Engineered exosome double-drug-loading system loaded with ergosterol as well as preparation method and application of engineered exosome double-drug-loading system

Abstract

The invention provides an ergosterol-loaded engineered exosome double-drug-loading system as well as a preparation method and application thereof, belongs to the field of nano materials and nano biological medicines, and is based on innovative ROS response type nano material CHPG design, a therapeutic active component ergosterol is loaded to play a role in treatment and repair, and the curative effect of the ergosterol is improved. Blood-brain barrier penetration and accurate drug release in a focus area are realized through targeted function modification; the Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG-M / Erg-CHPG- the nerve function defect and brain tissue damage after ischemic stroke are improved, and a new technical scheme is provided for precise treatment of central nervous system diseases such as cerebral stroke.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials and nanobiomedicine, and specifically relates to an engineered exosome dual-drug loading system loaded with ergosterol, and a preparation method and application thereof. Background Art

[0002] Ischemic stroke (IS) is an acute cerebrovascular disease caused by cerebral artery obstruction or stenosis, leading to hypoxic necrosis of brain tissue. It is associated with high disability and a complex pathological mechanism. Its pathological progression involves a multi-stage cascade: early blood flow interruption triggers neuronal energy metabolic failure and excitotoxicity, followed by mitochondrial dysfunction and a burst of reactive oxygen species (ROS). During the perfusion phase, free radical overload exacerbates blood-brain barrier (BBB) ​​damage, neuroinflammation, and gut-brain axis dysregulation. The latter manifests as gut microbiota disturbances, tight junction protein inactivation, and gut-derived proinflammatory cytokines (such as LPS and TNF-α) entering the brain via the vagus nerve or circulatory system, creating a positive feedback loop of "inflammation-secondary brain injury." These pathological characteristics make the efficacy of IS treatment highly dependent on targeted drug delivery, synergistic multi-pathway intervention, and holistic regulation of the neuro-immune-metabolic network.

[0003] In recent years, nano-drug delivery systems targeting neuroprotection and regeneration have become a research hotspot. For example, drugs are encapsulated in ROS-responsive materials to improve lesion enrichment. However, there are still the following limitations in practical applications: Although natural active ingredients such as ergosterol (Erg) have shown 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 nano-micelles can partially improve drug enrichment through passive targeting, but the carrier itself lacks responsiveness to the post-stroke oxidative stress microenvironment, making it difficult to achieve intelligent drug release. In addition, hydrophobic drugs such as ergosterol are prone to sudden release or leakage due to carrier instability. In addition, a single drug delivery mode cannot synergistically regulate the complex pathological network after stroke. For example, the interaction between central nervous system inflammation and brain-gut axis disorder (intestinal barrier destruction, dysbiosis) is often overlooked, and conventional delivery systems lack the ability to intervene in multiple targets. Therefore, there is an urgent need to develop a new drug delivery system that combines efficient targeting, multi-mechanism synergy and functional carriers to break through the existing technical bottleneck and provide a better solution for neurological function repair after ischemic stroke. Summary of the Invention

[0004] In response to the problems of low targeted delivery efficiency and insufficient brain-gut axis regulation in existing neurorepair treatments for ischemic stroke, this proposal provides an engineered exosome drug delivery system based on the brain-gut axis, its preparation method, and application. To address the problems of low blood-brain barrier penetration, poor microenvironment responsiveness, and insufficient synergistic intervention of the brain-gut axis in neurorepair for ischemic stroke, an engineered exosome-micelle hybrid system is proposed. This system combines the targeted drug release characteristics of ROS-responsive micelles with the brain-gut axis regulation function of exosomes, effectively solving the defects of traditional drugs' weak ability to cross the blood-brain barrier and single treatment.

[0005] To achieve the above objectives, this scheme first provides an engineered exosome dual-drug delivery system loaded with ergosterol, comprising ergosterol, caffeic acid phenyl borate, glycerol, and nasal mucosal stem cell-derived exosomes (EMSC-exo; Exo). The caffeic acid phenyl borate and glycerol encapsulate ergosterol to form ergosterol-caffeic acid phenyl borate-glycerol polymer micelles, which are then loaded into nasal mucosal stem cell-derived exosomes.

[0006] Preferably, 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.

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

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

[0009] Caffeic acid methyl ester and phenylboronic acid are dissolved in ethyl acetate and reacted with stirring at 70° C. After the reaction, an intermediate reaction product 1 is formed. The reaction intermediate 1 is added to a methanol aqueous solution and stirred to dissolve. After complete dissolution, sodium hydroxide is added and stirred at room temperature to react. After the reaction, an intermediate product 2 is obtained. 2-[(1-ethoxyethoxy)methyl]oxirane and potassium tert-butoxide are added to tetrahydrofuran to initiate a polymerization reaction. Dilute hydrochloric acid is then added dropwise to cause a hydrolysis reaction to obtain linear hydrophobic glycerol (LPG). The linear hydrophobic glycerol and the intermediate product 2 are mixed in N,N-dimethylformamide, and dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to catalyze an esterification reaction to obtain caffeic acid phenyl borate-glycerol. Anhydrous ether is added to the reaction solution for precipitation, and the reaction solution is washed three times with anhydrous ether. The precipitate is collected by centrifugation and freeze-dried under vacuum to obtain the carrier material caffeic acid phenyl borate-glycerol (CHPG).

[0010] Ergosterol and a carrier material are placed in a round-bottom flask, an organic solvent is added, and a magnetic stirrer is used to stir at room temperature until the carrier material and ergosterol are completely dissolved. The flask is connected to a rotary evaporator, and slowly rotary evaporated at 35-55° C. under vacuum conditions until the solvent is completely evaporated and a uniform film is formed on the inner wall of the flask. PBS buffer is added to the flask, and the flask is slowly shaken in a 37° C. water bath to completely hydrate the lipid film. After ultrasonic and dialysis purification, the flask is freeze-dried to obtain ergosterol-caffeic acid phenyl borate-glycerol polymer micelles (Erg-CHPG-M).

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

[0012] The ergosterol-caffeic acid phenyl borate-glycerol polymer micelles prepared in step (1) and the nasal mucosa stem cell-derived exosome solution were placed in a probe sonicator for ultrasonic treatment. The ultrasonicated 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 centrifugal precipitate was resuspended in pre-cooled PBS to obtain nasal mucosa stem cell-derived exosomes loaded with ergosterol-caffeic acid phenyl borate-glycerol polymer micelles (Exo@Erg-CHPG-M).

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

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

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

[0016] Preferably, the concentration of the ergosterol-encapsulating caffeic acid phenyl borate-glycerol polymer micelles in step (2) is 1 mg.mL-1, the volume ratio of the ergosterol-encapsulating caffeic acid phenyl borate-glycerol polymer micelles to the exosome solution is 1:1-15, the ultrasound parameters are: 20% amplitude, 30s on / off, 6 cycles, a total duration of 3 minutes, with a cooling time of 2 minutes between each cycle, and the centrifugation parameters are 12000×g, 4°C, and the centrifugation time is 70 minutes.

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

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

[0019] Based on the theory of food and medicine being of the same origin, this study, using bioinformatics screening methods, analyzed gut microbiota, metabolomics, and transcriptomics sequencing, revealed that the phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) / mTOR target of rapamycin (mTOR) signaling axis plays a significant regulatory role in the pathological progression of ischemic stroke. Ligand activation of the PI3K / AKT / mTOR pathway activates signaling: PI3K catalyzes the production of phosphatidylinositol triphosphate (PIP3), which promotes AKT membrane translocation and phosphorylation. This, in turn, effectively blocks mitochondrial cytochrome c leakage by inhibiting Bad protein dephosphorylation and caspase-9 activation, thereby suppressing ischemia-induced programmed cell death. This signaling network also maintains intracellular ion homeostasis by coordinating sodium-potassium pump and calcium channel activity, activating endothelial nitric oxide synthase (eNOS), enhancing vasodilation and endothelial repair, and regulating vascular endothelial growth factor (VEGF) biosynthesis to promote angiogenesis. Furthermore, mTOR-mediated neural stem cell proliferation provides a cellular basis for synaptic remodeling. Furthermore, studies have found that ergosterol can activate this signaling pathway by promoting AKT phosphorylation at Ser473, thus potentially being used 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 a therapeutic and repair effect. ROS-responsive micelles are used to achieve brain-targeted controlled release and central anti-inflammatory effects of ergosterol. The intestinal barrier and brain-gut interaction are regulated through exosome bioactive components, thereby promoting the recovery of neurological function after stroke.

[0021] Through targeted functional modification, it achieves blood-brain barrier penetration and precise drug release in the lesion area. It is further compounded with nasal mucosal stem cell exosomes to form a dual drug delivery system (Exo@Erg-CHPG-M), which combines the natural biological properties of exosomes with the precise targeting advantages of nanomicelles, significantly improving drug delivery efficiency. In vitro and animal model validation have shown 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 program are:

[0023] 1. The key carrier material selected by the present invention, caffeic acid methyl ester, has both anti-inflammatory and antioxidant pharmacological effects. The benzoate material generated by its reaction with benzoate breaks down in the brain environment with high ROS expression, leading to micelle breakage, ensuring effective drug protection and controlled release, forming an anti-inflammatory-repair synergistic effect, and achieving functional synergy and therapeutic enhancement.

[0024] 2. Breaking through the dual challenges of brain targeting and microenvironment response, based on a composite system of ROS-responsive micelles and exosomes (Exo@Erg-CHPG-M), the natural ability of exosomes to penetrate the blood-brain barrier and the responsiveness of micelles to the inflammatory high-ROS environment are utilized to achieve dual targeting of ergosterol across the barrier and precise release in lesions. This effectively solves the defects of ordinary preparations such as poor targeting and low bioavailability, significantly improves the lesion enrichment efficiency of ergosterol, and improves neurological function recovery after ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 This is the synthetic route of the carrier material CHPG in Example 1;

[0027] Figure 2 is the NMR H spectrum of intermediate 1 in Example 1 ( Figure 2 A) 2-[(1-ethoxyethoxy)methyl]oxirane H NMR spectrum of compound 3 ( Figure 2 B) H NMR spectrum of compound LPG ( Figure 2 C) H NMR spectrum of CHPG ( Figure 2 D);

[0028] Figure 3is the DLS particle size result of Erg-CHPG-M in Example 1;

[0029] Figure 4 TEM image of Erg-CHPG-M in Example 1;

[0030] Figure 5 The critical micelle concentration of CHPG-M in Example 1 is detected;

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

[0032] Figure 7 The particle size test results of the nanoformulation Exo@Erg-CHPG-M in Example 2;

[0033] Figure 8 The results of the investigation of the concentration gradient effects 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 inflammatory injury of BV2 microglia (B) in Example 3;

[0036] Figure 11 The Longa score results and comparison of post-stroke neurological deficits in rats in each group in Example 4 (sham operation group, model group, positive group, stem cell group, exosome group, ergosterol group, ergosterol-targeted nanomicelle group, and exosome@ergosterol-targeted nanomicelle group);

[0037] Figure 12 These are the TTC staining results of the rat brains of each group in Example 4 (sham operation 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 the cerebral infarction rate of rats in each group in Example 4 (sham operation 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 the rat brain tissues in each group in Example 4 (sham operation 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-targeted nanomicelles and exosomes@ergosterol-targeted nanomicelles in major organs of rats in Example 4;

[0041] Figure 16 Figure 5 is a diagram showing the mechanism of the nanoformulation Exo@Erg-CHPG-M in repairing neural function in Example 5. A is a bar graph showing the expression of PI3K / AKT signaling pathway-related proteins (AKT, p-AKT, PI3K, p-PI3K) in each group detected by Western Blot. B is a quantitative analysis of the relative expression levels of PI3K / AKT signaling pathway-related proteins (AKT, p-AKT, PI3K, p-PI3K) in each group (▲▲: 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, P < 0.01). DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0043] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

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

[0045] Example 1 Preparation and Characterization of Ergosterol-Caffeic Acid Phenylboronic Acid-Glycerol Polymer Micelles (Erg-CHPG-M)

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

[0047] 1) First, 1.6 g of caffeic acid methyl ester and 1.4 g of phenylboronic acid were accurately weighed and placed in a 50 mL double-necked flask. 10 mL of ethyl acetate was added and dissolved. The mixture was reacted at 70°C on a constant temperature magnetic stirrer for 8 hours to form an intermediate reaction product 1. After the reaction, 10 mL of methanol aqueous solution (methanol: water = 4:1, v / v) was added and stirred to dissolve. After complete dissolution, 0.85 g of NaOH was added and stirred at room temperature for 4 hours. After the reaction, an intermediate product 2 was obtained.

[0048] 2) Accurately weigh 1.0 g of 2-[(1-ethoxyethoxy)methyl]oxirane and 0.11 g of potassium tert-butoxide into 10 mL of THF. 1 M dilute hydrochloric acid was slowly added dropwise to hydrolyze for 2 hours to obtain LPG. 0.01 mol of LPG and an equal molar ratio of intermediate 2 were mixed into 10 mL of DMF. 2.6 g of DCC and 0.15 g of DMAP were added sequentially. The mixture was stirred at room temperature for 24 hours. Anhydrous ether was added to the reaction mixture to precipitate the mixture, which was then washed three times with anhydrous ether. The precipitate was collected by centrifugation and freeze-dried to obtain the carrier material, caffeic acid phenyl borate-glycerol polymer (CHPG).

[0049] 3) Weigh 0.1 g of ergosterol and 0.4 g of CHPG carrier material into a round-bottom flask. Add 10 mL of dichloromethane and stir at room temperature using a magnetic stirrer until the carrier material and drug are completely dissolved. Connect the flask to a rotary evaporator and slowly rotary evaporate the lipid solvent at 37°C under a vacuum of 0.08 MPa to completely volatilize, forming a uniform film on the inner wall of the flask. Add 6 mL of PBS buffer to the flask and slowly shake in a 37°C water bath to completely hydrate the lipid film. After sonication and dialysis purification, freeze-dry to obtain ergosterol-caffeic acid phenyl borate-glycerol polymer micelles (Erg-CHPG-M), hereinafter referred to as ergosterol-targeted nanomicelles.

[0050] The synthesis route of the carrier material caffeic acid phenyl borate-glycerol polymer (CHPG) in this embodiment is as follows: Figure 1 shown.

[0051] 2. Characterization of ergosterol-targeted nanomicelles (Erg-CHPG-M)

[0052] (1) H NMR spectroscopy characterization

[0053] Take an appropriate amount of nanomaterial CHPG, dissolve it in deuterated dimethyl sulfoxide to make the sample concentration reach 1-10mM, transfer it into a nuclear magnetic resonance tube, put the nuclear magnetic resonance tube into a nuclear magnetic resonance spectrometer, and perform nuclear magnetic H spectrum and C spectrum measurement.

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

[0055] (2) Particle size

[0056] Take an appropriate amount of ergosterol-CHPG micelles, dilute them with ultrapure water to an appropriate concentration, and add them to the sample pool to detect the particle size of the prepared ergosterol-CHPG micelles. The test results are as follows: Figure 3 As shown, according to the test results, 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 a copper grid dedicated to transmission electron microscopy, a dilute ergosterol-CHPG micelle solution of appropriate concentration was dripped onto the front of the copper grid; after standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was added, and the liquid on the copper grid was blotted dry with filter paper. The phosphotungstic acid dye was added again, and after standing for 2 minutes, the liquid was blotted dry with filter paper; after repeating the phosphotungstic acid staining once, the sample was dried under an incandescent lamp; and the copper grid was placed in a transmission electron microscope (TEM) to examine the sample morphology.

[0059] Test results such as Figure 4 shown.

[0060] (4) Critical micelle concentration detection

[0061] The critical micelle concentration (CMC) was determined by the fluorescent probe pyrene method, using pyrene as a hydrophobic environment-sensitive probe to characterize the self-assembly behavior of CHPG-M micelles. The specific operation was as follows: CHPG-M was dissolved in deionized water to prepare gradient concentration solutions (0.0001-1 mg / mL), and an equal amount of pyrene acetone solution was added to each tube (final concentration 6×10 -7 M), incubate in the dark with shaking, and then centrifuge to remove free pyrene; use a fluorescence spectrophotometer (excitation wavelength 335nm) to record the fluorescence intensity ratio (I1 / I3) of the first peak (I1, 373nm) and the third peak (I3, 384nm) of pyrene, and draw a curve of I1 / I3 versus the logarithm of CHPG-M concentration. The concentration corresponding to the inflection point of the curve is the CMC value. The test results are as follows Figure 5 As shown, the critical micelle concentration is 5.397 μg / mL. This low CMC indicates that the material can efficiently self-assemble into micelles at low concentrations. The hydrophobic interaction of the hydrophobic block helps improve the efficiency of micelle formation. It can also effectively resist the risk of micelle dissociation caused by environmental dilution during blood circulation in the body, ensuring the stability of the drug delivery system. In addition, the hydrophobic core advantage corresponding to the low CMC helps to stabilize the loading of fat-soluble 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 by sonication: 1 mL of ergosterol-CHPG micelle solution (1 mg / mL) and 3 mL of nasal stem cell-derived exosome solution were sonicated in a probe sonicator. The sonicator parameters were set as follows: 20% amplitude, 30-second on / off, 6 cycles for a total of 3 minutes, with a 2-minute cool-down period between cycles. The sonicated mixture was incubated 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 12,000 × g at 4°C, the supernatant removed, and the pellet resuspended in pre-chilled 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 hydrophilization treatment of a copper grid dedicated to transmission electron microscopy, dilute exosomes and exosome-loaded ergosterol-CHPG micelle solutions of appropriate concentrations were added to the front of the copper grid. After standing for 2 minutes, an appropriate amount of phosphotungstic acid dye was added, and the liquid on the copper grid was absorbed with filter paper. The phosphotungstic acid dye was added again, and after standing for 2 minutes, the liquid was absorbed with filter paper. After repeating the phosphotungstic acid staining once, the sample was dried under an incandescent lamp. The copper grid was placed in a transmission electron microscope (TEM) to examine the sample morphology.

[0068] TEM test results are as follows Figure 6 As shown in A and 6B, the SEM test 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 the exosome-loaded ergosterol-CHPG micelles were characterized using a dynamic light scattering particle size analyzer. Determination of particle size and PDI: After the instrument is preheated for 30 minutes, rinse the quartz cuvette 2-3 times with purified water, dilute the nanoformulation in proportion with purified water and add it to the cuvette. Set each sample to be measured three times in a cycle and set the single cycle time. Select "Water" as the test solvent. After the instrument stabilizes, start the measurement. The particle size test results are as follows: 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 is preheated for 30 minutes, the quartz cuvette is rinsed 2-3 times with purified water. Exosome-loaded ergosterol-CHPG micelles are diluted with purified water at a certain ratio and added to the cuvette. The electrode sheet is inserted into the cuvette and connected to the instrument. The cycle time and number of cycles are set. The test solvent is selected as "Water". After the instrument stabilizes, the measurement is started. The measured potential of the exosome-loaded ergosterol-CHPG micelles is -27.68±0.62mV. The absolute value of the zeta potential is relatively high, which effectively increases the stability of the exosome-loaded ergosterol-CHPG micelles and improves the transmembrane activity of the drug, thereby promoting the improvement of drug bioavailability.

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

[0073] After PC12 cells were cultured and counted normally, 5×10 4 / well, incubate for 24 hours until the cells are 80% confluent. 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 the wells respectively. Then incubate at 37°C for 48 hours. CCK-8 reagent (10 μL) was added to each well, and then the wells were placed in an incubator (5% CO2) and placed at 37°C for 4 hours. The culture medium was discarded, dimethyl sulfoxide (DMSO, 150 μL) was added, and the mixture was shaken vigorously for 10 minutes. Using a spectrophotometer, the absorbance (OD) of each well at 450 nm was read to calculate the cell viability value as follows. Each experiment was repeated 3 times. Cell activity = (OD experimental group) / (OD control group) × 100%

[0074] The results are as follows Figure 8 As shown in the results, the PC12 cell viability was less than 90% when ergosterol alone (Erg) was ≥200 μg / mL, while the cell viability of exosomes (Exo), exosome-drug complexes (Exo@Erg-CHPG-M) and targeted micelles (Erg-CHPG-M) was maintained at >90% in the concentration range of 1-200 μg / mL, confirming that the carrier system of the present invention overcomes the toxicity of free drugs through drug delivery and interface modification, effectively improving biosafety.

[0075] (4) Exosome-loaded targeted nanomicelles can be phagocytosed by neurons (Exo@CHPG-M)

[0076] Establishment of in vitro inflammatory cell model: PC12 cells were digested into single cell suspension and the suspension was 1×10 5 PC12 cells were seeded at a density of 100 cells / well in a 12-well plate, and 1 mL of cell culture medium was added to each well. After the cells were completely attached, lipopolysaccharide solution was added to a final concentration of 0.5 μg / mL. The cells were stimulated for 24 hours to activate PC12 cells, which was the in vitro inflammation model.

[0077] In vitro uptake efficiency: Fluorescently labeled coumarin (Coumarin) was co-dissolved with CHPG to prepare Coumarin-CHPG and Exo@Coumarin-CHPG-M loaded with Coumarin, and Coumarin-CHPG and Exo@Coumarin-CHPG-M were 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 Coumarin-CHPG and Exo@Coumarin-CHPG-M solution was added, respectively. Three replicate wells were set up for each group, and after incubation with cells for 2 hours, they were washed with PBS, fixed with paraformaldehyde, and stained with DAPI. The results were observed and analyzed using a confocal microscope. Figure 9 As shown in the fluorescence analysis, the exosome-targeted micelle complex (Exo@Coumarin-CHPG-M) significantly improved the intracellular delivery efficiency of coumarin in PC12 cells compared with free drugs and single modified bodies through synergistic membrane fusion effect and targeted recognition, thereby confirming that the targeted nanomicelles involved in the present invention have the function of neuronal cell transmission and can be taken up and phagocytosed by PC12 cells.

[0078] Example 3: Investigation of the efficacy of nanoformulations at the cellular level

[0079] The CCK-8 method was used to detect the effects of different drug groups on the cell survival rate in the PC12 neural cell hypoxia injury and BV2 microglial cell inflammation model.

[0080] (1) Cell culture and grouping

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

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

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

[0084] BV2 inflammation model: LPS (100 ng / mL) was stimulated for 24 h to induce inflammation, and various drugs were given at the same time.

[0085] (3) CCK-8 detection of 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. The cells were incubated in the dark for 2 h (37°C, 5% CO2). The absorbance at 450 nm (OD value) was measured by a microplate reader, and the cell survival rate was calculated (survival rate = experimental group OD / normal control OD × 100%).

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

[0088] Example 4: Application of nanoformulation in post-stroke neurological function repair in rats

[0089] A rat ischemic stroke model was established: Rats were anesthetized with sodium pentobarbital and fixed supine 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. A seton was prepared and the proximal ends of the ECA and CCA were ligated. The ICA was temporarily clamped with an artery clip. The CCA was lifted with ophthalmic forceps to create a small incision. A prepared seton was rapidly inserted from the CCA to the ICA. When slight resistance was felt at the tip of the seton, the seton was stopped. The seton was then tightened around the CCA to prevent dislodgement and bleeding. The seton was ligated and the occlusion time was recorded. After 1 hour of ischemia, the seton was removed and reperfusion was performed. The sham group underwent the same surgical procedures as above, but the seton was inserted only 15 mm deep, without occluding blood flow in the middle cerebral artery. When the rat's tail is lifted and suspended in the air, the left forelimb flexes and adducts, and a typical tail-chasing sign is shown during movement, indicating that the model is successfully established.

[0090] Experimental groups: sham operation 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 operation group and MCAO model group: 100 μL normal saline was administered via tail vein

[0092] Positive group: butylphthalide was administered by tail vein at 80 mg / kg / day for 2 weeks;

[0093] Stem cells: 1×10 6 Nasal mucosa-derived stem cells

[0094] Exosome group: 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 groups received ergosterol at a dose of 10 mg / kg / day via tail vein injection for 2 weeks.

[0096] (1) The Longa scoring method is used to quantify the degree of neurological deficit of animals through a series of behavioral observations. The scoring range is from 0 to 5 points, where: 0 points indicates no neurological deficit, 1 point indicates that the paralyzed front paw cannot be fully extended, 2 points indicates turning toward the paralyzed side when walking, 3 points indicates leaning toward the paralyzed side when walking, 4 points indicates inability to walk automatically and loss of consciousness, and 5 points indicates death. 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 can be used to preliminarily assess whether the model is successful. Usually, animals with scores of 1-3 are included in the next experiment, while animals with scores of 4-5 are excluded because this may indicate that the animal's condition is not suitable for participation in the experiment. Each group of mice was 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 rats with ischemic stroke, the exosome-loaded ergosterol-targeted nanomicelles (Exo@Erg-CHPG-M) of this protocol significantly reduced Longa scores compared with the model group (MCAO group) and the single-component formulations (Erg group and Erg-CHPG-M group) (P<0.01). Furthermore, the neurological function recovery effect was significantly superior to that of the positive drug butylphthalide 28 days after treatment (P<0.01). Compared with the sham-operated group, the difference gradually narrowed (with scores approaching baseline in the later stages), confirming that Exo@Erg-CHPG-M enhances neurorepair activity through the synergistic delivery mechanism of exosomes and targeted micelles.

[0097] (2) TTC staining: The rats were killed at the scheduled time and the brains were removed. The brains were quickly frozen at -20°C for 20 min and then sliced ​​into 2-mm thick sections along the coronal plane. The sections were immersed in 2% TTC staining solution and incubated at 37°C in the dark for 30 min. The sections were flipped during the incubation to ensure uniform staining and then fixed with 4% paraformaldehyde. Normal tissue appeared red while the ischemic infarct area appeared pale. ImageJ software was used to calculate the infarct area ratio of each section, and the percentage of total infarct volume was estimated by accumulating the infarct volume of each layer. The results are shown in Figure 2. Figure 12 、 Figure 13 shown. Figure 12 The brain tissue of rats in the model group showed a typical pale ischemic infarction area, confirming that the stroke model was successfully established; each treatment group showed some improvement, with the damaged area reduced. Among them, the infarction area of ​​the exosome-loaded ergosterol targeted nanomicelle group (Exo@Erg-CHPG-M) was the smallest, and its brain tissue staining was close to normal red, which was significantly better than other single-component treatment groups. Figure 13 According to the statistics of cerebral infarction rate, the nanoformulation effectively reduces the degree of damage in the ischemic area through the synergistic effect of exosome-mediated drug targeted delivery and ergosterol's neuroprotection, verifying the significant therapeutic effect of the targeted nanodelivery system described in this patent.

[0098] (3) The HE staining results of brain tissue directly reflect the brain damage. Figure 14 The results showed that compared with the normal tightly arranged cell structure of the sham operation group, the model group showed significant ischemic pathological damage such as increased intercellular space, nuclear condensation and uneven cytoplasmic staining, which confirmed that the construction of the stroke model was effective. The degree of brain tissue damage in the positive group and each single treatment group (stem cell group, exosome group, ergosterol group) was improved compared with the model group, but the cell morphology recovery of the ergosterol targeted nanomicelle group was more obvious. Among them, the exosome-loaded ergosterol targeted nanomicelle group (Exo@Erg-CHPG-M) had the highest cell arrangement regularity, the least necrotic cells, and the tissue gap was close to the normal level, indicating that the synergistic drug delivery mechanism of exosomes and targeted micelles significantly optimized the brain tissue regeneration effect in the pathological microenvironment by enhancing drug enrichment and neural repair efficacy in the ischemic area, verifying that the exosomes@ergosterol targeted nanomicelles provided by this patent have the best post-stroke neural repair effect.

[0099] (4) In vivo imaging experiments in rats

[0100] Through in vivo imaging experiments, the distribution of the drug preparation in the body parts of rats was detected, intuitively demonstrating the brain-targeting advantages of the exosomes@ergosterol targeted nanomicelles prepared by the present invention for post-stroke neural repair.

[0101] Rats were randomly divided into two groups and injected with Cy5-labeled ergosterol-targeted nanomicelles or exosomes@ergosterol-targeted nanomicelles via the tail vein. Two hours after administration, the animals were sacrificed, and the heart, liver, spleen, lung, kidney, and brain tissues were quickly removed. After rinsing with saline and removing any residual surface fluid, the organs were placed in an in vivo imaging device to collect fluorescence signals from each organ (see results). Figure 15 ). It is clearly visible that 2 hours after tail vein injection, the organ distribution characteristics of the exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M) showed significant differences compared to the ergosterol-targeted nanomicelle group. After exosome modification, the fluorescence intensity in the brain was significantly increased, confirming that the exosome biomimetic membrane actively enriched brain lesions by circumventing immune clearance and cross-barrier synergistic effects. In addition to the brain, the fluorescence signals of the exosome-loaded ergosterol-targeted nanomicelle group (Exo@Erg-CHPG-M) in organs such as the heart, liver, spleen, lungs, and kidneys were significantly stronger than those of the unmodified group, indicating that the introduction of exosome carriers comprehensively improved the in vivo stability of the targeted nanomicelles and effectively improved the bioavailability of the drug.

[0102] Example 5: Investigating the mechanism of the nanoformulation Exo@Erg-CHPG-M in repairing neural function

[0103] Based on Western Blot technology, the rat brain tissue samples were subjected to protein extraction, SDS-PAGE electrophoresis separation and membrane transfer. Specific antibodies against phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT) and total protein (PI3K, AKT) were used for hybridization and development. The expression levels of target proteins in each group were quantitatively analyzed, and the expression changes of key molecules of the PI3K / AKT signaling pathway in the brain tissue of rats with ischemic stroke were detected. Figure 16As shown, the protein phosphorylation levels of p-PI3K and p-AKT 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 these proteins was significantly upregulated compared with the model group (P < 0.05). The exosome@ergosterol-targeted nanomicelle (Exo@Erg-CHFC-M) group had the highest expression levels of p-PI3K and p-AKT, significantly higher than those in the model group (P < 0.01), and the increase was greater than that in the other intervention groups. There were no significant differences in total PI3K (PI3K) and total AKT (AKT) between the groups, suggesting that the intervention effect is primarily achieved by promoting the phosphorylation of key pathway molecules (rather than protein synthesis). This study, through animal-level Western blotting experiments combined with mechanistic analysis, has established the following core conclusions: Ischemic brain injury inhibits phosphorylation and activation of the PI3K / AKT pathway, and ergosterol can activate this pathway by promoting phosphorylation of AKT Ser473, exerting neuroprotection (inhibiting mitochondrial apoptosis), maintaining vascular homeostasis (ion channels, eNOS), and angiogenesis (VEGF regulation). Compared to free ergosterol, simple exosomes, or targeted nanomicelles, the exosome@ergosterol targeted nanomicelles (Exo@Erg-CHFC-M) constructed in this study, through the dual mechanisms of biocompatible carrier and targeted enrichment, effectively enhances the efficiency of ergosterol activation of the PI3K / AKT pathway.

[0104] In summary, this scheme successfully constructed a new type of ergosterol-caffeic acid phenyl borate-glycerol polymer micelle (Erg-CHPG-M) for targeted repair of post-stroke neural damage. Based on the innovative ROS-responsive nanomaterial CHPG design, the therapeutic active ingredient ergosterol (Erg) is loaded to exert a therapeutic and repair effect. It achieves blood-brain barrier penetration and precise drug release in the lesion area through targeted functional modification. It is further loaded on nasal mucosal stem cell exosomes to form a dual drug delivery system (Exo@Erg-CHPG-M), which combines the natural biological properties of exosomes with the precise targeting advantages of nanomicelles, significantly improving drug delivery efficiency. In vitro and animal model verification shows that this system can improve neurological function deficits and brain tissue damage after ischemic stroke by synergistically regulating inflammatory and neural repair pathways.

[0105] The present invention provides an innovative technical solution for the precise treatment of central nervous system diseases such as stroke. The embodiments described are preferred embodiments of the present invention, but the present 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 present invention are within the scope of protection of the present invention.

Claims

1. An engineered exosome dual-drug loading system loaded with ergosterol, characterized in that: The invention comprises ergosterol, caffeic acid phenyl borate, glycerol and nasal mucosal stem cell-derived exosomes. The caffeic acid phenyl borate and glycerol encapsulate ergosterol to form ergosterol-caffeic acid phenyl borate-glycerol polymer micelles, and the ergosterol-caffeic acid phenyl borate-glycerol polymer micelles are further loaded into the nasal mucosal stem cell-derived exosomes.

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

009.

3. A method for preparing the engineered exosome dual-drug loading system of ergosterol according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Preparation of ergosterol-caffeic acid phenylboronic acid ester-glycerol polymer micelles Caffeic acid methyl ester and phenylboric acid are dissolved in ethyl acetate and reacted with stirring at 70° C. After the reaction, an intermediate reaction product 1 is formed. The reaction intermediate 1 is added to a methanol aqueous solution and stirred to dissolve. After complete dissolution, sodium hydroxide is added and stirred at room temperature to react. After the reaction, an intermediate product 2 is obtained. 2-[(1-ethoxyethoxy)methyl]ethylene oxide and potassium tert-butoxide are added to tetrahydrofuran to initiate a polymerization reaction. Dilute hydrochloric acid is then added dropwise to cause a hydrolysis reaction to obtain linear hydrophobic glycerol. The linear hydrophobic glycerol and the intermediate product 2 are mixed in N,N-dimethylformamide, and dicyclohexylcarbodiimide and 4-dimethylaminopyridine are added to catalyze an esterification reaction to obtain caffeic acid phenyl borate-glycerol. Anhydrous ether is added to the reaction solution for precipitation, and the reaction solution is washed three times with anhydrous ether. The precipitate is collected by centrifugation and freeze-dried under vacuum to obtain the carrier material caffeic acid phenyl borate-glycerol. Ergosterol and a carrier material are placed in a round-bottom flask, an organic solvent is added, and a magnetic stirrer is used to stir at room temperature until the carrier material and ergosterol are completely dissolved. The flask is connected to a rotary evaporator, and slowly rotary evaporated at 35-55° C. under vacuum conditions until the solvent is completely evaporated and a uniform film is formed on the inner wall of the flask. PBS buffer is added to the flask, and the flask is slowly shaken in a 37° C. water bath to completely hydrate the lipid film. After ultrasonic and dialysis purification, the flask is freeze-dried to obtain ergosterol-caffeic acid phenyl borate-glycerol polymer micelles; (2) Preparation of exosome-loaded ergosterol-caffeic acid phenylboronic acid ester-glycerol polymer micelles The ergosterol-caffeic acid phenyl borate-glycerol polymer micelles prepared in step (1) and the nasal mucosal stem cell-derived exosome solution were placed in a probe sonicator for ultrasonic treatment. The ultrasonicated 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 centrifugal precipitate was resuspended in pre-cooled PBS to obtain nasal mucosal stem cell-derived exosomes loaded with ergosterol-caffeic acid phenyl borate-glycerol polymer micelles.

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

1.

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

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

7. The preparation method according to claim 3, characterized in that The concentration of the caffeic acid phenyl borate-glycerol polymer micelles containing ergosterol in step (2) is 1 mg.mL -1 The volume ratio of the ergosterol-encapsulated caffeic acid phenyl borate-glycerol polymer micelles to the exosome solution was 1:1-15, the ultrasound parameters were: 20% amplitude, 30s on / off, 6 cycles, a total duration of 3 minutes, with a 2-minute cooling time between each cycle, and the centrifugation parameters were 12,000×g, 4°C, and a centrifugation time of 70 minutes.

8. Use of the engineered exosome dual-drug delivery system loaded with ergosterol according to claims 1-2 or the engineered exosome dual-drug delivery system loaded with ergosterol prepared by the preparation method according to any one of claims 2-7 in the preparation of a drug for repairing post-stroke neurological function.

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