A brain-targeting delivery system of scutellarein based on angiopep-2 modified milk exosome, and a preparation method and application thereof

Abstract

The application relates to the technical field of medicines, and particularly discloses a brain-targeting delivery system of scutellarein loaded in milk exosome based on Angiopep-2 modification as well as a preparation method and application thereof. The delivery system comprises a carrier, which is the milk exosome modified by Angiopep-2, and the mass ratio of the total protein amount of the milk exosome to Angiopep-2 is 10:1-2; and a load, which is the neuroprotective agent scutellarein. The targeting delivery system has good biocompatibility and low immunogenicity, can improve the stability and bioavailability of scutellarein, can penetrate the blood-brain barrier, and can reach the lesion site of ischemic stroke. Furthermore, scutellarein is released in the brain, is taken by neurons, microglial cells and the like, plays the roles of removing active oxygen and regulating microglial cell polarization, and maximally improves the curative effect of anti-ischemic stroke.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine, its preparation method, and its application. Background Technology

[0002] Ischemic stroke is a severe neurological disease caused by cerebral vascular occlusion, leading to cerebral ischemia and hypoxia. It is characterized by high incidence, high disability rate, and high mortality rate. Currently, the core strategy for clinical treatment is to restore blood flow by recanalizing the blood vessels within a limited time window. However, the reperfusion process itself may trigger more complex "cerebral ischemia-reperfusion injury," which involves a cascade of multiple pathological mechanisms, including oxidative stress, neuroinflammation, and excitotoxicity. Traditional neuroprotective strategies targeting single pathological stages have repeatedly failed to achieve the expected results in clinical trials. Furthermore, the presence of the blood-brain barrier severely restricts the entry of most therapeutic drugs into brain tissue, resulting in insufficient drug concentrations at the lesion site. Therefore, developing intelligent delivery systems that can efficiently penetrate the blood-brain barrier and simultaneously act on multiple post-stroke injury stages has become an urgent need in current stroke treatment research.

[0003] Among numerous candidate drugs for stroke treatment, natural products have attracted considerable attention due to their multi-target effects and relatively good safety profile. Erigeron breviscapus extract, a major flavonoid component extracted from the traditional Chinese medicine *Erigeron breviscapus*, has been shown to possess significant antioxidant, anti-inflammatory, and neuroprotective activities. It can alleviate oxidative stress and regulate the reparative polarization of microglia, demonstrating promising potential for stroke treatment. However, erigeron breviscapus extract suffers from biopharmaceutical defects such as low oral bioavailability, rapid in vivo metabolism, poor stability, and difficulty in freely crossing the blood-brain barrier, severely limiting its clinical efficacy. Therefore, there is an urgent need to develop a novel carrier that can overcome these defects and achieve efficient brain-targeted delivery of erigeron breviscapus extract.

[0004] Exosomes, as endogenous nanovesicles, possess excellent biocompatibility, low immunogenicity, and the potential to naturally cross biological barriers, making them highly promising drug delivery carriers. Milk-derived exosomes, in particular, offer advantages such as abundant raw materials, simple preparation, and high safety, making them suitable for large-scale production and application. However, natural exosomes lack the ability to actively target specific diseased tissues and are easily cleared by the mononuclear phagocytic system in vivo, resulting in limited accumulation efficiency at lesion sites. To achieve precise delivery to ischemic brain regions, brain-targeting modification of the carrier is a key strategy. Angiopep-2, a targeting peptide that specifically binds to the LRP1 receptor (highly expressed on the blood-brain barrier), has been widely used in the brain-targeting engineering of delivery systems.

[0005] Based on this technical background, this invention studies a brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a brain-targeted delivery system for ligustrazine loaded on milk exosomes modified with Angiopep-2, along with its preparation method and applications. This delivery system utilizes the brain-targeting function of Angiopep-2 to guide the carrier across the blood-brain barrier, while leveraging the natural delivery characteristics of milk exosomes to efficiently load and protect ligustrazine, ultimately achieving precise enrichment of the drug in ischemic brain regions. This overcomes the shortcomings of natural ligustrazine, such as difficulty in penetrating the blood-brain barrier and insufficient concentration at lesion sites.

[0007] To achieve the above objectives, a first aspect of the present invention provides a brain-targeted delivery system based on Angiopep-2-modified milk exosomes loaded with ligustrazine, comprising: The carrier is a milk exosome modified with Angiopep-2, wherein the total protein content of the milk exosome is in a mass ratio of 10:1-2 to the Angiopep-2. The load is the neuroprotective agent, scutellarin.

[0008] A second aspect of the present invention provides a method for preparing the above-described delivery system, comprising: Milk exosomes were purified by differential centrifugation and sucrose density gradient centrifugation. The polyethylene glycol crosslinking agent Mal-PEG3500-SCM was dissolved in N,N-dimethylformamide and then added to the milk exosome suspension to obtain a mixed system; Under nitrogen protection, the mixture was stirred and reacted in phosphate buffer at room temperature, and then added to phosphate buffer containing Angiopep-2 to continue the reaction, removing free Mal-PEG3500-SCM and Angiopep-2, to obtain Angiopep-2 modified milk exosomes. Dissolve scutellarin in phosphate buffer and mix it with the Angiopep-2 modified milk exosomes to remove free scutellarin, thus obtaining the delivery system.

[0009] A third aspect of the present invention provides the application of the above-described delivery system, or the delivery system prepared by the above-described preparation method, in a drug for treating ischemic stroke.

[0010] The beneficial effects of this invention include: (1) The brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with scutellarin proposed in this invention utilizes the brain-targeting function of Angiopep-2 to guide the carrier to cross the blood-brain barrier. At the same time, it takes advantage of the natural delivery characteristics of milk exosomes to efficiently load and protect scutellarin, and finally achieves precise enrichment of the drug in the ischemic brain region, overcoming the defects of natural drug scutellarin being difficult to penetrate the blood-brain barrier and having insufficient concentration at the lesion site.

[0011] (2) The brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine uses naturally derived milk exosomes as carriers, which has excellent biocompatibility and low immunogenicity, and can effectively prolong the circulation time of drugs in the body; at the same time, by modifying the surface with the brain-targeting peptide Angiopep-2, the system can actively recognize and cross the blood-brain barrier to achieve precise delivery to ischemic brain lesions.

[0012] (3) The brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine is proposed in this invention. The lipid bilayer structure of milk exosomes can stably load ligustrazine, avoid its premature degradation, and significantly increase the concentration of the drug in the lesion area. Ligustrazine released in ischemic brain tissue can simultaneously exert multiple synergistic effects of scavenging free radicals, inhibiting neuroinflammation and regulating microglial cell polarization, thereby jointly intervening in the oxidative stress and inflammatory cascade response in ischemia-reperfusion injury. Through the above mechanism, the delivery system can significantly enhance the neuroprotective effect while reducing systemic side effects, providing a new, efficient, targeted and safe treatment strategy for ischemic stroke.

[0013] (4) The brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine proposed in this invention can not only improve the bioavailability of ligustrazine, but also achieve more effective intervention on ischemia-reperfusion injury through multiple synergistic mechanisms such as antioxidation, anti-inflammation and regulation of microglia, providing a new technical solution for targeted treatment of ischemic stroke.

[0014] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0015] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0016] Figure 1This is a transmission electron microscope (TEM) schematic diagram and particle size diagram of milk exosome particles in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine, as proposed in this invention.

[0017] Figure 2 This is a schematic diagram showing the changes in particle size, potential, and exosome surface marker proteins in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine.

[0018] Figure 3 This is a schematic diagram of in vitro release in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine proposed in this invention.

[0019] Figure 4 This is a schematic diagram illustrating the uptake of ligustrazine in bEnd.3 cells in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine proposed in this invention.

[0020] Figure 5 This is a schematic diagram illustrating the blood-brain barrier penetration efficiency in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine, as proposed in this invention.

[0021] Figure 6 This is a schematic diagram of the distribution of ligustrazine in the mouse brain in a specific embodiment of the brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine proposed in this invention. Detailed Implementation

[0022] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0023] This invention provides a brain-targeted delivery system for ligustrazine-based milk exosomes modified with Angiopep-2, comprising: The carrier is milk exosomes modified with Angiopep-2, and the total protein content of the milk exosomes to the mass ratio of Angiopep-2 is 10:1-2. The load is the neuroprotective agent, scutellarin.

[0024] This invention utilizes the brain-targeting function of Angiopep-2 to guide the carrier across the blood-brain barrier, while taking advantage of the natural delivery characteristics of milk exosomes to efficiently load and protect scutellarin, ultimately achieving precise enrichment of the drug in the ischemic brain region, overcoming the shortcomings of the natural drug scutellarin, which is difficult to penetrate the blood-brain barrier and has insufficient concentration at the lesion site.

[0025] According to the present invention, milk exosomes are purified by differential centrifugation combined with sucrose density gradient centrifugation, and have a typical vesicle structure with an average particle size of 130-140 nm and a particle size of 160-170 nm after targeting.

[0026] This invention uses milk exosomes of natural origin as carriers, which have excellent biocompatibility and low immunogenicity, and can effectively prolong the circulation time of drugs in the body; at the same time, by modifying the surface with the brain-targeting peptide Angiopep-2, the system can actively recognize and cross the blood-brain barrier to achieve precise delivery to ischemic brain lesions.

[0027] According to the present invention, Angiopep-2 is a ligand for the low-density lipoprotein LRP1 receptor and is coupled to milk exosomes via a polyethylene glycol crosslinking agent comprising maleimide groups and N-hydroxysuccinimide ester groups.

[0028] In this invention, the lipid bilayer structure of milk exosomes can stably load ligustrazine, preventing its premature degradation and significantly increasing the drug's concentration in the lesion area. Ligustrazine released in ischemic brain tissue can simultaneously exert multiple synergistic effects, including scavenging free radicals, inhibiting neuroinflammation, and regulating microglial cell polarization. This allows for joint intervention against the oxidative stress and inflammatory cascade in ischemia-reperfusion injury. Through the above mechanism, this delivery system significantly enhances neuroprotective efficacy while reducing systemic side effects, providing a highly efficient, targeted, and safe new treatment strategy for ischemic stroke.

[0029] According to the present invention, the polyethylene glycol crosslinking agent is Mal-PEG3500-SCM; The neuroprotective agent scutellarin contains flavonoid natural active ingredients.

[0030] According to the present invention, the average particle size of the delivery system is 150-200 nm.

[0031] This invention not only improves the bioavailability of ligustrazine, but also achieves more effective intervention in ischemia-reperfusion injury through multiple synergistic mechanisms such as antioxidation, anti-inflammation and regulation of microglia, providing a new technical solution for targeted therapy of ischemic stroke.

[0032] The present invention also provides a method for preparing the above-mentioned delivery system, comprising: Milk exosomes were purified by differential centrifugation and sucrose density gradient centrifugation. The polyethylene glycol crosslinking agent Mal-PEG3500-SCM was dissolved in N,N-dimethylformamide and then added to the milk exosome suspension to obtain a mixed system; Under nitrogen protection, the mixture was stirred in phosphate buffer at room temperature and then added to phosphate buffer containing Angiopep-2 to continue the reaction, removing free Mal-PEG3500-SCM and Angiopep-2, to obtain Angiopep-2 modified milk exosomes. Dissolve scutellarin in phosphate buffer and react it with Angiopep-2 modified milk exosomes to remove free scutellarin, thus obtaining the delivery system.

[0033] According to the present invention, the pH value of the phosphate buffer solution is 7.2-7.6; The stirring reaction at room temperature takes 16-20 hours. The reaction continues for 2-6 hours; The molar ratio of Mal-PEG3500-SCM to Angiopep-2 is 1:1-1.5; The mixing reaction was carried out at room temperature under magnetic stirring for 20-40 minutes.

[0034] According to the present invention, the removal of free Mal-PEG3500-SCM and Angiopep-2, as well as the removal of free scutellarin, are both carried out by transferring the corresponding reaction solutions into an ultrafiltration tube and centrifuging them.

[0035] The present invention also provides the application of the above-described delivery system, or the delivery system prepared by the above-described preparation method, in drugs for treating ischemic stroke.

[0036] According to the present invention, the drug is used to reduce oxidative stress and neuroinflammation in cerebral ischemia-reperfusion injury.

[0037] The present invention will be described in more detail below through embodiments.

[0038] Example 1 This embodiment provides a brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine. The delivery system consists of milk exosomes loaded with ligustrazine and the brain-targeting peptide Angiopep-2 modified on their surface. It can achieve efficient brain-targeted delivery of ligustrazine and simultaneously exert multiple neuroprotective effects such as anti-oxidation, anti-inflammation and regulation of microglia polarization. The specific implementation plan is as follows: The delivery system uses Angiopep-2 modified milk exosomes as carriers to carry the neuroprotective agent ligustrazine; the Angiopep-2 modified milk exosomes are prepared by coupling with the chemical cross-linking agent Mal-PEG3500-SCM. Among them, the neuroprotective agent scutellarin is a natural active ingredient of flavonoids; The brain-targeting peptide Angiopep-2 is a ligand for the low-density lipoprotein LRP1 receptor; Milk exosomes were obtained by differential centrifugation combined with sucrose density gradient purification. These milk exosomes were used to carry the neuroprotective agent ligustrazine. The milk exosomes have a typical vesicle structure with an average particle size of about 137 nm, which increases to about 165 nm after targeting.

[0039] In this embodiment, the method for preparing milk exosomes includes the following steps: Fresh milk is first centrifuged at 2000 × g for 20 min to remove impurities such as fat globules and larger cell debris. The resulting supernatant is then centrifuged at 5000 × g for 30 min to remove organelle debris, larger vesicles and other impurities. The resulting supernatant is then centrifuged at 10000 × g for 90 min to further remove large protein aggregates. The supernatant obtained in this centrifugation step is the final supernatant. The final supernatant was filtered through a 0.45 μm aqueous microporous membrane. The filtrate was centrifuged at 100,000 × g for 1-2 h. The precipitate was collected as a crude extract rich in milk exosomes and resuspended in an appropriate amount of phosphate buffer (PBS). To further purify, the resuspension was transferred to a gradient of sucrose solutions with concentrations of 30% (w / v), 45% (w / v), and 60% (w / v) from top to bottom, and then centrifuged at 100,000 × g for 2 h. After centrifugation, the liquid between the 30% and 45% sucrose layers was collected, diluted 1-2 times with PBS, and centrifuged at 100,000 × g for 1 h to wash away the sucrose. The resulting precipitate was the milk exosome. The precipitate was resuspended with an appropriate amount of PBS to obtain the milk exosome stock solution, which was stored at -80°C for experimental use. In this embodiment, the method for preparing Angiopep-2 modified milk exosomes includes the following steps: Dissolve 1.68 mg Mal-PEG3500-SCM in 200 μL N,N-dimethylformamide and add it to MExo suspension containing 10 mg total protein; The mixture was reacted with magnetic stirring at room temperature in 20 mL phosphate (PBS) buffer (pH 7.4) for 18 hours. Then, 1 mg of Angiopep-2 dissolved in 1 mL of PBS was added, and the reaction was continued for 4 hours. The entire reaction was carried out under nitrogen protection. After the reaction was completed, the solution was transferred to a 100 kDa ultrafiltration tube and centrifuged at 5000 × g for 10 min to remove free Mal-PEG3500-SCM and Angiopep-2, thus obtaining Angiopep-2-grafted milk exosomes. In this embodiment, the preparation method of the Angiopep-2 modified milk exosome brain-targeting delivery system involves first coupling milk exosomes with the brain-targeting peptide Angiopep-2, and then loading them with the neuroprotective agent ligustrazine to prepare drug-loaded nanoparticles. Specifically, this includes: Dissolve 10 mg of ligustrazine in 10 mL of PBS, mix it with Angiopep-2-grafted milk exosomes (total protein 10 mg), stir magnetically at room temperature for 30 min, transfer to a 100 kDa ultrafiltration tube, centrifuge at 5000 × g for 10 min to remove free ligustrazine, and obtain the delivery system (drug-loaded nanoparticles).

[0040] Test Example 1 This test case describes the preparation and characterization of milk exosomes prepared in Example 1, specifically including: The morphology of the milk exosomes prepared in Example 1 was characterized by dynamic light scattering particle size analyzer (DLS) and high-resolution transmission electron microscopy (TEM); Figure 1 The left image shows milk exosomes observed under a transmission electron microscope, exhibiting a typical vesicle appearance, uniform size, and a particle size of approximately 100 nm; DLS measurements of the hydrated particle size of milk exosomes are as follows... Figure 1 The right image shows approximately 137 nm.

[0041] Test Example 2 This test case characterizes the targeted delivery system prepared in Example 1, specifically including: The particle size, potential, and exosome surface protein markers of the delivery system (drug-loaded nanoparticles) were characterized, such as... Figure 2 In (1), the particle size of milk exosomes slightly increased after loading with ligustrazine, and the particle size increased to about 165 nm after Angiopep-2 modification; since Angiopep-2 is positively charged in PBS (pH 7.4), the zeta potential of the resulting co-delivery system increased to -6.9 mV compared to -12.7 mV of milk exosomes. Figure 2 As shown in (2) of the text; Figure 2 (3) shows that milk exosomes still contain abundant exosome-related proteins after being coupled with the targeting peptide and loaded with drugs, indicating that drug loading and targeting peptide loading have almost no effect on the structure of exosomes.

[0042] Test Example 3 This test case examines the in vitro release behavior of the targeted delivery system prepared in Example 1, specifically including: The in vitro release behavior of SCU@AMExo was investigated using dialysis. Two mL of the 0.5 mg / mL SCU@AMExo delivery system sample was placed in a dialysis bag with a molecular weight cutoff of 3,000 kDa, sealed, and immersed in 50 mL of artificial cerebrospinal fluid (aCSF) release medium at pH 7.4. The release system was kept at a constant temperature of 37°C and gently oscillated at 100 rpm to simulate the in vivo physiological environment and maintain the leak conditions. At preset time points of 25 min, 50 min, 1 h, 2 h, 6 h, 8 h, and 12 h, 1.0 mL of sample solution was accurately transferred from the external medium, and an isothermal and equal volume of fresh aCSF was immediately added to maintain a constant total volume. The collected samples were filtered through a 0.22 μm microporous membrane and then quantitatively analyzed for the concentration of scutellarin using high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 reversed-phase column (4.6 × 150 mm, 5...). The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution (25:75, v / v), the flow rate was 1.0 mL / min, the column temperature was 30℃, the detection wavelength was 335 nm, and the injection volume was 20 μL; the sample was operated in triplicate, and the cumulative release percentage of scutellarin (%) was calculated; Figure 3 As shown, the cumulative release rate of ligustrazine reached 80% within the first eight hours, indicating that under physiological conditions, ligustrazine can be successfully released from milk exosomes and exert a neuroprotective effect.

[0043] Test Example 4 This test case utilizes bEnd.3 cells to conduct an uptake experiment of the targeted delivery system to evaluate its cellular targeting ability, specifically including: Coumarin-6 was loaded onto milk exosomes modified with Angiopep-2 (C6@AMExo), with untargeted milk exosomes loaded with coumarin-6 (C6@MExo) serving as a control; logarithmically cultured bEnd.3 cells were cultured at 2×10⁻⁶ cells per cell line. 4Cells were evenly seeded at a density of 100 μg / well in 24-well plates. After 24 hours of culture, C6@AMExo and C6@MExo were diluted to coumarin-6 concentrations of 1, 2, and 4 μg / mL in DMEM medium without serum and antibiotics. 500 μL of each solution was added to each well of the 24-well plates and incubated at 37°C for 2 h. After incubation, the cells were washed three times with PBS, and 200 μL of 4% (w / v) paraformaldehyde solution was added to each well for fixation at room temperature for 15 min. After fixation, the cells were washed three times with PBS, and 100 μL of DAPI staining solution was added to each well for nuclear staining at room temperature for 10 min. After incubation, the staining solution was discarded, and the cells were washed three times with PBS and photographed under a fluorescence microscope. Cell uptake results are shown below. Figure 4 As shown, the uptake of both types of exosomes by bEnd.3 cells was concentration-dependent; increased coumarin-6 concentration significantly enhanced intracellular green fluorescence. At the same concentration, the C6@AMExo group with the Angiopep-2 targeting peptide exhibited stronger green fluorescence than the C6@MExo group. This indicates that modification with the Angiopep-2 targeting peptide can significantly promote the uptake of exosomes by bEnd.3 cells, thereby promoting their intracellular distribution.

[0044] Test Example 5 This test case demonstrates the blood-brain barrier penetration efficiency of Angiopep-2 modified milk exosomes (C6@AMExo in Test Case 4), specifically including: bEnd.3 cells were loaded at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 1.0 μm in Transwell cell culture chambers (pore size 1.0 μm, surface area 0.33 cm²). 2 Cells were cultured in 24-well plates for 7 days using standard methods. Then, both chambers were replaced with DMEM medium containing hydrocortisone (1 μM). After 3 days of culture, cell junctions were tightly bound, forming a dense monolayer. Monolayer integrity was assessed by measuring transepithelial / transendothelial electrical resistance (TEER) using a Millicell@-ERS voltmeter. A TEER value higher than 200 Ω·cm was considered optimal. 2 The monolayer of cells was used as a blood-brain barrier model for subsequent research. After establishing the model, C6@MExo and C6@AMExo (10 μg / mL) prepared in DMEM medium were added, and DMEM medium was added to another 24-well plate. The Transwell chamber was then transferred into the plate and incubated at 37 °C. At 2 h, 4 h, and 6 h, the lower chamber culture medium was aspirated, and the absorbance was measured at an emission wavelength of 504 nm using a microplate reader to investigate the LPR-1-mediated in vitro blood-brain barrier penetration of C6@AMExo. Figure 5 As shown, at each time point, the C6@AMExo group exhibited stronger blood-brain barrier permeability than the unmodified Angiopep-2 C6@MExo group. After 8 hours, the blood-brain barrier permeability of C6@AMExo reached 11.09%, which was 100% higher than that of the C6@MExo group. This indicates that in vitro, Angiopep-2 modified milk exosomes can not only be successfully taken up by bEnd.3 cells, but also successfully cross the blood-brain barrier through the secretion of bEnd.3 cells.

[0045] Test Example 6 This test case compares the distribution of the targeted delivery system (SCU@AMExo) and the non-targeted delivery system (SCU@MExo) prepared in the mouse brain, specifically including: A mouse MCAO model was constructed using the Dir-labeled targeted scutellarin delivery system (SCU@AMExo) and the non-targeted delivery system (SCU@MExo) prepared according to the embodiments. One hour after reperfusion, Dir-labeled SCU@AMExo and SCU@MExo were injected via the tail vein. Two and eight hours after administration, the distribution of these substances in the major organs of the mice was observed using a small animal in vivo imaging system. After eight hours of in vivo circulation, the mice were sacrificed, and the major tissues (heart, liver, spleen, lung, kidney, and brain) were removed. The fluorescence distribution of the isolated tissues and mouse brain slices was observed using an in vivo imaging system. Figure 6 As shown, the SCU@AMExo group exhibited higher efficiency in brain-targeted delivery compared to the SCU@MExo group; fluorescence in the brains of mice in the SCU@MExo group decreased over time and became very weak after 8 hours, while the SCU@AMExo group maintained stronger fluorescence; mice were sacrificed at 8 hours for fluorescence analysis of isolated tissues; the SCU@MExo group accumulated more in other tissues than the SCU@AMExo group, but the fluorescence intensity in the brain was higher in the SCU@AMExo group than in the SCU@MExo group; these results demonstrate that the targeted delivery system prepared in Example 1 has the ability to actively target brain tissue.

[0046] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A brain-targeted delivery system based on Angiopep-2 modified milk exosomes loaded with ligustrazine, characterized in that, include: The carrier is a milk exosome modified with Angiopep-2, wherein the total protein content of the milk exosome is in a mass ratio of 10:1-2 to the Angiopep-2. The load is the neuroprotective agent, scutellarin.

2. The delivery system according to claim 1, characterized in that, The milk exosomes were purified by differential centrifugation combined with sucrose density gradient centrifugation and had a typical vesicle structure with an average particle size of 130-140 nm and a particle size of 160-170 nm after targeting.

3. The delivery system according to claim 1, characterized in that, The Angiopep-2 is a ligand for the low-density lipoprotein LRP1 receptor and is coupled to the milk exosomes via a polyethylene glycol crosslinking agent containing maleimide and N-hydroxysuccinimide ester groups.

4. The delivery system according to claim 4, characterized in that, The polyethylene glycol crosslinking agent is Mal-PEG3500-SCM; The neuroprotective agent, scutellarin, contains flavonoid natural active ingredients.

5. The delivery system according to any one of claims 1-4, characterized in that, The average particle size of the delivery system is 150-200 nm.

6. A method for preparing the delivery system according to any one of claims 1-5, characterized in that, include: Milk exosomes were purified by differential centrifugation and sucrose density gradient centrifugation. The polyethylene glycol crosslinking agent Mal-PEG3500-SCM was dissolved in N,N-dimethylformamide and then added to the milk exosome suspension to obtain a mixed system; Under nitrogen protection, the mixture was stirred and reacted in phosphate buffer at room temperature, and then added to phosphate buffer containing Angiopep-2 to continue the reaction, removing free Mal-PEG3500-SCM and Angiopep-2, to obtain Angiopep-2 modified milk exosomes. Dissolve scutellarin in phosphate buffer and mix it with the Angiopep-2 modified milk exosomes to remove free scutellarin, thus obtaining the delivery system.

7. The preparation method according to claim 6, characterized in that, The pH value of the phosphate buffer solution is 7.2-7.6; The stirring reaction at room temperature is carried out for 16-20 hours. The continued reaction time is 2-6 hours; The molar ratio of Mal-PEG3500-SCM to Angiopep-2 is 1:1-1.5; The mixing reaction is carried out at room temperature and under magnetic stirring for 20-40 minutes.

8. The preparation method according to claim 6, characterized in that, The removal of free Mal-PEG3500-SCM and Angiopep-2, as well as the removal of free scutellarin, involved transferring the corresponding reaction solutions to an ultrafiltration tube and centrifuging them.

9. The use of a delivery system according to any one of claims 1-5, or a delivery system prepared by the preparation method according to any one of claims 6-8, in a drug for treating ischemic stroke.

10. The application according to claim 9, characterized in that, The drug is used to reduce oxidative stress and neuroinflammation in cerebral ischemia-reperfusion injury.

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