prepared can be used to prepare the drug for treating ischemic disease, restore the energy homeostasis of ischemic cell, significantly promote angiogenesis and muscle fiber regeneration, and have good clinical conversion prospect in ischemic disease treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical nanomaterials and targeted delivery technology, and in particular relates to a mitochondrial-targeted carbon dot engineered exosome, its preparation method and application. Background Technology

[0002] Severe ischemic diseases (including severe lower extremity ischemia (CLI), myocardial infarction, and ischemic stroke) pose a significant clinical challenge with high morbidity and mortality worldwide. The microenvironment of these diseases is characterized by persistent hypoxia and nutrient deprivation, leading to the excessive production of local reactive oxygen species (ROS) and severe inflammatory responses. Macrophage-derived extracellular vesicles (especially M2-type, M2-EVs) have emerged as a promising natural nanotherapy platform due to their inherent immunomodulatory and tissue repair capabilities.

[0003] However, the clinical translation of natural EVs in ischemic diseases is hindered by two major microenvironmental barriers. First, severe oxidative stress at the lesion site damages the lipid bilayer and destroys the bioactive macromolecules within EVs, leading to a significant decrease in their actual utilization. Second, after natural EVs enter the cell, their therapeutic contents are mainly confined to the cytoplasm, lacking the ability to precisely deliver them at the subcellular level to core damaged organelles (especially mitochondria).

[0004] During ischemic injury, mitochondria are not only the main source of excessive pathogenic ROS production, easily triggering a vicious cycle of "ROS-induced ROS release (RIRR)," but their structural damage can also disrupt the cell's ATP metabolism network. Current exosome-based interventions mainly focus on passively neutralizing free ROS in the cytoplasm, failing to address the root cause of mitochondrial oxidative stress. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a mitochondrial-targeted carbon dot engineered exosome, its preparation method, and its application.

[0006] The technical solution adopted in this invention is: a method for preparing mitochondrial-targeted carbon dot engineered exosomes, which uses Panax notoginseng saponins as a carbon source to prepare carbon dots, then covalently grafts triphenylphosphine to construct mitochondrial-targeted carbon dots, and combines the mitochondrial-targeted carbon dots with M2 macrophage exosomes to form mitochondrial-targeted carbon dot engineered exosomes.

[0007] Preferably, the specific steps are as follows:

[0008] Step 1: Dissolve Panax notoginseng saponins and citric acid in water and prepare Panax notoginseng saponin carbon dots (PNS-CDs) through hydrothermal reaction.

[0009] Step 2: Activate the oxygen-containing functional groups on the surface of PNS-CDs with a condensing agent, and covalently react with a triphenylphosphine derivative containing an amino group to prepare mitochondrial-targeted carbon dots (CDs-TPP).

[0010] Step 3: Isolate M2 macrophage exosomes (M2-EVs) from M2 polarized macrophages;

[0011] Step 4: Mix mitochondrial-targeting carbon dots with M2 macrophage exosomes and self-assemble them to obtain mitochondrial-targeting carbon dot engineered exosomes (M2-EVs@CDs-TPP).

[0012] Preferably, in step four, the mass ratio of mitochondrial targeting carbon dots to M2 macrophage exosomes is 1-5:1.

[0013] Preferably, after the mitochondrial-targeting carbon dots are mixed with M2 macrophage exosomes, the self-assembly process is carried out under ice bath conditions with ultrasonic treatment. The ultrasonic treatment parameters are: ultrasonic amplitude 10-30%, 10 s on and 5 s off, and a total ultrasonic time of 2-5 min.

[0014] Preferably, in step two, the condensing agents are EDC and NHS; the covalent reaction is carried out at pH 6.5-7.5, at room temperature with light protection and magnetic stirring for 4-12 h.

[0015] Preferably, in step one, the mass ratio of notoginsenosides to citric acid is 5-15:1, the hydrothermal reaction temperature is 160-200℃, and the time is 4-8 h.

[0016] A method for preparing mitochondrial-targeted carbon dot-engineered exosomes.

[0017] Preferably, mitochondrial-targeting carbon dots are encapsulated within M2 macrophage exosomes and anchored to the lipid bilayer on the outer side of the M2 macrophage exosomes.

[0018] Application of mitochondrial-targeted carbon dot engineered exosomes in the preparation of drugs for treating ischemic diseases.

[0019] The advantages and positive effects of this invention are as follows: the constructed mitochondrial-targeted carbon dot engineered exosome M2-EVs@CDs-TPP has a unique dual loading configuration inside and outside; the positively charged CDs-TPP anchored on the outer leaflet of the exosome not only acts as a "nanoprotective shield" to resist external oxidative degradation, but the depolarization effect caused by neutralizing the negative potential of the vesicle also greatly weakens the electrostatic repulsion with the cell membrane, overcomes the environmental degradation and internalization barrier, and significantly promotes the uptake of the engineered vesicle by the target cell;

[0020] M2-EVs@CDs-TPP overcomes the limitation of natural exosomes to the cytoplasm. After undergoing vesicle endocytosis and endosome escape, the TPP group modified on the carbon dots utilizes the negative potential gradient of the mitochondrial inner membrane under pathological conditions to mediate the active ingredient to reach the damaged mitochondria directly. Attached Figure Description

[0021] Figure 1 Physicochemical properties of M2-EVs@CDs-TPP; (A) UV-Vis absorption spectra of CDs and CDs-TPP; (B) X-ray photoelectron spectroscopy (XPS) scan of CDs-TPP; (C) High-resolution P 2p XPS spectrum of CDs-TPP; (D) High-resolution N 1s XPS spectrum of CDs-TPP; (E) Hydrodynamic diameters of CDs, CDs-TPP, M2-EVs and M2-EVs@CDs-TPP (DLS test results); (F) Zeta potentials of CDs, CDs-TPP, M2-EVs and M2-EVs@CDs-TPP;

[0022] Figure 2 CLSM detection of dual fluorescence colocalization and fluorescence distribution analysis of CDs-TPP and M2-EVs; (A) CLSM image of DiD-labeled M2-EVs (red) and FITC-labeled CDs-TPP (green) after co-incubation, with the merged channel showing high colocalization (yellow signal); (B) Fluorescence intensity distribution curve along the white dashed line in (A), quantitative analysis of the spatial overlap of red and green fluorescence signals in the selected area;

[0023] Figure 3 Flow cytometry and CLSM analysis were used to analyze the endothelial cell uptake efficiency and mitochondrial targeting of different formulations; (A) Flow cytometry histograms of fluorescence intensity of different treatment groups (from bottom to top: blank cell control group used to define background autofluorescence, and M2-EVs, M2-EVs@CDs, and M2-EVs@CDs-TPP groups that were actually intervened); (B) Quantitative statistical comparison of mean fluorescence intensity (MFI) of each group that were actually intervened; (C) Localization of M2-EVs@CDs-TPP in mitochondria of endothelial cells after treatment with M2-EVs@CDs-TPP.

[0024] Figure 4Regulation of mitochondrial oxidative stress and energy metabolism by M2-EVs@CDs-TPP; (A) Fluorescence images of intracellular mtROS levels after different treatments; (B) Quantitative statistical analysis of mtROS fluorescence intensity; (C) Detection of mitochondrial membrane potential by JC-1 staining (red fluorescence represents high membrane potential, green fluorescence represents membrane depolarization); (D) Quantitative statistical analysis of red / green fluorescence ratio; (E) Relative quantification of intracellular ATP production in each group.

[0025] Figure 5 Western blot analysis of the effects of M2-EVs@CDs-TPP on the expression of Nrf2 / PGC-1α signaling axis and its downstream antioxidant and mitochondrial biosynthesis-related proteins; (A) Protein expression bands of Nrf2, PGC-1α, HO-1, SOD2, NRF1, TFAM, Caspase-9 and internal reference protein GAPDH after different treatments; (B) Quantitative statistical analysis of the relative expression levels of each protein using gray values.

[0026] Figure 6 Evaluation of the in vitro angiogenesis and myoblast differentiation activity of M2-EVs@CDs-TPP; (A) Representative images of tube formation of HUVECs on matrix gel after different treatments; (B) Quantitative statistics of the number of tubes formed; (C) Representative images of MF20 immunofluorescence staining after differentiation of C2C12 myoblasts after different treatments; (D) Quantitative statistics of relative fluorescence intensity of MF20. Detailed Implementation

[0027] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0028] This invention relates to a mitochondrial-targeted carbon-dot engineered exosome, its preparation method, and its applications. Carbon dots are prepared using natural Panax notoginseng saponins as a carbon source, and triphenylphosphine (TPP) is covalently grafted onto them to construct a mitochondrial-targeted carbon dot module. Subsequently, using mild sonication and electrostatic self-assembly, this module is combined with M2-type macrophage exosomes (M2-EVs). This system forms a unique dual-load configuration, where the carbon dots act both as an antioxidant shield on the exosome surface to resist degradation in the ischemic microenvironment and mediate its precise targeting to damaged mitochondria after entry into the cell. The prepared carbon-dot engineered exosomes can be used to prepare drugs for treating ischemic diseases, restore energy homeostasis in ischemic cells, and significantly promote angiogenesis and myofibril regeneration.

[0029] The preparation of mitochondrial-targeted carbon dot-engineered exosomes includes the following steps:

[0030] Step 1: Synthesis and modification of carbon dots. Carbon dots with intrinsic antioxidant activity (PNS-CDs) are synthesized by hydrothermal method using Panax notoginseng saponins and citric acid. Specifically, Panax notoginseng saponin powder and citric acid are mixed at a mass ratio of 5-15:1, dissolved in ultrapure water, and clarified by sonication. The mixture is placed in a reaction vessel and hydrothermally reacted at 160-200℃ for 4-8 h. After the reaction, the supernatant is collected by cooling and centrifugation, dialyzed through a dialysis bag with a molecular weight cutoff of 500 Da for 6-8 h, and freeze-dried to obtain yellowish-brown solid PNS-CDs.

[0031] Step 2: Activate the carboxyl groups on the surface of the carbon dots using a condensing agent, and then perform an amidation reaction with TPP-NH2 to prepare mitochondrial-targeted carbon dots (CDs-TPP). Disperse PNS-CDs in MES buffer and sonicate to dissolve. Add activators EDC and NHS to activate the surface carboxyl groups, and stir magnetically at room temperature in the dark for 15-30 min. Then adjust the pH to 6.5-7.5 using 0.1 M NaOH. Slowly add TPP-NH2 dissolved in DMSO to the above solution, and stir at room temperature in the dark for 4-12 h. The molar ratio of mitochondrial-targeted carbon dots to TPP-NH2 is 2-10:1. Dialyze the reaction solution through a 1 kDa dialysis bag for 24 h, and freeze-dry for 24-36 h to obtain targeted carbon dot CDs-TPP.

[0032] Step 3: Isolation of exosomes, induction of RAW 264.7 macrophages to M2 polarization, collection of supernatant, purification by ultracentrifugation and ultrafiltration to obtain M2-EVs.

[0033] Step 4: Assembly of engineered exosomes; CDs-TPP and M2-EVs were mixed in buffer solution and subjected to gentle sonication and electrostatic self-assembly under ice bath conditions. After purification, M2-EVs@CDs-TPP was obtained. The targeted carbon dot CDs-TPP and M2-EVs were mixed and dissolved in PBS buffer at pH 7.4 at a mass ratio of 1:1-5:1. Under ice bath conditions, a sonicator with a power set to 10-30% was used for gentle sonication at a "10 s on, 5 s off" mode for a total of 2-5 min to promote electrostatic assembly. The treated mixture was centrifuged at 12000 g for 15 min through a 100 kDa ultrafiltration tube to remove unbound free carbon dots, thus obtaining mitochondrial targeted carbon dot engineered exosomes (M2-EVs@CDs-TPP).

[0034] Using the above method, natural saponin-derived carbon dots with intrinsic ROS scavenging ability were grafted with the mitochondrial targeting molecule triphenylphosphine (TPP) and loaded onto M2-EVs to construct a multifunctional "bio-nano hybrid platform". Some of the mitochondrial targeting carbon dots were encapsulated inside the exosome vesicles, while the other carbon dots were stably anchored to the surface of the lipid bilayer on the outside of the exosome through electrostatic interactions, forming a spatial configuration with dual internal and external loading.

[0035] Mitochondrial-targeted carbon-dot engineered exosomes effectively restore energy homeostasis in ischemic cells and significantly promote angiogenesis and myofiber regeneration through a dual mechanism of "in-situ chemical quenching of mitochondrial ROS to block the vicious cycle of ROS-induced ROS release (RIRR)" and "activation of the Nrf2-PGC-1α axis to drive biological reprogramming." Furthermore, these exosomes can activate the cellular Nrf2-PGC-1α signaling axis, constructing a biphasic antioxidant defense line and promoting mitochondrial biosynthesis. Based on this dual repair mechanism of "chemical quenching + biological reprogramming," these exosomes can implement precise dual-effect intervention at the subcellular level. First, CDs-TPP chemically quench pathogenic mtROS in situ, blocking the RIRR vicious cycle at its source. The relief of microenvironmental stress synergizes with the bioactivity of M2-EVs, thereby effectively activating the upstream metabolic regulatory center centered on Nrf2-PGC-1α. This not only constructs a durable biphasic antioxidant defense line composed of HO-1 and SOD2, but also effectively restores ATP homeostasis in ischemic cells by powerfully driving mitochondrial biosynthesis through NRF1 / TFAM. Experimental verification shows that mitochondrial-targeted carbon-dot engineered exosomes have excellent performance in targeted regulation of mitochondrial metabolism and treatment of ischemic diseases, especially showing good clinical translation prospects in the treatment of ischemic diseases.

[0036] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0037] Example 1: Preparation and physicochemical characterization of M2-EVs@CDs-TPP

[0038] (1) Synthesis of PNS-CDs: 2 g of Panax notoginseng saponin (PNS) powder and 0.2 g of citric acid were dissolved in 200 mL of deionized water and sonicated for 10 min to make it transparent. The solution was transferred to a polytetrafluoroethylene high-pressure reactor and reacted at 180 °C for 6 h. After cooling, the solution was centrifuged at 10,000 g for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and dialyzed using a 500 Da dialysis bag for 8 h. The solution was then lyophilized to obtain PNS-CDs powder.

[0039] (2) CDs-TPP synthesis: 20 mg of PNS-CDs were suspended in MES buffer and sonicated for 10 min. Activators EDC and NHS were added, and the mixture was magnetically stirred at room temperature in the dark for 30 min. The pH was adjusted to 7.0 with 0.1 M NaOH. The targeting ligand (3-aminopropyl)triphenylphosphine bromide (TPP-NH2) dissolved in DMSO was slowly added dropwise; the molar ratio of PNS-CDs to TPP-NH2 was 3:1. After reacting for 12 h, the mixture was dialyzed for 24 h using a 1 kDa dialysis bag, and finally lyophilized to obtain targeted carbon dot CDs-TPP. The prepared carbon dot CDs-TPP was tested, and the results are as follows: Figure 1 As shown, the characteristic peak of the TPP aromatic ring at 230 nm was detected by UV-vis, and the covalent grafting was confirmed by high-resolution peak separation of P 2p and N 1s by XPS.

[0040] (3) Engineered exosome complex: RAW 264.7 macrophages were induced to M2 polarized state with 20 ng / mL IL-4. The supernatant of the culture medium was collected and purified by ultracentrifugation to obtain M2-EVs. CDs-TPP and M2-EVs were mixed in PBS at a mass ratio of 3:1 and placed in an ice bath. The mixture was sonicated at 20% amplitude (10 s on, 5 s off) for a total of 3 min. The mixture was washed with a 100 kDa ultrafiltration tube (centrifuged at 12000 g for 15 min) to remove free carbon dots.

[0041] The physicochemical verification of the product prepared above was performed, and the results are as follows: Figure 1 As shown, dynamic light scattering (DLS) measurements indicate that the particle size of pure M2-EVs is approximately 110 nm, while the particle size of the composite M2-EVs@CDs-TPP increases to approximately 130 nm. Zeta potential measurements show that the original natural M2-EVs are strongly negatively charged (-25.6 mV), and after assembly with high-positive-potential CDs-TPP, the surface charge of the composite system reverses and neutralizes to near neutral (+1.9 mV), proving that the carbon dots not only enter the exosome interior but also stably bind to the outer membrane through electrostatic interactions.

[0042] Example 2: Internalization and membrane localization verification of engineered exosomes (M2-EVs@CDs-TPP)

[0043] To visually demonstrate the successful loading of CDs-TPP onto exosomes, purified M2-EVs were labeled with a lipid membrane by incubation at 37°C for 15 min using the lipophilic red fluorescent probe DiD, followed by ultrafiltration to remove free dye. Similarly, the green fluorescent dye FITC (FITC-CDs-TPP) was grafted onto the targeted carbon dots rich in primary amino groups on the surface.

[0044] The two materials were mixed and assembled according to the ultrasonic co-incubation method described in Example 1. A small amount of the assembled suspension was dropped into a confocal culture dish for observation. Figure 2 As shown, the imaging results of laser confocal scanning microscopy (CLSM) show that the DiD-labeled exosomal lipid membrane (red fluorescence) and the FITC-labeled carbon dots (green fluorescence) exhibit strong yellow fused fluorescence in the microscopic space, with an extremely high Pearson correlation coefficient, directly confirming the highly stable integration of the two.

[0045] Example 3: Efficient endocytosis of engineered exosomes (M2-EVs@CDs-TPP) by endothelial cells and precise mitochondrial localization

[0046] HUVECs were seeded in confocal dishes and cultured for 24 h, then co-incubated with the dual fluorescently labeled M2-EVs@CDs-TPP from Example 2 for 4 h. After cell digestion, flow cytometry analysis showed that the mean fluorescence intensity (MFI) of the targeted exosome group was significantly higher than that of the unmodified native M2-EVs group. This is because the carbon dots neutralized the negative charge of the exosome membrane, weakening the electrostatic repulsion with the cell membrane and thermodynamically lowering the endocytosis barrier, thereby significantly increasing cellular uptake.

[0047] Furthermore, MitoTracker Red was added to specifically stain the mitochondria of live HUVECs cells. For example... Figure 3 As shown, CLSM observation revealed that free, non-targeted carbon dots mostly remained in the cytoplasm; while after M2-EVs@CDs-TPP was taken up by endothelial cells and escaped from the endosome, its green fluorescence signal (derived from the released CDs-TPP) highly overlapped with the red mitochondrial probe network. This result validates that the engineered platform endows therapeutic components with the ability to break through cytoplasmic constraints and directly reach damaged mitochondria.

[0048] Example 4: Engineered exosomes (M2-EVs@CDs-TPP) in situ quench mtROS, interrupting the vicious cycle and activating Nrf2-PGC-1α to reshape steady state.

[0049] HUVECs were treated with CoCl2 to simulate an in vitro ischemia / oxidative stress injury model. The experiment included a control group (NC group) of untreated normal cells and a model group treated with only CoCl2 (CoCl2 group). The intervention groups were treated with equivalent doses of PBS, CDs, M2-EVs, M2-EVs@CDs, and M2-EVs@CDs-TPP, respectively, to examine the in situ quenching of mtROS and the restoration of mitochondrial membrane potential (JC-1) in the model cells.

[0050] MitoSOX-specific probes were used to detect mitochondrial reactive oxygen species (mtROS). Figure 4 As shown in AB, CLSM observation revealed that the control group, lacking mitochondrial targeting capability, could not prevent the continuous leakage of electrons from mitochondria. However, M2-EVs@CDs-TPP, with its precise penetration into the mitochondrial matrix, exhibited effective mtROS quenching capability, cutting off the storm source of ROS cascade amplification from a physicochemical perspective. This demonstrates that the mitochondrial-targeting carbon dot-engineered exosome has the effect of blocking the vicious cycle of RIRR.

[0051] like Figure 4 As shown in CE, JC-1 staining results confirmed that M2-EVs@CDs-TPP treatment effectively rescued ischemia-induced mitochondrial depolarization collapse (MMP recovery). Subsequent luminescence assays demonstrated that ATP production in the targeted treatment group of endothelial cells significantly increased, thereby achieving membrane potential repolarization and energy recovery in the mitochondrial energy factory.

[0052] Biological cascade reprogramming network: Extracting total cellular proteins after intervention for Western blotting analysis, such as... Figure 5 As shown, in a microenvironment where the in situ oxidative storm was relieved, M2-EVs@CDs-TPP significantly activated the upstream master switch of the oxidative stress response—transcription factor Nrf2 and its core coactivator PGC-1α. This signaling axis drove two parallel pathways downstream: on the one hand, it upregulated heme oxygenase (HO-1) and mitochondrial-specific antioxidant enzyme (SOD2), constructing a long-lasting cellular defense wall; on the other hand, it strongly activated the downstream nuclear respiratory factor NRF1 and mitochondrial transcription factor TFAM. TOMM20 immunofluorescence morphology confirmed that the mitochondrial network, which was originally severely disrupted due to ischemia, was regenerated in large quantities and assembled into a healthy, long, tubular interconnected network under the drive of NRF1 / TFAM, verifying the de novo biosynthesis of mitochondria.

[0053] Example 5: M2-EVs@CDs-TPP promotes the in vitro synergistic regeneration of blood vessels and muscle tissue.

[0054] The core phenotype of repair in ischemic diseases is the regeneration of microvascular networks and damaged muscle fibers.

[0055] M2-EVs@CDs-TPP can promote endothelial tubule formation. In a CoCl2-induced damaged HUVECs model, Transwell migration and matrix gelation experiments were conducted, and the results are as follows: Figure 6As shown in Figures AB, cells in the M2-EVs@CDs-TPP intervention group not only showed a significant recovery in migration ability but also self-assembled into a coherent and dense capillary-like network structure on the matrix gel. The total tube length and the number of tube-forming nodes were significantly superior to those treated with carbon dots alone or natural EVs. This indicates that the restoration of ATP production in the underlying mitochondria provides the driving force for endothelial chemotaxis and cytoskeleton rearrangement.

[0056] M2-EVs@CDs-TPP can also promote myogenic differentiation, and its myogenic activity was evaluated using a C2C12 myoblast model. Figure 6 As shown in CD, the ability of C2C12 cells to form myotubes is severely impaired under ischemic stimulation. After intervention with M2-EVs@CDs-TPP, differentiation and fusion barriers were effectively relieved: early marker myopoietin (MyoG) was highly expressed on day 3 of differentiation induction, and by day 7, robust muscle fibers with regular arrangement, multinucleate fusion, and high expression of the maturation marker myosin heavy chain (MF20) were formed. This phenotypic experiment confirms that this targeted platform possesses significant transformative potential for efficiently rescuing multi-tissue stem / progenitor cell regeneration disorders by correcting underlying metabolic and energy imbalances.

[0057] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for preparing mitochondrial-targeted carbon dot-engineered exosomes, characterized in that: Carbon dots were prepared using Panax notoginseng saponins as a carbon source, and then triphenylphosphine was covalently grafted onto them to construct mitochondrial-targeting carbon dots. These mitochondrial-targeting carbon dots were then combined with M2 macrophage exosomes to form mitochondrial-targeting carbon dot engineered exosomes.

2. The method for preparing mitochondrial-targeted carbon dot-engineered exosomes according to claim 1, characterized in that: The specific steps are as follows: Step 1: Dissolve Panax notoginseng saponins and citric acid in water and prepare carbon dots PNS-CDs through a hydrothermal reaction; Step 2: Activate the oxygen-containing functional groups on the surface of PNS-CDs with a condensing agent, and covalently react with a triphenylphosphine derivative containing an amino group to prepare mitochondrial-targeting carbon dots; Step 3: Isolate M2 macrophage exosomes from M2 polarized macrophages; Step 4: Mix mitochondrial-targeting carbon dots with M2 macrophage exosomes and self-assemble them to obtain mitochondrial-targeting carbon dot engineered exosomes.

3. The method for preparing mitochondrial-targeted carbon dot-engineered exosomes according to claim 2, characterized in that: In step four, the mass ratio of mitochondrial targeting carbon dots to M2 macrophage exosomes is 1-5:

1.

4. The method for preparing mitochondrial-targeted carbon dot-engineered exosomes according to claim 3, characterized in that: After mitochondrial-targeted carbon dots were mixed with M2 macrophage exosomes, the self-assembly process was carried out under ice bath conditions with sonication. The sonication parameters were: sonicator amplitude 10-30%, 10 s on and 5 s off, and a total sonication time of 2-5 min.

5. The method for preparing mitochondrial-targeted carbon dot-engineered exosomes according to claim 2, characterized in that: In step two, the condensing agents are EDC and NHS; the covalent reaction is carried out at pH 6.5-7.5, at room temperature with light protection and magnetic stirring for 4-12 h.

6. The method for preparing mitochondrial-targeted carbon dot-engineered exosomes according to claim 2, characterized in that: In step one, the mass ratio of notoginsenosides to citric acid is 5-15:1, the hydrothermal reaction temperature is 160-200℃, and the time is 4-8 h.

7. Mitochondrial-targeted carbon dot-engineered exosomes prepared by the method described in any one of claims 1-6.

8. The mitochondrial-targeted carbon dot-engineered exosome according to claim 7, characterized in that: Mitochondrial-targeting carbon dots are encapsulated within M2 macrophage exosomes and anchored to the lipid bilayer on the outer side of the M2 macrophage exosomes.

9. The use of the mitochondrial-targeted carbon dot engineered exosomes according to claim 7 or 8 in the preparation of drugs for treating ischemic diseases.

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