Angiopep-2 / LK5 modified targeted delivery drug-loaded exosome and preparation method thereof

Abstract

The invention relates to the technical field of biological medicines, and provides an Angiopep-2 / LK5 modified drug-loaded exosome targeting delivery system. According to the invention, a human mesenchymal stem cell exosome is used as a carrier, and rapamycin and a CRISPR-Cas9 / sgRNA compound targeting SNCA are respectively loaded through a pH gradient method and electroporation; further, Angiopep-2 and LK5 peptides are co-modified on the surface by utilizing chemical crosslinking, and an MMP-2 response lipid material is embedded through ultrasound. Experimental results show that the system can efficiently break through the blood brain barrier, specifically capture alpha-synuclein aggregates and accurately release drugs in an MMP-2 high expression region, and targeted brain provides a new strategy for multi-mechanism synergistic treatment of Parkinson's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to targeted delivery drug-loaded exosomes modified with Angiopep-2 / LK5 and their preparation methods. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and the formation of Lewy bodies from the abnormal aggregation of α-synuclein. Currently, clinical treatment of PD still faces several key challenges: First, the low penetration efficiency of the blood-brain barrier (BBB) ​​severely limits drug delivery—existing small-molecule drugs such as levodopa, while possessing some BBB penetration ability, are prone to reduced efficacy and motor complications with long-term use; while large-molecule drugs (such as antibodies targeting SNCA) are difficult to effectively cross the blood-brain barrier, with bioavailability typically below 1%. Second, traditional drug delivery systems lack specific targeting capabilities to the lesion site, leading to drug accumulation in non-target tissues (such as the peripheral vascular system), potentially causing systemic side effects such as orthostatic hypotension. Furthermore, pathological α-synuclein can spread between neurons via exosomes, but current exosome-based drug delivery systems mainly rely on their natural penetrating properties and have not yet been engineered to enhance their specific affinity for α-synuclein aggregates, thus limiting the effective clearance of abnormal proteins. Summary of the Invention

[0003] The purpose of this invention is to address the above-mentioned technical problems by providing a drug-loaded exosome targeted delivery system modified with Angiopep-2 / LK5.

[0004] The exosomes of this invention comprise exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs-EXO), rapamycin, a CRISPR-Cas9 / sgRNA complex, and MMP-2 responsive lipid material. The ligands modified on the surface of the exosomes comprise angiopep-2 peptide that binds to the LRP1 receptor and an LK5 peptide that specifically captures α-synuclein.

[0005] The mass ratio of the HUC-MSCs-EXO, Rapamycin, CRISPR-Cas9 / sgRNA complex, Angiopep-2 peptide, LK5 peptide, and MMP-2 responsive lipid material was 1:10:50:0.24:0.08:0.0625. ；

[0006] The MMP-2 responsive lipid material is DSPE-PEG-GPLGVRGD.

[0007] This invention also provides a method for preparing an Angiopep-2 / LK5 modified drug-loaded exosome targeted delivery system, specifically including the following steps:

[0008] (4) Preparation of exosomes HUC-MSCs-EXO: Exosomes HUC-MSCs-EXO were extracted from the culture supernatant of human umbilical cord mesenchymal stem cells using differential ultracentrifugation. The specific steps were as follows: differential centrifugation was performed at 4℃ for 1 h to remove cells and debris; finally, ultracentrifugation was performed at 120000×g for 70 min, the exosome precipitate was collected, resuspended in PBS, and the protein concentration was determined by the BCA method and adjusted to 1 mg / mL. The precipitate was then stored at -80℃ for later use.

[0009] (5) Preparation of drug-loaded exosomes HUC-MSCs-EXO@R-SNCA: Rapamycin was actively loaded using a pH gradient method: exosomes HUC-MSCs-EXO were mixed with Rapamycin and incubated in HEPES buffer at pH 7.4 for 30 min; then the pH of the system was adjusted to 5.0 with citric acid, and heated in a 45°C water bath for 15 min to induce efficient drug loading; finally, the pH was restored to 7.4 to obtain Rapamycin-loaded exosomes. Subsequently, in order to introduce the CRISPR-Cas9 / sgRNA complex targeting the SNCA gene into the Rapamycin-loaded exosomes, electroporation was used for loading, wherein the weight ratio of the complex to the Rapamycin-loaded exosomes was 50:1, and the electroporation conditions were set as follows: voltage 150V, capacitance 100μF, single pulse with a pulse duration of 5ms; after transfection, the system was incubated at room temperature for 30 min to obtain drug-loaded exosomes HUC-MSCs-EXO@R-SNCA.

[0010] (6)Angiopep -2 / LK5 co-modification and MMP-2 responsive membrane embedding: Angiopep-2 peptide was covalently modified onto the surface of HUC-MSCs-EXO@R-SNCA with 100 μM using a carbodiimide chemical cross-linking method, wherein the mass ratio of Angiopep-2 peptide to HUC-MSCs-EXO@R-SNCA was 0.24:1. The reaction was carried out in MES buffer at pH 6.0 for 2 h, and then unreacted cross-linking agent and free peptides were removed by PBS dialysis to obtain HUC-MSCs-EXO@R-SNCA@Angiopep-2. Similarly, the D-type LK5 peptide was covalently linked to HUC-MSCs-EXO@R-SNCA@Angiopep-2 using a carbodiimide chemical cross-linking method, with a mass ratio of D-type LK5 peptide to HUC-MSCs-EXO@R-SNCA@Angiopep-2 of 0.08:1. This enhanced its specific recognition and binding ability to α-synuclein fiber structures, resulting in HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5. Finally, the MMP-2 responsive lipid material DSPE-PEG-GPLGVRGD was mixed with HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5 and embedded into the exosome membrane via ultrasonic co-extrusion, thus obtaining the exosome HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5@DPG with MMP-2 enzyme-triggered drug release function.

[0011] Further, in step (2), the exosomes HUC-MSCs-EXO and Rapamycin are mixed at a mass ratio of 1:10.

[0012] Furthermore, in step (2), the molar ratio of CRISPR-Cas9 to sgRNA in the CRISPR-Cas9 / sgRNA complex is 1:1.

[0013] Furthermore, the sequence of the MMP-2 responsive lipid material in step (3) is GPLGVRGD.

[0014] Furthermore, the concentration of the Angiopep-2 peptide in step (3) is 100 μM.

[0015] Furthermore, the sequence of the D-type LK5 peptide in step (3) is D-LKLLLKL, and the working concentration is 100 μM.

[0016] Further, the MMP-2 responsive lipid material described in step (3) is mixed with the drug-loaded exosomes co-modified with Angiopep-2 / LK5 at a molar ratio of 1:20.

[0017] Furthermore, the ultrasonic co-extrusion method described in step (3) is achieved under ice bath conditions, with 200W ultrasonic treatment for 5 seconds followed by a 5-second interval, for a total of 10 cycles.

[0018] The present invention also provides the use of an Angiopep-2 / LK5 modified drug-loaded exosome targeted delivery system in the preparation of products for the treatment of neurodegenerative diseases:

[0019] Furthermore, the neurodegenerative disease mentioned is Parkinson's disease;

[0020] Furthermore, the product is administered via nasal administration.

[0021] The advantages of this invention are:

[0022] 1. This invention constructs a bifunctional ligand synergistic system. By co-modifying the exosome surface with Angiopep-2, which targets the blood-brain barrier, and a modified LK5 peptide that specifically captures α-synuclein, the limitations of single-function modification are overcome, and the targeting efficiency and lesion binding ability of the delivery system are significantly enhanced.

[0023] 2. This invention innovatively introduces MMP-2 responsive membrane material to achieve precise drug release in the microenvironment of α-synuclein aggregation region, effectively improving the controllability of the enrichment and release of therapeutic components at the lesion site.

[0024] 3. This invention integrates the triple therapeutic mechanisms of rapamycin drug inhibition, Angiopep-2-mediated BBB penetration, and LK5-guided α-synuclein clearance, which show a significant synergistic effect compared to single-mechanism approaches, providing a new approach for the comprehensive treatment of Parkinson's disease. Attached Figure Description

[0025] Figure 1 Here is a physical characterization diagram of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in this invention: Figure 1 (a) is a topographic diagram; Figure 1 (b) is the Zeta potential.

[0026] Figure 2 This invention relates to the responsive drug release behavior of exosomes.

[0027] Figure 3 This is a characterization of the BBB penetration performance of the exosomes of this invention.

[0028] Figure 4 This is a characterization of the α-synuclein capture performance of exosomes in this invention.

[0029] Figure 5 This invention describes the brain distribution of exosomes.

[0030] Figure 6 This relates to the biosafety of the exosomes of this invention. Detailed Implementation

[0031] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0032] Example 1: Preparation of drug-loaded exosomes modified with Angiopep-2 / LK5

[0033] (1) Preparation of exosomes HUC-MSCs-EXO: Exosomes HUC-MSCs-EXO were extracted from the culture supernatant of human umbilical cord mesenchymal stem cells using differential ultracentrifugation. The specific steps were as follows: differential centrifugation was performed at 4℃ for 1 h to remove cells and debris; finally, ultracentrifugation was performed at 120000×g for 70 min, the exosome precipitate was collected, resuspended in PBS, and the protein concentration was determined by the BCA method and adjusted to 1 mg / mL. The precipitate was then stored at -80℃ for later use.

[0034] (2) Preparation of drug-loaded exosomes HUC-MSCs-EXO@R-SNCA: Rapamycin was actively loaded using the pH gradient method: exosomes HUC-MSCs-EXO and Rapamycin were mixed at a drug / exosome mass ratio of 10:1 and incubated in HEPES buffer at pH 7.4 for 30 min. Then, the pH of the system was adjusted to 5.0 with citric acid and heated in a 45°C water bath for 15 min to induce efficient drug loading. Finally, the pH was restored to 7.4 to obtain Rapamycin drug-loaded exosomes. Subsequently, to introduce the CRISPR-Cas9 / sgRNA complex (CRISPR-Cas9 to sgRNA molar ratio of 1:1) targeting the SNCA gene into Rapamycin-loaded exosomes, electroporation was used for loading. The weight ratio of the complex to the Rapamycin-loaded exosomes was 50:1. The electroporation conditions were set as follows: voltage 150V, capacitance 100μF, single pulse, and pulse duration 5ms. After transfection, the cells were incubated at room temperature for 30 minutes to obtain drug-loaded exosomes HUC-MSCs-EXO@R-SNCA.

[0035] (3) Angiopep-2 / LK5 co-modification and MMP-2 responsive membrane embedding: Angiopep-2 peptide was covalently modified onto the surface of HUC-MSCs-EXO@R-SNCA with 100 μM using a carbodiimide chemical cross-linking method, wherein the mass ratio of Angiopep-2 peptide to HUC-MSCs-EXO@R-SNCA was 0.24:1. The reaction was carried out in MES buffer at pH 6.0 for 2 h. After that, unreacted cross-linking agent and free peptide fragments were removed by PBS dialysis to obtain HUC-MSCs-EXO@R-SNCA@Angiopep-2. Similarly, D-type LK5 peptide (sequence: D-LKLLLKL, working concentration 100 μM) was covalently linked to HUC-MSCs-EXO@R-SNCA@Angiopep-2 using a carbodiimide chemical cross-linking method, with a mass ratio of D-type LK5 peptide to HUC-MSCs-EXO@R-SNCA@Angiopep-2 of 0.08:1, enhancing its specific recognition and binding ability to α-synuclein fiber structures, resulting in HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5. Finally, MMP-2 responsive lipid material (sequence: GPLGVRGD) DSPE-PEG-GPLGVRGD was linked to HUC-MSCs-EXO@R-SNCA@Angiopep-2. - 2 / LK5 were mixed at a molar ratio of 1:20 and embedded into the exosome membrane by ultrasonic co-extrusion (under ice bath conditions, 200W ultrasonic treatment for 5s, interval for 5s, for a total of 10 cycles), thus obtaining exosomes HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5@DPG with MMP-2 enzyme-triggered drug release function.

[0036] Comparative Example 1: Preparation of Free Drug Complex

[0037] The difference between this comparative example and Example 1 is that in step (2), only Rapamycin and the CRISPR-Cas9 / sgRNA complex were physically mixed at a mass ratio of 1:5, and they were not encapsulated into exosomes HUC-MSCs-EXO.

[0038] Comparative Example 2: Preparation of Unmodified Drug-Loaded Exosomes

[0039] The difference between this comparative example and Example 1 is that the drug-loaded exosomes are only embedded in the MMP-2 responsive membrane, but are not modified with Angiopep-2 and LK5 peptides.

[0040] Comparative Example 3: Preparation of drug-loaded exosomes with monomodified Angiopep-2

[0041] The difference between this comparative example and Example 1 is that only the drug-loaded exosomes were modified with Angiopep-2, and the LK5 peptide was not modified.

[0042] Comparative Example 4: Preparation of drug-loaded exosomes with single-modified LK5 peptide

[0043] The difference between this comparative example and Example 1 is that only the drug-loaded exosomes were modified with the LK5 peptide, and Angiopep-2 was not modified.

[0044] Comparative Example 5: Preparation of Non-responsive Drug-Loaded Exosomes

[0045] The difference between this comparative example and Example 1 is that the lipid material does not contain MMP-2 responsiveness and DSPE-PEG is used.

[0046] Comparative Example 6: Preparation of low-density Angiopep-2 modified drug-loaded exosomes

[0047] The difference between this comparative example and Example 1 is that the concentration of Angiopep-2 modified with drug-loaded exosomes is 50 μM.

[0048] Comparative Example 7: High-density Angiopep - 2. Preparation of modified drug-loaded exosomes

[0049] The difference between this comparative example and Example 1 is that the concentration of Angiopep-2 modified with drug-loaded exosomes is 200 μM.

[0050] Experimental Example 1: Physical characterization of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1

[0051] Microstructure: The exosomes prepared in Example 1 were diluted and dropped onto a copper grid and allowed to dry. Their microstructure was characterized using transmission scanning electron microscopy (TEM). The results are as follows: Figure 1 As shown in (a), the exosomes are spherical with a diameter of about 60 nm.

[0052] Zata potential: Angiopep for characterizing exosomes - To determine whether 2 / LK5 modification was successful, the surface charge of the particles was characterized. The microspheres were ultrasonically dispersed in deionized water, and the zeta potential of the exosomes was evaluated using a zeta potential meter. Results are as follows: Figure 1 As shown in (b), the Zeta potentials before and after modification are -20.9 and -14.3 mV, respectively. This is because surface modification may partially shield the negative charge, causing the potential to shift towards neutral, but a certain negative value should still be maintained to avoid particle aggregation. Therefore, the exosomes prepared in Example 1 were successfully modified with Angiopep-2 / LK5.

[0053] Experimental Example 2: Characterization of drug release behavior of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1

[0054] To evaluate the drug release behavior of the exosomes prepared in Example 1, Comparative Example 1, and Comparative Example 5, exosomes were placed in 50 mL of PBS (containing 100 ng / mL MMP-2 enzyme) at 37°C for drug release experiments. 3 mL samples were collected at 1, 2, 6, 12, 24, and 48 hours, with an equal volume of fresh medium added simultaneously. The concentration of rapamycin in the collected samples was determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated accordingly.

[0055] The results are as follows Figure 2 As shown, Comparative Example 1 released 78% of its drug rapidly within 2 hours, Comparative Example 5 released 43% within 48 hours, while Example 1 released 68% within 24 hours and reached 99% within 48 hours. This is because Comparative Example 1, being a free drug complex, did not undergo exosome encapsulation and therefore lacked sustained-release effect. Comparative Example 5, due to the lack of MMP-2 responsiveness in its carrier material, was difficult to achieve responsive release in the MMP-2 enzyme environment. Therefore, the exosomes prepared in Example 1 exhibited excellent MMP-2 responsive release behavior.

[0056] Experimental Example 3: Characterization of BBB penetration performance of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1.

[0057] To evaluate the BBB penetration ability of delivery systems with different Angiopep-2 ligand densities, an in vitro blood-brain barrier model was constructed using a Transwell co-culture system. Human brain microvascular endothelial cells and astrocytes were co-cultured to induce the formation of a tight junction barrier, and the transendothelial resistance (TEER > 200 Ω·cm) was measured. 2 To verify the integrity of the model, DiR fluorescently labeled exosomes prepared in Examples 1, 2, 3, 6, and 7 were added to the upper chamber and incubated at 37°C for a specific time. The culture medium from the lower chamber was then collected. The fluorescence intensity was quantitatively detected using a microplate reader, and the apparent permeability coefficient (Papp) was calculated accordingly.

[0058] The results are as follows Figure 3 As shown, the Papp values ​​for Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 6, and Comparative Example 7 are 6.0 × 10⁻⁶. -6 cm / s, 0.1×10 -6 cm / s, 0.15×10 -6 cm / s, 0.6×10 -6 cm / s and 1.1×10 -5cm / s. This is because Angiopep-2 modification efficiently enables exosomes to cross the blood-brain barrier, and its permeability increases with increasing Angiopep-2 concentration, but a ligand concentration of 100 μM already provides the exosomes with good BBB penetration ability. In summary, the exosomes prepared in Example 1 have good BBB penetration ability.

[0059] Experimental Example 4: Characterization of α-synuclein capture performance of Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1

[0060] The binding ability of exosomes to pathological α-synuclein was quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). Pre-formed α-synuclein fibers were coated onto 96-well plates and incubated overnight at 4°C, followed by blocking of non-specific sites with 5% BSA solution. A series of exosomes at concentration gradients (0-100 μg / mL) measured by BCA (Example 1, Comparative Example 2, and Comparative Example 4) were added to the wells and incubated at room temperature for 2 hours to allow for sufficient binding of the exosomes to the immobilized α-synuclein. After washing, mouse anti-human CD63 primary antibody and horseradish peroxidase (HRP)-labeled secondary antibody were added sequentially for incubation. Finally, TMB chromogenic substrate was added, and the reaction was terminated with 2M H2SO4. The absorbance (OD value) of each well was immediately measured at 450 nm using a microplate reader.

[0061] The results are as follows Figure 4 As shown, the absorbances of Example 1, Comparative Example 2, and Comparative Example 4 were 1.3, 0.1, and 0.18 au, respectively. This is because the LK5 peptide can bind to α-synuclein, while the unmodified LK5 peptides in Comparative Examples 2 and 4 have difficulty binding to α-synuclein. In summary, the exosomes prepared in Example 1 exhibit good binding efficiency to α-synuclein.

[0062] Experimental Example 5: Brain distribution characterization of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1.

[0063] To investigate the distribution of exosomes prepared in Example 1 and Comparative Example 4 in the cerebellum after intranasal administration, this study employed an in vivo real-time fluorescence imaging system. Mice were randomly divided into three groups, receiving intranasal administration of DiR solution (control group), DiR-labeled Exosome 1, and DiR-labeled Exosome 4, respectively. The DiR dosage was 0.25 mg / kg body weight for all groups. All mice were anesthetized by intraperitoneal injection of chloral hydrate prior to administration. Intranasal administration was performed by gently inserting a polyethylene PE10 catheter connected to a microsyringe into the nostril to a depth of approximately 10 mm. Whole-body fluorescence imaging was performed on anesthetized mice at 0.5, 1, 2, 4, and 6 hours post-administration. To further verify the brain targeting of NP and Lf-NP, mice were sacrificed 4 hours post-administration, and major organs such as the whole brain, heart, liver, spleen, lungs, and kidneys were isolated. Fluorescence images of each organ were acquired using an in vivo imaging system.

[0064] The results are as follows Figure 5 As shown, no signal was observed in the control group, and the fluorescence signals of Example 1 and Comparative Example 4 were detected only in the brain, with a strong signal in the brains of mice in the Example 1 group. This is because the exosomes of Example 1, modified with the LK5 peptide, achieved specific capture of α-synuclein aggregates, highlighting the preferential accumulation of the exosomes prepared in Example 1 in the brain, indicating that after intranasal administration, Example 1 targets the brain and is poorly absorbed in other non-target organs. Therefore, the exosomes prepared in Example 1 have a good brain-targeting effect.

[0065] Experimental Example 6: In vivo biosafety characterization of the Angiopep-2 / LK5 modified drug-loaded exosomes prepared in Example 1.

[0066] To evaluate the Angiopep-2 / LK5-modified drug-loaded exosomes prepared in Example 1, we euthanized mice on the last day of the experiment and harvested vital organs (spleen, liver, heart, kidney, lung, and brain). After H&E staining, the vital organs were fixed, embedded, and subjected to histological examination. In any treatment group, such as Figure 6 As shown, we did not detect necrosis or significant tissue damage (including apoptosis, nuclear cleavage, pyknosis, or inflammatory cell infiltration). This indicates that the dosage used was safe and there was no apparent toxicity.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. Angiopep-2 / LK5 modified targeted delivery drug-loaded exosomes, characterized in that, The exosome comprises human umbilical cord mesenchymal stem cell-derived exosome, rapamycin, CRISPR-Cas9 / sgRNA complex, MMP-2 responsive lipid material, and a ligand for surface modification of the exosome comprises Angiopep-2 peptide combined with LRP1 receptor and LK5 peptide specific to capture alpha-synuclein; The mass ratio of the human umbilical cord mesenchymal stem cell-derived exosome, rapamycin, CRISPR-Cas9 / sgRNA complex, Angiopep-2 peptide, LK5 peptide and MMP-2 responsive lipid material is 1:10:50:0.24:0.08:0.0625; The MMP-2 responsive lipid material is DSPE-PEG-GPLGVRGD.

2. A method of preparing Angiopep-2 / LK5 modified targeted delivery drug-loaded exosomes according to claim 1, characterized in that, The preparation method comprises the following steps: (1) Preparation of exosome HUC-MSCs-EXO: The exosome HUC-MSCs-EXO is extracted from the culture supernatant of human umbilical cord mesenchymal stem cells by differential ultracentrifugation method; the specific steps are as follows: differential centrifugation for 1 h at 4 DEG C to remove cells and debris; finally, ultracentrifugation at 120000xg for 70 min, collect the exosome precipitate, resuspend with PBS, and determine the protein concentration by BCA method, adjust to 1 mg / mL, and store at -80 DEG C for standby; (2) Preparation of drug-loaded exosome HUC-MSCs-EXO@R-SNCA: rapamycin is loaded by pH gradient method: the exosome HUC-MSCs-EXO is mixed with rapamycin, and incubated in HEPES buffer at pH 7.4 for 30 min; then, the pH of the system is adjusted to 5.0, heated in a 45 DEG C water bath for 15 min to induce high-efficiency drug loading; finally, the pH is restored to 7.4 to obtain rapamycin-loaded exosome; then, in order to introduce the CRISPR-Cas9 / sgRNA complex targeting SNCA gene into the rapamycin-loaded exosome, an electroporation method is used for loading, wherein the feeding weight ratio of the complex to the rapamycin-loaded exosome is 50:1, and after transfection, the temperature is restored to room temperature for incubation for 30 min, to obtain the drug-loaded exosome HUC-MSCs-EXO@R-SNCA; (3) Angiopep-2 / LK5 co-modification and MMP-2-responsive membrane embedding: adopt carbodiimide chemical cross-linking method, covalently modify Angiopep-2 peptide on the surface of HUC-MSCs-EXO@R-SNCA at 100 μM, wherein the mass ratio of Angiopep-2 peptide and HUC-MSCs-EXO@R-SNCA is 0.24:1, react in MES buffer solution at pH 6.0 for 2 h, and then use PBS to dialyze to remove unreacted cross-linking agent and free peptide segment, to obtain HUC-MSCs-EXO@R-SNCA@Angiopep-2; similarly, use carbodiimide chemical cross-linking method to covalently connect D-type LK5 peptide to HUC-MSCs-EXO@R-SNCA@Angiopep-2, wherein the mass ratio of D-type LK5 peptide and HUC-MSCs-EXO@R-SNCA@Angiopep-2 is 0.08:1, to obtain HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5; finally, mix MMP-2-responsive lipid material DSPE-PEG-GPLGVRGD with HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5, and embed it into the exosome membrane by ultrasonic co-extrusion method, to obtain exosomes HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5@DPG with MMP-2 enzyme triggered drug release function.

3. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of, The exosomes HUC-MSCs-EXO in step (2) are mixed with rapamycin at a mass ratio of 1:

10.

4. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of: The molar ratio of CRISPR-Cas9 to sgRNA in the CRISPR-Cas9 / sgRNA complex in step (2) is 1:

1.

5. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of: The sequence of the MMP-2-responsive lipid material in step (3) is GPLGVRGD.

6. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of: The concentration of the Angiopep-2 peptide in step (3) is 100 μM.

7. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of: The sequence of the D-type LK5 peptide in step (3) is D-LKLLLKL, and the working concentration is 100 μM.

8. The method of claim 2, wherein the exosome is prepared by the method comprising the steps of: The molar ratio of the MMP-2-responsive lipid material to HUC-MSCs-EXO@R-SNCA@Angiopep-2 / LK5 in step (3) is 1:

20.

9. Use of the exosome of claim 1 in the manufacture of a product for the treatment of a neurodegenerative disease, characterized in that, The product use method is nasal administration.

10. Use of the exosome of claim 9 in the manufacture of a product for the treatment of a neurodegenerative disease, characterized in that, The neurodegenerative disease is Parkinson's disease.

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