An engineered exosome dual drug-loaded system loaded with ergosterol and a preparation method and application thereof

Abstract

The scheme provides a kind of engineered exosome double drug delivery system loaded with ergosterol and its preparation method and application, belongs to the field of nanomaterials and nanobiomedicine, based on the innovative ROS-responsive nanomaterial CHPG design, load the active ingredient ergosterol to play the role of treatment and repair, which realizes the blood-brain barrier penetration and precise drug release in lesion area through targeted functional modification;It is wrapped in the nasal mucosa-derived stem cell exosome to form a drug delivery system Exo@Erg-CHPG-M, which combines the natural biological characteristics of exosome and the precise targeting advantage of nanomicelle, significantly improves the drug delivery efficiency, in vitro and animal model verification show that, the system can improve the neurological impairment and brain tissue injury after ischemic stroke by synergistically regulating inflammation and neural repair pathways, the scheme provides a new technical scheme for the precise treatment of central nervous system diseases such as cerebral stroke.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and nanobiomedicine, specifically relating to an engineered exosome dual-drug delivery system loaded with ergosterol, its preparation method, and its application. Background Technology

[0002] Ischemic stroke (IS) is an acute cerebrovascular disease caused by cerebral artery occlusion or stenosis, leading to hypoxic necrosis of brain tissue. It has a high disability rate and complex pathological mechanisms. Its pathological process involves a multi-stage cascade reaction: early blood flow interruption triggers neuronal energy metabolism exhaustion and excitotoxicity, followed by mitochondrial dysfunction and reactive oxygen species (ROS) bursts; during perfusion, free radical overload exacerbates blood-brain barrier (BBB) ​​damage, neuroinflammatory activation, and gut-brain axis dysregulation. The latter manifests as gut microbiota dysbiosis, inactivation of tight junction proteins, and the entry of gut-derived pro-inflammatory factors (such as LPS and TNF-α) into the brain via the vagus nerve or circulatory system, forming a positive feedback loop of "inflammation-secondary brain injury." These pathological characteristics make the efficacy of IS treatment highly dependent on the targeted delivery capability of drugs, multi-pathway synergistic intervention, and overall regulation of the neuro-immune-metabolic network.

[0003] In recent years, nanomedicine delivery systems targeting neuroprotection and regeneration have become a research hotspot. For example, drugs can be encapsulated in ROS-responsive materials to improve lesion accumulation. However, practical applications still face limitations: Although natural active ingredients such as ergosterol (Erg) show potential in inhibiting neuroinflammation and promoting synaptic regeneration, their clinical application is limited by low blood-brain barrier (BBB) ​​penetration and insufficient targeting of lesion areas. Existing delivery systems, such as traditional nanomicelles, can partially improve drug accumulation through passive targeting, but the carrier itself lacks responsiveness to the post-stroke oxidative stress microenvironment, making intelligent drug release difficult. Furthermore, hydrophobic drugs like ergosterol are prone to burst release or leakage due to carrier instability. In addition, single drug delivery modalities cannot synergistically regulate the complex post-stroke pathological network. For example, the interaction between central nervous system inflammation and gut-brain axis disorders (intestinal barrier disruption, dysbiosis) is often overlooked, and conventional delivery systems lack multi-target intervention capabilities in this regard. Therefore, there is an urgent need to develop a novel drug delivery system that combines efficient targeting, multi-mechanism synergy, and functional carriers to overcome existing technological bottlenecks and provide a better solution for neurological function repair after ischemic stroke. Summary of the Invention

[0004] To address the issues of low targeted delivery efficiency and insufficient brain-gut axis regulation in existing neurorehabilitation treatments for ischemic stroke, this solution provides an engineered exosome drug delivery system based on the brain-gut axis, along with its preparation method and application. Addressing the problems of low blood-brain barrier penetration, poor microenvironment responsiveness, and insufficient synergistic intervention of the brain-gut axis in neurorehabilitation for ischemic stroke, this system proposes an engineered exosome-micelle hybrid system. This system combines the targeted drug release characteristics of ROS-responsive micelles with the brain-gut axis regulation function of exosomes, effectively solving the shortcomings of traditional drugs' weak ability to cross the blood-brain barrier and their limited therapeutic scope.

[0005] To achieve the above objectives, this solution first provides an engineered exosome dual-drug delivery system loaded with ergosterol, comprising ergosterol, caffeoyl phenylboronic acid ester, glycerol, and nasal mucosal stem cell-derived exosomes (EMSC-exo; Exo). The caffeoyl phenylboronic acid ester and glycerol encapsulate ergosterol to form ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles, and further loads the ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles into nasal mucosal stem cell-derived exosomes.

[0006] Preferably, the engineered exosome dual-drug delivery system has a potential of -27.68±0.62mV, a particle size of 137.26±2.61nm, and a PDI of 0.219±0.009.

[0007] Based on a general inventive concept, this invention also provides a method for preparing an engineered exosome dual-drug delivery system loaded with ergosterol, comprising the following steps:

[0008] (1) Preparation of ergosterol-caffeic acid phenylboronic acid ester-glycerol polymer micelles

[0009] Methyl caffeate and phenylboronic acid were dissolved in ethyl acetate and stirred at 70°C. After the reaction was completed, intermediate 1 was formed. Intermediate 1 was added to an aqueous methanol solution and stirred to dissolve. After complete dissolution, sodium hydroxide was added and stirred at room temperature. After the reaction was completed, intermediate 2 was obtained. 2-[(1-ethoxyethoxy)methyl]ethylene oxide and potassium tert-butoxide were added to tetrahydrofuran to initiate a polymerization reaction. Then, dilute hydrochloric acid was added dropwise to cause a hydrolysis reaction to obtain linear hydrophobic glycerol (LPG). The linear hydrophobic glycerol and intermediate 2 were mixed in N,N-dimethylformamide. Dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to catalyze the esterification reaction to obtain caffeic acid phenylboronic acid ester-glycerol. Anhydrous diethyl ether was added to the reaction solution to precipitate the precipitate. The precipitate was washed three times with anhydrous diethyl ether, centrifuged to collect the precipitate, and freeze-dried under vacuum to obtain the carrier material caffeic acid phenylboronic acid ester-glycerol (CHPG).

[0010] Ergosterol and carrier material were placed in a round-bottom flask, and organic solvent was added. The mixture was stirred with a magnetic stirrer at room temperature until the carrier material and ergosterol were completely dissolved. The flask was then connected to a rotary evaporator and slowly rotated and evaporated under vacuum at 35–55 °C until the solvent was completely evaporated, forming a uniform film on the inner wall of the flask. PBS buffer was added to the flask, and the lipid film was slowly shaken under a 37 °C water bath to completely hydrate it. After purification by sonication and dialysis, the ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles (Erg-CHPG-M) were obtained by freeze-drying.

[0011] (2) Preparation of exosome-loaded ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles

[0012] The ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles and the nasal mucosal stem cell-derived exosome solution prepared in step (1) were placed in a probe sonicator for sonication. The sonicated mixture was allowed to stand at 37°C for 0.5 h and at 4°C for 1 h. The supernatant was removed by centrifugation, and the centrifuged precipitate was resuspended in pre-cooled PBS to obtain nasal mucosal stem cell exosomes (Exo@Erg-CHPG-M) loaded with ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles.

[0013] Preferably, in step (1), the amount of ethyl acetate is 10 mL, the amount of methyl caffeate is 1.0-2.0 g, the amount of phenylboronic acid is 1.0-1.5 g, the stirring reaction time is 8 h, and the amount of methanol-water solution is 10 mL, wherein the volume ratio of methanol to water is 4:1.

[0014] Preferably, in step (1), the amount of sodium hydroxide is 0.2-1.0 g, the amount of 2-[(1-ethoxyethoxy)methyl]ethylene oxide is 0.5-2.0 g, the amount of potassium tert-butoxide is 0.11 g, the amount of tetrahydrofuran solvent is 10 mL, the amount of dicyclohexylcarbodiimide (DCC) is 2.0-3.0 g, the amount of 4-dimethylaminopyridine (DAMP) is 0.05-0.2 g, the amount of linear hydrophobic glycerol (LPG) and solvent N,N-dimethylformamide (DMF) is 0.01 mol and 10 mL, respectively, the esterification reaction temperature is room temperature, and the esterification reaction time is 24 h.

[0015] Preferably, in step (1), the mass ratio of ergosterol and caffeoyl phenylboronic acid ester-glycerol polymer is 1:4 to 12, and the organic solvent is selected from any one or more of methanol, dichloromethane, and trichloromethane.

[0016] Preferably, in step (2), the concentration of the caffeoyl phenylboronic acid ester-glycerol polymer micelles loaded with ergosterol is 1 mg·mL⁻¹, the volume ratio of the caffeoyl phenylboronic acid ester-glycerol polymer micelles loaded with ergosterol to the exosome solution is 1:1 to 15, the ultrasonic parameters are: 20% amplitude, 30s on / off, 6 cycles, lasting for 3 min, with a 2 min cooling time between each cycle, and the centrifugation parameters are 12000×g, 4℃, and centrifugation time of 70 min.

[0017] Based on a general inventive concept, this invention also provides the application of an engineered exosome dual-drug delivery system loaded with ergosterol in the preparation of drugs for repairing post-stroke neurological function.

[0018] The working mechanism of the engineered exosome dual-drug delivery system loaded with ergosterol in this scheme is as follows:

[0019] Based on the theory of food and medicine sharing the same origin, this study used gut microbiota, metabolomics, and transcriptomics sequencing technologies, along with bioinformatics methods, to screen and discover that the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) / target of rapamycin (mTOR) signaling axis plays a significant regulatory role in the pathological process of ischemic stroke. The PI3K / AKT / mTOR pathway, after ligand activation, activates signal transduction: PI3K catalyzes the production of phosphatidylinositol triphosphate (PIP3), promoting AKT membrane translocation and phosphorylation. By inhibiting Bad protein dephosphorylation and caspase-9 activation pathways, it effectively blocks mitochondrial cytochrome c leakage and inhibits ischemia-induced programmed cell death. This signaling network can also maintain intracellular ion homeostasis by coordinating the activity of sodium-potassium pumps and calcium ion channels, and activate endothelial nitric oxide synthase (eNOS) to enhance vasodilation and endothelial repair capacity, while simultaneously regulating vascular endothelial growth factor (VEGF) biosynthesis to promote angiogenesis. Furthermore, mTOR-mediated neural stem cell proliferation provides the cellular basis for synaptic remodeling. Additionally, studies have found that ergosterol can activate this signaling pathway by promoting phosphorylation of serine residue 473 (Ser473) in AKT, thus potentially finding applications in stroke prevention and treatment.

[0020] Based on the innovative ROS-responsive nanomaterial CHPG design, the therapeutic active ingredient ergosterol (Erg) is loaded to exert therapeutic and repair effects. ROS-responsive micelles enable brain-targeted controlled release of ergosterol and central anti-inflammatory effects. Exosomal bioactive components regulate the intestinal barrier and brain-gut interaction, promoting the recovery of neurological function after stroke.

[0021] This invention achieves blood-brain barrier penetration and precise drug release at the lesion site through targeted functional modification. It is further combined with nasal mucosal stem cell exosomes to form a dual drug delivery system (Exo@Erg-CHPG-M), integrating the natural biological characteristics of exosomes with the precise targeting advantages of nanomicelles, significantly improving drug delivery efficiency. In vitro and animal model validations show that this system can improve neurological deficits and brain tissue damage after ischemic stroke by synergistically regulating inflammatory and neural repair pathways. This invention provides an innovative technical solution for the precision treatment of central nervous system diseases such as stroke.

[0022] The beneficial effects of this plan are:

[0023] 1. The key carrier material selected in this invention, methyl caffeate, has both anti-inflammatory and antioxidant pharmacological effects. The methyl caffeate ester material generated by its reaction with benzoic acid ester breaks down in the brain in a high ROS expression environment, resulting in micelle breakage. This ensures effective protection and controlled release of the drug, forming an anti-inflammatory-repair synergistic effect, and achieving functional synergy and therapeutic enhancement.

[0024] 2. Overcoming the dual challenges of brain targeting and microenvironment response, based on the ROS-responsive micelle and exosome composite system (Exo@Erg-CHPG-M), the system utilizes the natural blood-brain barrier penetration ability of exosomes and the response characteristics of micelles to the high ROS environment of inflammation to achieve dual targeting of ergosterol across the barrier and precise release at the lesion. This effectively solves the defects of poor targeting and low bioavailability of ordinary formulations, significantly improves the lesion enrichment efficiency of ergosterol, and improves the recovery of neurological function after ischemic stroke. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The synthetic route for the carrier material CHPG in Example 1 is shown below;

[0027] Figure 2 The NMR spectrum of intermediate 1 in Example 1 (H2Nm) Figure 2 A) The 1H NMR spectrum of compound 3 in 2-[(1-ethoxyethoxy)methyl]ethylene oxide ( Figure 2 B) The 1H NMR spectrum of compound LPG ( Figure 2 C) 1H NMR spectrum of CHPG ( Figure 2 D);

[0028] Figure 3The DLS particle size results for Erg-CHPG-M in Example 1;

[0029] Figure 4 This is a transmission electron microscope image of Erg-CHPG-M in Example 1;

[0030] Figure 5 The results of the CHPG-M critical micelle concentration detection in Example 1;

[0031] Figure 6 Transmission electron microscopy (TEM) images of exosomes and exosomes loaded with Erg-CHPG-M from Example 2. Figure 6 A, Figure 6 B) Scanning electron microscope image ( Figure 6 C Figure 6 D);

[0032] Figure 7 The particle size distribution of the nano-formulation Exo@Erg-CHPG-M in Example 2 is shown below.

[0033] Figure 8 The results of the study on the effects of concentration gradients of exosomes, ergosterol, Erg-CHPG-M and Exo@Erg-CHPG-M on the survival rate of PC12 cells in Example 2 are as follows;

[0034] Figure 9 The uptake of Coumarin, Coumarin-CHPG, and Exo@Coumarin-CHPG-M by PC12 cells in Example 2;

[0035] Figure 10 The therapeutic effects of exosomes, ergosterol, Erg-CHPG-M and Exo@Erg-CHPG-M on hypoxic injury of PC12 neurons (A) and on inflammatory injury of BV2 microglia (B) in Example 3;

[0036] Figure 11 The results and comparisons of Longa scores for neurological deficits after stroke in each group of rats in Example 4 (sham-operated group, model group, positive group, stem cell group, exosome group, ergosterol group, ergosterol-targeted nanomicelle group, exosome@ergosterol-targeted nanomicelle group);

[0037] Figure 12 The results of TTC staining in the brains of rats in Example 4 are as follows: (sham-operated group, model group, positive group, stem cell group, exosome group, ergosterol group, ergosterol-targeted nanomicelle group, and exosome@ergosterol-targeted nanomicelle group).

[0038] Figure 13Comparison of cerebral infarction rates in rats in Example 4 (sham-operated group, model group, positive group, stem cell group, exosome group, ergosterol group, ergosterol-targeted nanomicelle group, exosome@ergosterol-targeted nanomicelle group);

[0039] Figure 14 HE staining results of rat brain tissue in each group in Example 4 (sham-operated group, model group, positive group, stem cell group, exosome group, ergosterol group, ergosterol-targeted nanomicelle group, exosome@ergosterol-targeted nanomicelle group);

[0040] Figure 15 The fluorescence distribution imaging results of Cy5-labeled ergosterol-targeting nanomicelles and exosomes@ergosterol-targeting nanomicelles in major organs of rats in Example 4;

[0041] Figure 16 This is a diagram illustrating the mechanism by which the nano-formulation Exo@Erg-CHPG-M repairs nerve function in Example 5. A shows the expression bands of PI3K / AKT signaling pathway-related proteins (AKT, p-AKT, PI3K, p-PI3K) in each group detected by Western blotting. B shows the quantitative analysis of the relative expression levels of PI3K / AKT signaling pathway-related proteins (AKT, p-AKT, PI3K, p-PI3K) in each group (▲▲: P < 0.01 compared with the control group; **: P < 0.01 compared with the model group; ##: P < 0.01 compared with the stem cell group; △△: P < 0.01 compared with Erg; &&: P < 0.01 compared with Erg-CHPG-M; ★★: P < 0.01 compared with the Exo group). Detailed Implementation

[0042] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0043] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0044] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0045] Example 1: Preparation and characterization of ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles (Erg-CHPG-M)

[0046] 1. Preparation of Erg-CHPG-M

[0047] 1) First, accurately weigh 1.6g of methyl caffeate and 1.4g of phenylboronic acid, place them in a 50mL double-necked flask, add 10mL of ethyl acetate and let them dissolve. Then, react at 70℃ for 8 hours on a constant temperature magnetic stirrer to form intermediate reaction 1. After the reaction is completed, add 10mL of methanol aqueous solution (methanol:water = 4:1, v / v) and stir to dissolve. After complete dissolution, add 0.85g of NaOH and stir at room temperature for 4 hours. After the reaction is completed, intermediate product 2 is obtained.

[0048] 2) Accurately weigh 1.0 g of 2-[(1-ethoxyethoxy)methyl]ethylene oxide and 0.11 g of potassium tert-butoxide, add them to 10 mL of THF solvent, and slowly add 1 M dilute hydrochloric acid dropwise for 2 hours to hydrolyze and obtain LPG. Weigh 0.01 mol of LPG and mix it with an equimolar ratio of intermediate 2 in 10 mL of LMF, add 2.6 g of DCC and 0.15 g of DMAP sequentially, stir the reaction at room temperature for 24 h, add anhydrous diethyl ether to precipitate the reaction, wash three times with anhydrous diethyl ether, centrifuge to collect the precipitate, and freeze-dry to obtain the carrier material caffeoyl phenylboronic acid ester-glycerol polymer (CHPG).

[0049] 3) Weigh 0.1g of ergosterol and 0.4g of CHPG carrier material and place them in a round-bottom flask. Add 10mL of dichloromethane and stir with a magnetic stirrer at room temperature until the carrier material and drug are completely dissolved. Connect the flask to a rotary evaporator and slowly evaporate the lipid solvent at 37℃ and a vacuum of 0.08Mpa until a uniform film forms on the inner wall of the flask. Add 6mL of PBS buffer to the flask and slowly shake at 37℃ to completely hydrate the lipid film. After sonication and dialysis purification, freeze-dry to obtain ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles (Erg-CHPG-M), hereinafter referred to as ergosterol-targeted nanomicelles.

[0050] In this embodiment, the synthetic route of the carrier material caffeoyl phenylboronic acid ester-glycerol polymer (CHPG) is as follows: Figure 1 As shown.

[0051] 2. Characterization of Ergosterol-Targeted Nanomicelles (Erg-CHPG-M)

[0052] (1) Nuclear magnetic resonance (NMR) characterization

[0053] Take an appropriate amount of CHPG nanomaterial, dissolve it in deuterated dimethyl sulfoxide to achieve a sample concentration of 1-10 mM, transfer it to an NMR tube, place the NMR tube in an NMR spectrometer, and perform NMR H- and C- spectra measurements.

[0054] The NMR spectrum of intermediate 1 is as follows: Figure 2 As shown in Figure A, the 1H NMR spectrum of compound 3,2-[(1-ethoxyethoxy)methyl]ethylene oxide is as follows: Figure 2As shown in B, the hydrogen NMR spectrum of compound LPG is as follows: Figure 2 As shown in C, the CHPG 1H NMR spectrum is as follows: Figure 2 As shown in Figure D, the above characterization results confirm the successful synthesis of the nanomaterial CHPG.

[0055] (2) Particle size

[0056] Take an appropriate amount of ergosterol-CHPG micelles, dilute them with ultrapure water to a suitable concentration, and add them to the sample cell. Detect the particle size of the prepared ergosterol-CHPG micelles. The detection results are as follows: Figure 3 As shown, the test results indicate that the average particle size of the prepared nanomaterial is 53.26±1.21nm, the PDI is 0.195±0.007, and the particle size distribution is uniform.

[0057] (3) Morphological characterization

[0058] After hydrophilizing the copper mesh for transmission electron microscopy, dilute ergosterol-CHPG micelle solution of appropriate concentration was dropped onto the front side of the copper mesh. After standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was added, and the liquid on the copper mesh was blotted dry with filter paper. Phosphotungstic acid dye was added again, and after standing for 2 minutes, the liquid was blotted dry with filter paper. The above phosphotungstic acid staining was repeated once, and then the mesh was dried under an incandescent lamp. The morphology of the sample was examined in a transmission electron microscope (TEM).

[0059] Test results as follows Figure 4 As shown.

[0060] (4) Critical micelle concentration detection

[0061] The critical micelle concentration (CMC) was determined using the pyrene fluorescent probe method, with pyrene serving as a hydrophobic environment-sensitive probe to characterize the self-assembly behavior of CHPG-M micelles. The specific procedure was as follows: CHPG-M was dissolved in deionized water to prepare gradient concentration solutions (0.0001–1 mg / mL), and an equal volume of pyrene-acetone solution (final concentration 6 × 10⁻⁶) was added to each tube. -7 After incubation in the dark with shaking, free pyrene was removed by centrifugation. The fluorescence intensity ratio (I1 / I3) of the first peak (I1, 373 nm) and the third peak (I3, 384 nm) of pyrene was recorded using a fluorescence spectrophotometer (excitation wavelength 335 nm). A curve was plotted showing the change of I1 / I3 with the logarithm of CHPG-M concentration; the concentration corresponding to the inflection point of the curve is the CMC value. The detection results are as follows: Figure 5 As shown, the critical micelle concentration (CMC) is 5.397 μg / mL. This low CMC indicates that the material can efficiently self-assemble into micelles even at low concentrations. The hydrophobic interactions of the hydrophobic blocks help improve the efficiency of micelle formation and effectively resist the risk of micelle dissociation due to dilution during blood circulation, ensuring the stability of the drug delivery system. Furthermore, the hydrophobic core advantage corresponding to the low CMC helps stabilize the loading of lipophilic drugs.

[0062] Example 2: Preparation and Detection of Exosome-Loaded Ergosterol-Targeted Nanomicelles (Exo@Erg-CHPG-M)

[0063] 1. Preparation of exosome-loaded ergosterol-CHPG micelles (Exo@Erg-CHPG-M)

[0064] Exosome-loaded ergosterol-CHPG micelles (Exo@Erg-CHPG-M) were prepared using an ultrasonic method: 1 mL of ergosterol-CHPG micelle solution (1 mg / mL) and 3 mL of nasal mucosal stem cell-derived exosome solution were placed in a probe sonicator for sonication. The sonication parameters were set as follows: 20% amplitude, 30 s on / off, 6 cycles, for a total duration of 3 min, with a 2 min cooling time between each cycle. The sonicated mixture was allowed to stand at 37 °C for 0.5 h and then at 4 °C for 1 h to restore the exosome membrane structure. The mixture was then centrifuged at 12000 × g at 4 °C to remove the supernatant. The precipitate was resuspended in pre-cooled PBS to obtain Exo@Erg-CHPG-M.

[0065] 2. Basic characterization and detection of exosome-loaded ergosterol-targeted nanomicelles (Exo@Erg-CHPG-M)

[0066] (1) Morphological characterization

[0067] After hydrophilizing the copper mesh for transmission electron microscopy, dilute exosomes and exosome-loaded ergosterol-CHPG micelle solutions of appropriate concentrations were dropped onto the front side of the copper mesh. After standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was added, and the liquid on the copper mesh was blotted dry with filter paper. Phosphotungstic acid dye was added again, and after standing for 2 minutes, the liquid was blotted dry with filter paper. The above phosphotungstic acid staining was repeated once, and then the mesh was dried under an incandescent lamp. The morphology of the sample was examined in a transmission electron microscope (TEM).

[0068] TEM test results as follows Figure 6 As shown in A and 6B, the SEM detection results are as follows: Figure 6 As shown in C and 6D.

[0069] (2) Determination of particle size, potential and PDI

[0070] The particle size, potential, and PDI of exosome-loaded ergosterol-CHPG micelles were characterized using a dynamic light scattering particle size analyzer. Particle size and PDI determination: After the instrument warmed up for 30 min, the quartz cuvettes were rinsed 2-3 times with purified water. The nano-formulation was then diluted with purified water according to the specified ratio and added to the cuvettes. Each sample was cycled 3 times, and the cycle time was set. The test solvent was selected as "Water". Measurements were started after the instrument stabilized. The particle size test results are shown below. Figure 7As shown, the average particle size is 137.26±2.61nm, the PDI is 0.219±0.009, and the particle size distribution is uniform.

[0071] Potential determination: After the instrument was preheated for 30 minutes, the quartz cuvette was rinsed 2-3 times with purified water. Ergosterol-CHPG micelles loaded with exosomes were diluted with purified water at a certain ratio and added to the cuvette. The electrode was inserted into the cuvette and connected to the instrument. The cycle time and number of cycles were set, and "Water" was selected as the test solvent. After the instrument stabilized, the measurement was started. The measured potential of the ergosterol-CHPG micelles loaded with exosomes was -27.68±0.62mV. The absolute value of the zeta potential was relatively high, effectively increasing the stability of the ergosterol-CHPG micelles loaded with exosomes, improving the transmembrane activity of the drug, and thus promoting the improvement of drug bioavailability.

[0072] (3) Safety study of exosome-loaded ergosterol-targeted nanomicelles (Exo@Erg-CHPG-M)

[0073] After normal culture and counting of PC12 cells, add 5×10⁶ cells of cell suspension. 4 / well, incubated for 24 h until cells reached 80% confluence. Then, different concentrations of nasal mucosal stem cell-derived exosomes, ergosterol, ergosterol-targeted micelles (Erg-CHPG-M), and Exo@Erg-CHPG-M (100 μL, 1, 10, 100, 200 μg / mL) were added to each well. Incubation was then carried out at 37°C for 48 h. CCK-8 reagent (10 μL) was added to each well, and the wells were placed in an incubator (5% CO2) at 37°C for 4 h. The culture medium was discarded, and dimethyl sulfoxide (DMSO, 150 μL) was added, followed by vigorous shaking for 10 min. The absorbance (OD) of each well was read at 450 nm using a spectrophotometer, and cell viability was calculated as follows. Each experiment was repeated three times. Cell viability = (OD experimental group) / (OD control group) × 100%

[0074] The results are as follows Figure 8 As shown, the survival rate of PC12 cells was less than 90% when ergosterol monotherapy (Erg) was ≥200 μg / mL, while the cell viability of exosomes (Exo), exosome-loaded complex (Exo@Erg-CHPG-M), and targeted micelles (Erg-CHPG-M) remained >90% in the concentration range of 1-200 μg / mL. This confirms that the vector system of the present invention overcomes the toxicity of free drugs through drug delivery and interface modification, and effectively improves biosafety.

[0075] (4) The phagocytosis of (Exo@CHPG-M) neural cells by exosome-loaded targeted nanomicelles was investigated.

[0076] Establishment of an in vitro inflammatory cell model: PC12 cells were digested into a single-cell suspension and then subjected to a 1×10⁻⁶ cell suspension. 5 The cells were seeded at a density of 1 cell per well in 12-well plates, with 1 mL of cell culture medium added to each well. After the cells were fully adhered, lipopolysaccharide solution was added to bring the final concentration to 0.5 μg / mL. The cells were stimulated for 24 h to activate the PC12 cells, thus creating an in vitro inflammation model.

[0077] In vitro uptake efficiency: Coumarin-CHPG and Exo@Coumarin-CHPG-M, respectively, were prepared by co-dissolving fluorescently labeled coumarin with CHPG. Coumarin-CHPG and Exo@Coumarin-CHPG-M were then diluted with culture medium to a coumarin concentration of 0.5 μg / mL. After treating PC12 cells, they were washed with blank culture medium, and then 1 mL of coumarin-CHPG and Exo@Coumarin-CHPG-M solutions were added to each well (three replicates per group). After incubation with cells for 2 h, the cells were washed with PBS, fixed with paraformaldehyde, stained with DAPI, and observed and analyzed using a confocal microscope. The results are as follows: Figure 9 As shown, based on fluorescence analysis, the exosome-targeted micelle complex (Exo@Coumarin-CHPG-M) significantly improves the intracellular delivery efficiency of coumarin in PC12 cells compared to free drugs and single modifiers through synergistic membrane fusion effect and targeted recognition, thus confirming that the targeted nanomicelles involved in this invention have neuronal transmission function and can be taken up and phagocytosed by PC12 cells.

[0078] Example 3: Cellular-level efficacy evaluation of nano-formulations

[0079] The effects of different drug groups on the hypoxic injury of PC12 neurons and cell survival in BV2 microglia inflammation models were detected using the CCK-8 assay.

[0080] (1) Cell Culture and Grouping

[0081] PC12 cells (DMEM high glucose medium + 10% FBS) and BV2 microglia (RPMI-1640 + 10% FBS) were routinely cultured to the logarithmic growth phase and then divided into 8 groups for treatment: control group, model group (hypoxia / inflammation), positive control group, stem cell intervention group, nasal mucosal stem cell-derived exosome group, ergosterol group, ergosterol-targeted micelle group (Erg-CHPG-M), and Exo@Erg-CHPG-M group. Each group had 3 replicates and was independently repeated 3 times.

[0082] (2) Model construction and drug administration:

[0083] PC12 hypoxia model: CoCl2 (300μM) induced chemical hypoxia for 6 h, and each drug was added 2 h before modeling;

[0084] BV2 inflammation model: LPS (100ng / mL) stimulation for 24h induced inflammation, while various drugs were administered simultaneously.

[0085] (3) CCK-8 assay for survival rate

[0086] After the intervention, the two model cells were replaced with fresh culture medium, and CCK-8 reagent (10% by volume) was added to each well. They were incubated in the dark for 2 hours (37℃, 5% CO2). The absorbance (OD value) at 450 nm was measured by an ELISA reader, and the cell viability was calculated (viability = experimental group OD / normal control OD × 100%).

[0087] Test results as follows Figure 10 A, Figure 10 As shown in Figure B: In the PC12 hypoxic injury and BV2 microglial inflammation model, compared with the model group and each single-factor drug group, the exosome-loaded complex (Exo@Erg-CHPG-M) in this regimen showed a concentration-dependent increase in cell survival, with the most significant protective effect at high concentrations. After CoCl2 / LPS induction, the neuroprotective and anti-inflammatory activities of the Exo@Erg-CHPG-M group were significantly better than those of free ergosterol (Erg) and targeted micelles (Erg-CHPG-M) (P<0.01), verifying the synergistic effect of the exosome delivery system and the targeted micelles.

[0088] Example 4: Application of nano-formulation in the repair of neurological function after stroke in rats

[0089] A rat model of ischemic stroke was established: Rats were anesthetized with sodium pentobarbital and fixed in a supine position on a rat board. The right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were bluntly dissected using ophthalmic forceps. Sutures were prepared for use. The proximal ends of the ECA and CCA were ligated, and the ICA was temporarily clamped with an arterial clamp. A small incision was made in the CCA using ophthalmic forceps, and the prepared suture was quickly inserted from the CCA to the ICA. The insertion was stopped when slight resistance was felt at the tip of the suture. At this point, the suture on the CCA was tightened to prevent suture movement, dislodgement, and bleeding. The suture was ligated, and the occlusion time was recorded. After 1 hour of ischemia, the suture was removed, and reperfusion was performed. The sham-operated group underwent the same procedure, but the suture was inserted only 15 mm deep, without occluding blood flow to the middle cerebral artery. When the rat's tail is lifted and suspended in the air, its left forelimb flexes and adducts, exhibiting a typical tail-chasing sign during movement, indicating successful model establishment.

[0090] Experimental groups: sham surgery group, MCAO model group, positive group, stem cell group, exosome group, ergosterol group (Erg), ergosterol-targeted micelle group (Erg-CHPG-M), and exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M).

[0091] Sham surgery group and MCAO model group: 100 μL of normal saline was administered via the tail vein.

[0092] Positive group: Butylphthalide was administered intravenously via tail vein at a dose of 80 mg / kg / day for 2 weeks;

[0093] Stem cells: 1×10⁻⁶ via tail vein injection 6 nasal mucosa-derived stem cells

[0094] Exosome group: tail vein 1×10 6 Nasal mucosa-derived stem cell exosomes

[0095] Ergosterol (Erg), ergosterol-targeted micelles (Erg-CHPG-M), and exosome-loaded ergosterol-targeted nanomicelles (Exo@Erg-CHPG-M): all were administered ergosterol 10 mg / kg / day via tail vein injection for 2 weeks.

[0096] (1) The Longa scoring method was used to quantify the degree of neurological deficit in animals through a series of behavioral observations. The scoring range was from 0 to 5 points, where: 0 points indicated no neurological deficit; 1 point indicated that the forepaw on the paralyzed side could not be fully extended; 2 points indicated that the animal turned in circles on the paralyzed side while walking; 3 points indicated that the animal leaned towards the paralyzed side while walking; 4 points indicated that the animal could not walk automatically and had lost consciousness; and 5 points indicated mortality. The higher the score, the worse the animal's neurobehavioral condition and the more severe the neurological deficit. In the experiment, the Longa scoring method could be used to preliminarily assess whether the model was successfully established. Animals with scores of 1-3 were usually included in the next stage of the experiment, while animals with scores of 4-5 were excluded because this may indicate that the animal's condition was not suitable for participation in the experiment. Mice in each group were scored before treatment and on days 1, 3, 7, 14, and 28 after treatment. The scoring results are as follows: Figure 11 As shown, in a rat model of ischemic stroke, the exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M) significantly reduced the Longa score (P<0.01) compared to the model group (MCAO group) and the single-component formulations (Erg group, Erg-CHPG-M group), and the neurological function recovery effect was significantly superior to that of the positive control drug butylphthalide at 28 days after treatment (P<0.01). The difference compared with the sham-operated group gradually narrowed (the score approached baseline in the later stage), confirming that Exo@Erg-CHPG-M enhances neurorepair activity through the synergistic delivery mechanism of exosomes and targeted micelles.

[0097] (2) TTC staining: After sacrificing rats at a predetermined time, brains were harvested and flash-frozen at -20℃ for 20 min. 2 mm thick sections were then continuously sliced ​​along the coronal plane. The sections were immersed in 2% TTC staining solution and incubated at 37℃ in the dark for 30 min, rotating occasionally to ensure uniform staining. The sections were then fixed with 4% paraformaldehyde. Normal tissue appeared red, while ischemic infarct areas appeared pale white. ImageJ software was used to calculate the infarct area ratio of each section, and the total infarct volume percentage was assessed by summing the infarct volumes of each layer. The results are as follows: Figure 12 , Figure 13 As shown. Figure 12 The model group rats showed typical pale white ischemic infarct areas in their brain tissue, confirming the successful establishment of the stroke model. All treatment groups showed some improvement, with a reduction in the damaged area. The exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M) had the smallest infarct area, and its brain tissue staining was close to normal red, significantly superior to other single-component treatment groups. Figure 13 According to statistics on cerebral infarction rates, this nano-formulation effectively reduces the degree of damage to ischemic areas through the synergistic effect of exosome-mediated targeted drug delivery and the neuroprotective effect of ergosterol, verifying the significant therapeutic effect of the targeted nano-delivery system described in this patent.

[0098] (3) HE staining results of brain tissue directly reflect the extent of brain damage. Figure 14 As a result, compared with the normally tightly packed cell structure in the sham-operated group, the model group showed significant ischemic pathological damage, including increased intercellular spaces, nuclear condensation, and uneven cytoplasmic staining, confirming the effectiveness of the stroke model. The degree of brain tissue damage in the positive group and each single treatment group (stem cell group, exosome group, and ergosterol group) was improved compared to the model group, but the ergosterol-targeted nanomicelle group showed more significant cell morphology recovery. Among them, the exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M) exhibited the highest cell arrangement regularity, the fewest necrotic cells, and intercellular spaces approaching normal levels. This indicates that the synergistic drug delivery mechanism of exosomes and targeted micelles significantly optimizes brain tissue regeneration in the pathological microenvironment by enhancing drug accumulation and neural repair efficacy in the ischemic area, verifying that the exosome@ergosterol-targeted nanomicelles provided in this patent have the best post-stroke neural repair effect.

[0099] (4) Rat in vivo imaging test

[0100] In vivo imaging experiments were conducted to detect the distribution of the drug formulation in rat body parts, visually demonstrating the brain-targeting advantages of the exosomes@ergosterol-targeting nanomicelles prepared in this invention for post-stroke nerve repair.

[0101] Rats were randomly divided into two groups, receiving intravenous injections via tail vein of Cy5-labeled ergosterol-targeting nanomicelles and exosomes@ergosterol-targeting nanomicelles, respectively. Two hours after administration, the animals were sacrificed, and the heart, liver, spleen, lungs, kidneys, and brain tissue were rapidly dissected. After rinsing with physiological saline and aspirating any residual fluid, the organs were placed in an in vivo imaging system to collect fluorescence signals (results are shown in [see details]). Figure 15 It was clearly observed that, 2 hours after tail vein injection, the organ distribution characteristics of the exosome-loaded ergosterol-targeting nanomicelle group (Exo@Erg-CHPG-M) differed significantly from those of the unmodified ergosterol-targeting nanomicelle group. After exosome modification, the fluorescence intensity in the brain was significantly enhanced, confirming that the exosome-mimetic membrane achieved active enrichment of brain lesions through synergistic effects of immune clearance and cross-barrier action. In addition to the brain, the fluorescence signals of the exosome-loaded ergosterol-targeting nanomicelle group (Exo@Erg-CHPG-M) in organs such as the heart, liver, spleen, lungs, and kidneys were significantly stronger than those in the unmodified group, indicating that the introduction of the exosome carrier comprehensively improved the in vivo stability of the targeted nanomicelles and effectively enhanced drug bioavailability.

[0102] Example 5: Investigating the mechanism of the nano-formulation Exo@Erg-CHPG-M in repairing nerve function

[0103] Based on Western blotting, proteins were extracted from rat brain tissue samples, separated by SDS-PAGE electrophoresis, and transferred to a membrane. Hybridization and imaging were then performed using specific antibodies against phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT), and total proteins (PI3K, AKT). The expression levels of target proteins in each group were quantitatively analyzed, and the expression changes of key molecules in the PI3K / AKT signaling pathway in the brain tissue of rats with ischemic stroke were detected. Results are as follows: Figure 16As shown, the phosphorylation levels of p-PI3K and p-AKT proteins in the brain tissue of rats in the ischemic stroke model group were significantly lower than those in the blank control group (P<0.05, P<0.01). After intervention with stem cells, exosomes, ergosterol, or ergosterol-targeted nanomicelles, the phosphorylation expression of the above proteins was significantly upregulated compared with the model group (P<0.05). Among them, the expression levels of p-PI3K and p-AKT in the exosome@ergosterol-targeted nanomicelles (Exo@Erg-CHFC-M) group were the highest, significantly higher than those in the model group (P<0.01), and the increase was greater than that in other intervention groups. There were no significant differences in total PI3K (PI3K) and total AKT (AKT) among the groups, suggesting that the intervention effect was mainly achieved by promoting the phosphorylation of key molecules in the pathway (rather than protein synthesis). This invention, through animal-level Western blotting experiments combined with mechanistic analysis, clarifies the following core conclusions: Ischemic brain injury can inhibit the phosphorylation activation of the PI3K / AKT pathway, while ergosterol can activate this pathway by promoting phosphorylation at the Ser473 site of AKT, exerting neuroprotective effects (inhibition of mitochondrial apoptosis), maintenance of vascular homeostasis (ion channels, eNOS), and angiogenesis (VEGF regulation). Compared to free ergosterol, exosomes alone, or targeted nanomicelles, the exosome@ergosterol targeted nanomicelles (Exo@Erg-CHFC-M) constructed in this invention effectively enhances the activation efficiency of the PI3K / AKT pathway by ergosterol through a dual mechanism of biocompatible carrier and targeted enrichment.

[0104] In summary, this approach successfully constructed a novel ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelle (Erg-CHPG-M) for targeted repair of post-stroke neurological damage. Based on the innovative ROS-responsive nanomaterial CHPG, the therapeutically active ingredient ergosterol (Erg) is loaded to exert its therapeutic and repairing effects. Targeted functional modification enables blood-brain barrier penetration and precise drug release at the lesion site. Further loading onto nasal mucosal stem cell exosomes forms a dual drug delivery system (Exo@Erg-CHPG-M), combining the natural biological characteristics of exosomes with the precise targeting advantages of nanomicelles, significantly improving drug delivery efficiency. In vitro and animal model validations demonstrate that this system can improve neurological deficits and brain tissue damage after ischemic stroke by synergistically regulating inflammatory and neurological repair pathways.

[0105] This invention provides an innovative technical solution for the precision treatment of central nervous system diseases such as stroke. The embodiments described are preferred embodiments of this invention, but the invention is not limited to the above embodiments. Any obvious improvements, substitutions, or modifications that can be made by those skilled in the art without departing from the essence of the invention are within the scope of protection of this invention.

Claims

1. An engineered exosome dual-drug delivery system loaded with ergosterol, characterized in that, The exosomes include ergosterol, caffeoyl phenylboronic acid ester, glycerol, and nasal mucosal stem cell-derived exosomes. The caffeoyl phenylboronic acid ester and glycerol encapsulate ergosterol to form ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles. The ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles are further loaded into nasal mucosal stem cell-derived exosomes. The preparation method of the engineered exosome dual-drug delivery system loaded with ergosterol includes the following steps: (1) Preparation of ergosterol-caffeic acid phenylboronic acid ester-glycerol polymer micelles Caffeic acid methyl ester and phenylboronic acid were dissolved in ethyl acetate and stirred at 70°C. After the reaction was completed, intermediate reaction body 1 was formed. Intermediate reaction body 1 was added to methanol aqueous solution and stirred to dissolve. After complete dissolution, sodium hydroxide was added and stirred at room temperature. After the reaction was completed, intermediate product 2 was obtained. 2-[(1-ethoxyethoxy)methyl]ethylene oxide and potassium tert-butoxide were added to tetrahydrofuran to initiate a polymerization reaction. Then, dilute hydrochloric acid was added dropwise to cause a hydrolysis reaction to obtain linear hydrophobic glycerol. The linear hydrophobic glycerol and intermediate product 2 were mixed in N,N-dimethylformamide. Dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to catalyze the esterification reaction to obtain caffeic acid phenylboronic acid ester-glycerol. Anhydrous diethyl ether was added to the reaction solution to precipitate the product. The product was washed three times with anhydrous diethyl ether, centrifuged to collect the precipitate, and freeze-dried under vacuum to obtain the carrier material caffeic acid phenylboronic acid ester-glycerol. Ergosterol and carrier material were placed in a round-bottom flask, and organic solvent was added. The mixture was stirred with a magnetic stirrer at room temperature until the carrier material and ergosterol were completely dissolved. The flask was then connected to a rotary evaporator and slowly rotated and evaporated under vacuum at 35–55 °C until the solvent was completely evaporated, forming a uniform film on the inner wall of the flask. PBS buffer was added to the flask, and the lipid film was slowly shaken under a 37 °C water bath to completely hydrate it. After purification by sonication and dialysis, the ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles were obtained by freeze-drying. (2) Preparation of exosome-loaded ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles The ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles prepared in step (1) and the nasal mucosal stem cell-derived exosome solution were placed in a probe sonicator for sonication. The sonicated mixture was allowed to stand at 37°C for 0.5 h and at 4°C for 1 h. The supernatant was removed by centrifugation, and the centrifuged precipitate was resuspended in pre-cooled PBS to obtain exosomes loaded with ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles.

2. The engineered exosome dual-drug delivery system loaded with ergosterol according to claim 1, characterized in that, The engineered exosome dual-drug delivery system has a potential of -27.68±0.62 mV, a particle size of 137.26±2.61 nm, and a PDI of 0.219±0.

009.

3. A method for preparing an engineered exosome dual-drug delivery system loaded with ergosterol as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Preparation of ergosterol-caffeic acid phenylboronic acid ester-glycerol polymer micelles Caffeic acid methyl ester and phenylboronic acid were dissolved in ethyl acetate and stirred at 70°C. After the reaction was completed, intermediate reaction body 1 was formed. Intermediate reaction body 1 was added to methanol aqueous solution and stirred to dissolve. After complete dissolution, sodium hydroxide was added and stirred at room temperature. After the reaction was completed, intermediate product 2 was obtained. 2-[(1-ethoxyethoxy)methyl]ethylene oxide and potassium tert-butoxide were added to tetrahydrofuran to initiate a polymerization reaction. Then, dilute hydrochloric acid was added dropwise to cause a hydrolysis reaction to obtain linear hydrophobic glycerol. The linear hydrophobic glycerol and intermediate product 2 were mixed in N,N-dimethylformamide. Dicyclohexylcarbodiimide and 4-dimethylaminopyridine were added to catalyze the esterification reaction to obtain caffeic acid phenylboronic acid ester-glycerol. Anhydrous diethyl ether was added to the reaction solution to precipitate the product. The product was washed three times with anhydrous diethyl ether, centrifuged to collect the precipitate, and freeze-dried under vacuum to obtain the carrier material caffeic acid phenylboronic acid ester-glycerol. Ergosterol and carrier material were placed in a round-bottom flask, and organic solvent was added. The mixture was stirred with a magnetic stirrer at room temperature until the carrier material and ergosterol were completely dissolved. The flask was then connected to a rotary evaporator and slowly rotated and evaporated under vacuum at 35–55 °C until the solvent was completely evaporated, forming a uniform film on the inner wall of the flask. PBS buffer was added to the flask, and the lipid film was slowly shaken under a 37 °C water bath to completely hydrate it. After purification by sonication and dialysis, the ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles were obtained by freeze-drying. (2) Preparation of exosome-loaded ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles The ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles prepared in step (1) and the nasal mucosal stem cell-derived exosome solution were placed in a probe sonicator for sonication. The sonicated mixture was allowed to stand at 37°C for 0.5 h and at 4°C for 1 h. The supernatant was removed by centrifugation, and the centrifuged precipitate was resuspended in pre-cooled PBS to obtain exosomes loaded with ergosterol-caffeoyl phenylboronic acid ester-glycerol polymer micelles.

4. The preparation method according to claim 3, characterized in that, In step (1), the amount of ethyl acetate is 10 mL, the amount of methyl caffeate is 1.0-2.0 g, the amount of phenylboronic acid is 1.0-1.5 g, the stirring reaction time is 8 h, and the amount of methanol-water solution is 10 mL, wherein the volume ratio of methanol to water is 4:

1.

5. The preparation method according to claim 3, characterized in that, In step (1), the amount of sodium hydroxide used is 0.2–1.0 g, the amount of 2-[(1-ethoxyethoxy)methyl]ethylene oxide is 0.5–2.0 g, the amount of potassium tert-butoxide is 0.11 g, the amount of tetrahydrofuran solvent is 10 mL, the amount of dicyclohexylcarbodiimide is 2.0–3.0 g, the amount of 4-dimethylaminopyridine is 0.05–0.2 g, the amount of linear hydrophobic glycerol and solvent N,N-dimethylformamide is 0.01 mol and 10 mL, respectively, the esterification reaction temperature is room temperature, and the esterification reaction time is 24 h.

6. The preparation method according to claim 3, characterized in that, In step (1), the mass ratio of ergosterol and caffeic acid phenylboronic acid ester-glycerol polymer is 1:4 to 12, and the organic solvent is selected from any one or more of methanol, dichloromethane, and trichloromethane.

7. The preparation method according to claim 3, characterized in that, In step (2), the concentration of ergosterol caffeoyl phenylboronic acid ester-glycerol polymer micelles is 1 mg / mL. -1 The volume ratio of the ergosterol caffeoyl phenylboronic acid ester-glycerol polymer micelles to the exosome solution is 1:1 to 15. The ultrasonic parameters are: 20% amplitude, 30 s on / off, 6 cycles, lasting for 3 min in total, with a 2 min cooling time between each cycle. The centrifugation parameters are: 12000×g, 4℃, and centrifugation time of 70 min.

8. The use of an engineered exosome dual-drug delivery system loaded with ergosterol as described in any one of claims 1-2, or an engineered exosome dual-drug delivery system loaded with ergosterol prepared by the preparation method described in any one of claims 3-7, in the preparation of drugs for repairing post-stroke neurological function.

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