A nanodelivery system for treating ischemic stroke

Abstract

This invention discloses a nanomedicine delivery system for treating ischemic stroke and its preparation method. The nanomedicine delivery system consists of hollow mesoporous silica nanoparticles, ROS-sensitive ketothiols, quercetin, bacterial outer membrane vesicles, and the CXCR4 targeting ligand AMD3100. It can treat ischemic stroke through a cascade mechanism of "neutrophil delivery - CXCR4 active targeting - ROS-responsive drug release", which solves the problems of drugs being unable to cross the blood-brain barrier and unable to be precisely released in time and space in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a brain-targeted nanodelivery system, its preparation method and application. It is a nanodelivery system that integrates three functions: "neutrophil loading", "CXCR4 active targeting" and "ROS-responsive drug release". By loading quercetin, it can be used to treat ischemic stroke. Background Technology

[0002] Ischemic stroke, a serious central nervous system disease, is characterized by high incidence, high disability rate, and high mortality rate worldwide. Essentially, it is a clinical syndrome caused by cerebral vascular occlusion leading to interruption of blood supply to brain tissue, resulting in ischemic hypoxic necrosis and neurological deficits. Epidemiological data shows that ischemic stroke accounts for more than 80% of all stroke cases. The primary strategy for treating ischemic stroke is to restore blood flow within the time window. While intravenous administration of recombinant tissue plasminogen activator (rt-PA) is a commonly used thrombolytic method, its therapeutic window is narrow (usually within 4.5 hours of symptom onset) and carries the risk of hemorrhagic transformation and reperfusion injury. Therefore, how to effectively regulate neuroinflammatory responses, reduce reperfusion injury, and promote neurological repair while achieving blood flow restoration has become a key focus and challenge in current research.

[0003] Quercetin (Que) is a naturally occurring flavonoid compound that has attracted considerable attention in the biomedical field due to its multiple bioactivities, including anti-inflammatory, anti-apoptotic, and antioxidant effects. Preclinical studies have revealed that quercetin possesses potential neuroprotective effects in patients with ischemic stroke, specifically through mechanisms including inhibiting inflammatory thrombus formation, alleviating oxidative stress damage, reducing apoptosis, and promoting autophagy. Furthermore, related research has shown that quercetin can activate the NRF2 / HO1 signaling pathway, promoting microglia to M2 polarization, thereby effectively reducing inflammatory responses and oxidative stress damage. However, the clinical application of quercetin is limited by its low bioavailability, chemical instability, and difficulty in effectively crossing the blood-brain barrier, which severely restricts its expected therapeutic efficacy in the treatment of central nervous system diseases.

[0004] In recent years, nanobiotechnology, as an emerging approach, has provided a new drug delivery platform for the treatment of ischemic stroke. Rational design and modification of nanoplatforms can improve blood-brain barrier crossing efficiency, reduce intracerebral drug accumulation, and decrease the need for repeated drug administration. Therefore, developing a nanomedicine delivery system capable of efficiently crossing the blood-brain barrier, precisely targeting ischemic lesions, and achieving intelligent drug release is of significant scientific and clinical value for the treatment of ischemic stroke. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a nano-drug delivery system for treating ischemic stroke. This system achieves efficient brain-targeted delivery of quercetin and precise drug release to ischemic lesions through a cascade mechanism of "neutrophil loading - CXCR4 active targeting - ROS-responsive drug release". This solves the problems of drugs being unable to cross the blood-brain barrier and being unable to release drugs precisely in time and space in existing technologies, and can effectively improve the treatment effect of ischemic stroke.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to protect a nanomedicine delivery system for treating ischemic stroke, which uses hollow mesoporous silica nanoparticles (HMSN) as the core carrier, modifies the surface of the HMSN with ROS-sensitive ketithiolide (TK), loads quercetin (Que) onto the HMSN, and then co-incubates it with bacterial outer membrane vesicles (OMV) to coat the surface of the nanoparticles with OMV. Finally, AMD3100 targeting CXCR4 is modified on the surface of the OMV to form a nanoassembly with a "shell-core" structure.

[0007] The mechanisms of action of each structural component in the nano-drug delivery system are as follows: (1) Core drug loading layer: Quercetin is loaded into the mesoporous cavity of hollow mesoporous silica nanoparticles (HMSN) by physical impregnation to form quercetin-loaded nanoparticles. (2) ROS-responsive linking layer: The ketithiolide (TK) linking bonds fixed on the surface of HMSN by chemical coupling can be broken under the action of ROS, so as to achieve controlled drug release; (3) Bionic coating layer: The bacterial outer membrane vesicles (OMV) coated on the surface of nanoparticles can be specifically recognized and internalized by the Toll-like receptors on the surface of neutrophils, the immune cells that infiltrate the brain earliest after ischemia, thereby carrying the drug-loaded nanosystem to the ischemic lesion and realizing "in vivo loading". (4) Active targeting layer: AMD3100 modified on the outer layer of OMV has a high affinity for CXCR4 and can specifically recognize the CXCR4 receptor highly expressed in the ischemic brain region, thereby achieving secondary targeting and retention enhancement.

[0008] Therefore, the quercetin loaded in the nano-drug delivery system can treat ischemic stroke through a cascade mechanism of "neutrophil loading - CXCR4 active targeting - ROS-responsive drug release".

[0009] The second objective of this invention is to protect the preparation method of the aforementioned nano-drug delivery system, which includes the following steps: (1) Hollow mesoporous silica nanoparticles (HMSN) were synthesized using a template method. (2) HMSN-TK was obtained by chemically coupling ROS-sensitive ketethioglycolate (TK) to the surface of hollow mesoporous silica nanoparticles. (3) Quercetin (Que) was loaded into the mesoporous cavity of HMSN-TK by impregnation method to obtain Q@HMSN-TK; (4) Q@HMSN-TK was co-incubated with bacterial outer membrane vesicles (OMV) to coat the surface of the nanoparticles with bacterial outer membrane vesicles, thus obtaining Q@HMSN-O; (5) By modifying AMD3100 on the outer layer of Q@HMSN-O using bioconjugation technology, a nano-drug delivery system Q@HMSN-O@A was obtained.

[0010] Further, step (2) involves a two-step method to chemically couple ROS-sensitive ketithiolide (TK) linkages onto the surface of hollow mesoporous silica nanoparticles (HMSN). Specifically, HMSN is first dispersed in a mixture of anhydrous ethanol and ultrapure water, and 3-aminopropyltriethoxysilane (APTES) is slowly added under stirring at 45°C. After reacting for 8 hours, the mixture is centrifuged and washed to obtain amino-modified HMSN-NH2. Then, the TK molecules 3,3'-(propane-2,2-diylbis(thioalkyldiyl))dipropane Acid, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in DMSO and ultrasonically mixed to activate the carboxyl groups of TK. HMSN-NH2 was dispersed in DMSO and triethanolamine was added as a catalyst. The activated TK solution was then slowly added dropwise to the reaction system. After stirring at room temperature for 24 hours, the mixture was washed thoroughly with DMSO and ultrapure water to remove unreacted reagents, and finally, the carrier HMSN-TK with TK linkages on its surface was obtained.

[0011] Furthermore, the ratio of the hollow mesoporous silica nanoparticles, 3-aminopropyltriethoxysilane, 3,3'-(propane-2,2-diylbis(thionidyl))dipropionic acid, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and triethanolamine is 50 mg:50 μL:32 mg:22.4 mg:36 mg:10 μL.

[0012] Furthermore, the volume ratio of anhydrous ethanol to ultrapure water in the mixture of anhydrous ethanol and ultrapure water is 1:1.

[0013] Further, in step (3), HMSN-TK is dispersed in anhydrous ethanol, and then quercetin is added at a mass ratio of 1:1 to HMSN-TK. After stirring at 4°C in the dark for 8 hours, the precipitate is collected by centrifugation and washed with PBS to remove free quercetin, thereby obtaining drug-loaded Q@HMSN-TK.

[0014] Furthermore, the mass ratio of Q@HMSN-TK to bacterial outer membrane vesicles used in step (4) is 1:2; the co-incubation is carried out at 4°C with shaking for 1 hour.

[0015] Further, in step (5), the CXCR4 antagonist AMD3100 is first synthesized into the targeting molecule DSPE-PEG-AMD3100 through the Michael addition reaction of maleimide-thiol. Then, Q@HMSN-O and DSPE-PEG-AMD3100 are mixed in PBS at a mass ratio of 50:1 and incubated at 4°C for 2 hours to obtain the complete nano-drug delivery system Q@HMSN-O@A.

[0016] A third objective of this invention is to protect the application of the aforementioned nano-drug delivery system in the preparation of drugs for treating ischemic stroke.

[0017] Specifically, after intravenous administration, the nano-drug delivery system can be endocytosed and carried by neutrophils via OMV, and cross the blood-brain barrier using the natural pathway of neutrophil migration to ischemic brain regions. Upon reaching the brain, it achieves precise targeting of ischemic areas by binding to the CXCR4 receptor via AMD3100. In the high ROS microenvironment of ischemic lesions, the TK linkage breaks, the OMV shell detaches, and quercetin is precisely released, exerting the effects of clearing ROS, regulating microglia polarization towards the M2 phenotype, inhibiting neuroinflammation, reducing oxidative stress damage, and promoting nerve repair.

[0018] (1) By integrating the “neutrophil-carrying” strategy, this invention utilizes the natural chemotaxis of neutrophils as a “living transport vehicle”, effectively solving the key bottleneck that nanomedicines cannot cross the blood-brain barrier and achieving efficient brain-targeted delivery; (2) This invention achieves secondary precise localization and enhanced retention of nanomedicines in ischemic brain regions through active targeted modification of CXCR4, significantly increasing the local drug concentration in the lesion. (3) This invention uses ROS-responsive drug release design to control the intelligent release of drugs at the target site by using excessive ROS generated in ischemic lesions as a trigger signal, thereby maximizing the local therapeutic concentration and reducing system exposure. (4) This invention utilizes the multiple biological activities of quercetin to simultaneously clear ROS and regulate the polarization of microglia toward the M2 phenotype, thereby inhibiting oxidative stress and neuroinflammation while promoting endogenous repair and recovery of nerve function, achieving synergistic treatment with "one drug, multiple effects". Attached Figure Description

[0019] Figure 1 The image shows a transmission electron microscope (TEM) image of the Q@HMSN-O@A nanodrug delivery system prepared in Example 1.

[0020] Figure 2 The hydrated particle size distribution of different nanoparticles prepared in Example 1 is shown.

[0021] Figure 3 This is a fluorescence colocalization map of Q@HMSN-O@A being endocytosed by neutrophils at different time points.

[0022] Figure 4 Fluorescence thermography images of in vitro blood-brain barrier (BBB) ​​penetration experiments of different nanoparticles.

[0023] Figure 5 Immunofluorescence images showing the regulation of M2 polarization in microglia by different nanoparticles.

[0024] Figure 6 TTC staining images of cerebral infarction volumes in MCAO model mice treated with different nanoparticles.

[0025] Figure 7 HE staining images of the heart, liver, spleen, lungs, kidneys, and brain of mice treated with Q@HMSN-O@A. Detailed Implementation

[0026] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0027] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0028] Example 1: Preparation of the nano-drug delivery system Q@HMSN-O@A 1) Synthesis of SiO2 template: At 50℃, 3.15 mL of ammonia solution (38wt%) and 10 mL of ultrapure water were added to 74 mL of anhydrous ethanol and stirred at 800 rpm for 20 min. Then, 6 mL of tetraethyl orthosilicate was slowly added dropwise and the reaction was continued for 3 h. The product was washed three times each with anhydrous ethanol and ultrapure water (10000 rpm, 10 min) to obtain monodisperse SiO2 nanospheres.

[0029] 2) Preparation of SiO2@MSN core-shell structure: 160 mg of the prepared SiO2 nanospheres were dispersed in 30 mL of ultrapure water and sonicated for 15 min. Then, the mixture was added to a mixture containing 225 mg of hexadecyltrimethylammonium bromide, 45 mL of ultrapure water, 30 mL of anhydrous ethanol and 300 μL of ammonia solution (38 wt%). The mixture was stirred at 800 rpm for 30 min at room temperature. Then, 375 μL of tetraethyl orthosilicate was slowly added and the reaction was continued for 6 h. The product was washed to obtain SiO2@MSN.

[0030] 3) Preparation of HMSN: Take 50 mg SiO2@MSN, add 10 mL of ultrapure water and 232 mg of sodium carbonate, and stir slowly at 50 °C for 12 h to selectively etch the SiO2 core; after washing, disperse the product in a mixture of 70 mL of anhydrous ethanol and 7 mL of concentrated hydrochloric acid, and reflux at 80 °C for 24 h to remove the CTAB template. The final product is washed to obtain silica nanoparticles (HMSN) with a hollow mesoporous structure.

[0031] 4) Amination of HMSN-NH2: 50 mg of HMSN was dispersed in a mixture of 25 mL of anhydrous ethanol and 25 μL of ultrapure water. 50 μL of 3-aminopropyltriethoxysilane (APTES) was slowly added under stirring at 45 °C and 800 rpm. After reacting for 8 h, the mixture was centrifuged and washed to obtain amino-modified silica nanoparticles (HMSN-NH2).

[0032] 5) Linkage of HMSN-TK response bonds: 50 mg HMSN-NH2 was dispersed in 10 mL DMSO, and 10 μL triethanolamine was added; at the same time, 32 mg 3,3'-(propane-2,2-diylbis(thioalkyldiyl))dipropionic acid (TK-COOH), 22.4 mg N-hydroxysuccinimide (NHS) and 36 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in 800 μL LDMSO, ultrasonically mixed, and then slowly added dropwise to the reaction system containing HMSN-NH2. The reaction was stirred at 800 rpm for 24 h at room temperature. The product was thoroughly washed with DMSO and ultrapure water to obtain a support (HMSN-TK) with ketethiolated (TK) linkage bonds on the surface.

[0033] 6) Loading of quercetin (Que): 10 mg of HMSN-TK was dispersed in anhydrous ethanol, 10 mg of quercetin was added, and the mixture was stirred at 4°C in the dark for 8 h. The precipitate was then collected by centrifugation and washed with PBS to remove the drug adsorbed on the surface, thus obtaining drug-loaded nanoparticles (Q@HMSN-TK).

[0034] 7) OMV Extraction and Coating: *E. coli* DH5α was cultured to the late logarithmic growth phase. After centrifugation at 4°C and 4000 g for 20 min, bacterial cells were collected, resuspended in PBS, and then disrupted using ultrasonication (80 W, 10 s operation, 10 s interval, 30 cycles). The disrupted solution was centrifuged at 4°C and 14000 g for 20 min to remove cell debris. The resulting supernatant was further centrifuged at 4°C and 150000 g for 2 h. The resulting precipitate was resuspended in PBS and filtered through a 0.22 μm filter to obtain a bacterial outer membrane vesicle (OMV) suspension, which was stored at 4°C. Q@HMSN-TK was mixed with the OMV suspension at a mass ratio of 1:2 and incubated under gentle shaking conditions (4°C, 1 h) to achieve OMV coating of the nanoparticles. Unbound OMV was removed by centrifugation to obtain Q@HMSN-O.

[0035] 8) Modification of the CXCR4 targeting ligand: 12.57 mg AMD3100 and 50 mg distearate phosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-Mal) were dissolved in 2 mL of methanol / water (7:3, v / v). The pH was adjusted to 7.4 with NaOH solution, and the reaction was carried out at 37°C with stirring for 24 hours (pH was checked every 3 hours during the reaction to ensure that the solution pH was maintained at 7.4) to form a stable thioether bond between the two. The reaction solution was then transferred to a dialysis bag (MWCO 2000Da) and dialyzed with PBS buffer (pH=7.4) at 4°C for 24 hours (the buffer was changed every 6 hours) to remove unreacted small molecules and solvent. After lyophilization, a white solid of the targeting molecule DSPE-PEG-AMD3100 was obtained. 1 H-NMR confirmed its structure; then Q@HMSN-O and DSPE-PEG-AMD3100 were mixed in PBS at a mass ratio of 50:1 and incubated at 4°C for 2 hours to allow the hydrophobic end of DSPE to insert into the phospholipid bilayer of OMV, thereby exposing AMD3100 on the surface of the nanosystem, and finally obtaining the complete nano-drug delivery system Q@HMSN-O@A.

[0036] 1. Take a sample solution containing 100 μg / mL Q@HMSN-O@A, drop it onto a copper grid, and after the sample dries naturally, observe the morphology of the nanoparticles under a transmission electron microscope. The results are shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that the particle size of Q@HMSN-O@A is about 200nm, with uniform distribution and good dispersibility.

[0037] 2. The hydrated particle size and distribution of SiO2, SiO2@MSN core-shell structure, HMSN, HMSN-NH2, HMSN-TK, Q@HMSN-TK, and Q@HMSN-O@A nanoparticles were determined using a Malvern particle size analyzer. The results are shown in [Figure number missing]. Figure 2 .Depend on Figure 2 It can be seen that its particle size distribution is relatively uniform, with the size of Q@HMSN-O@A being approximately 220 nm.

[0038] Example 2: Efficiency Verification of Neutrophil Loading in Q@HMSN-O@A Neutrophils were isolated from the peripheral blood of healthy C57BL / 6 mice. FITC-labeled Q@HMSN-O@A was co-incubated with neutrophils at 37°C for 0, 30, 60, 90, and 120 min, respectively. Fluorescence co-localization detection was then performed, and the results are shown below. Figure 3 .Depend on Figure 3 It is evident that after 90 minutes of co-incubation, most Q@HMSN-O@A cells had been phagocytosed by neutrophils.

[0039] Example 3 Blood-brain barrier (BBB) ​​penetration experiment A 24-well Transwell in vitro blood-brain barrier model was constructed (bEND.3 cells were seeded in the upper chamber). Cy5-labeled Q@HMSN-TK, Q@HMSN-O, and Q@HMSN-O@A were pre-incubated with or without neutrophils for 90 minutes according to Table 1. Then, these were added to the upper chamber of the model (the lower chamber was supplemented with serum-free DMEM medium, and some groups received FMLP chemokine). After incubation at 37°C for 4 hours, Cy5 fluorescence intensity was detected using a small animal imaging system. The results are shown in Table 1. Figure 4 .

[0040] Table 1

[0041] Figure 4The results showed that, without FMLP, the lower chamber fluorescence intensity was low in all groups regardless of whether they were pre-incubated with neutrophils, indicating that the materials themselves and neutrophils had difficulty actively crossing the BBB in a resting state. With FMLP chemotactic stimulation, the lower chamber fluorescence intensity of the Q@HMSN-O+neutrophil group (3+N+f) and the Q@HMSN-O@A+neutrophil group (4+N+f) was significantly higher than that of the other groups. This demonstrates that neutrophils can effectively phagocytose nanomaterials (Q@HMSN-O and Q@HMSN-O@A) with bacterial outer membrane vesicles on their surface. Under the induction of inflammatory chemotactic signals simulated by FMLP, neutrophils carrying these materials can cross the BBB model in vitro and enter the lower chamber. This provides experimental evidence that neutrophils can serve as live-cell carriers to deliver therapeutic drugs to ischemic brain regions (with inflammatory chemotactic environments).

[0042] Example 4: Immunofluorescence images of Q@HMSN-O@A regulating microglial M2 polarization: First, BV2 cells were subjected to oxygen-glucose-deficiency (OGD) treatment, i.e., cultured in glucose-free DMEM for 6 hours, then replaced with DMEM containing different materials (HMSN, Q@HMSN-TK, Q@HMSN-O, and Q@HMSN-O@A, all at a concentration of 100 μg / mL), and reoxygenated for 12 hours. After treatment, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes; then permeabilized with 0.1% Triton X-100 for 10 minutes, followed by blocking with 5% BSA for 30 minutes; then, the cells were incubated overnight at 4°C with primary antibodies against Iba1, anti-CD86, and anti-CD206, followed by incubation with fluorescently labeled secondary antibodies and DAPI at 37°C for 1 hour. Finally, images were acquired using a fluorescence microscope, and the fluorescence intensity of each group was analyzed using ImageJ software. The results are shown in [Figure number missing]. Figure 5 .

[0043] Figure 5 The results showed that Q@HMSN-TK, Q@HMSN-O, and Q@HMSN-O@A could significantly inhibit microglia polarization to M1 type (decreased CD86 expression) and promote microglia polarization from M1 type to M2 type (increased CD206 expression).

[0044] Example 5: Treatment of cerebral infarction in MCAO model mice with Q@HMSN-O@A A mouse model of cerebral ischemia due to midbrain artery occlusion / reperfusion (MCAO / R) was first established. Then, different nanoparticles (Q@HMSN-TK, Q@HMSN-O, and Q@HMSN-O@A, at a concentration of 2 mg / mL and a dose of 15 mg / kg) were injected via the tail vein. Mice were sacrificed 24 hours after administration, and brain tissue was harvested, frozen at -80℃ for 5 minutes, and cut into 1 mm thick sections along the coronal plane. The sections were stained in 37℃, 2% TTC solution (prepared with PBS), and photographed. The infarct volume was calculated using ImageJ software. The results are shown below. Figure 6 .

[0045] Figure 6 The results showed that the cerebral infarction volume of mice in the Q@HMSN-O@A group was significantly smaller than that in the Q@HMSN-TK group and the Q@HMSN-O group, indicating that it had the best therapeutic effect on this cerebral ischemia model.

[0046] Example 6 Material Safety Verification Male C57BL / 6 mice were acclimatized for one week and then divided into a normal control group and a treatment group, with 6 mice in each group. The treatment group received a tail vein injection of Q@HMSN-O@A (concentration 2 mg / mL, dose 15 mg / kg) every 48 hours. Seven days later, the mice were euthanized by cervical dislocation, and their hearts, livers, spleens, lungs, kidneys, and brains were collected. After fixation in paraformaldehyde for 24 hours, the samples were embedded in paraffin and analyzed by HE staining. The results are shown below. Figure 7 .

[0047] Figure 7 The results showed that Q@HMSN-O@A treatment did not cause significant organ damage in mice.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A nanomedicine delivery system for treating ischemic stroke, characterized in that, The nano-drug delivery system uses hollow mesoporous silica nanoparticles as the core carrier, modifies the surface of the nanoparticles with ketethiol, loads quercetin onto the nanoparticles, and then co-incubates them with bacterial outer membrane vesicles, so that the bacterial outer membrane vesicles coat the surface of the nanoparticles. Then, AMD3100 targeting CXCR4 is modified on the surface of the nanoparticles, thus forming a nano-assembly with a "shell-core" structure.

2. A method for preparing the nano-drug delivery system as described in claim 1, characterized in that, Includes the following steps: (1) Hollow mesoporous silica nanoparticles were synthesized using a template method; (2) HMSN-TK was obtained by chemically coupling ketethiol to modify the surface of hollow mesoporous silica nanoparticles; (3) Quercetin was loaded into the mesoporous cavity of HMSN-TK by impregnation method to obtain Q@HMSN-TK; (4) Q@HMSN-TK was co-incubated with bacterial outer membrane vesicles to coat the surface of the nanoparticles with bacterial outer membrane vesicles, thus obtaining Q@HMSN-O; (5) By modifying AMD3100 on the outer layer of Q@HMSN-O using bioconjugation technology, a nano-drug delivery system Q@HMSN-O@A was obtained.

3. The method for preparing the nano-drug delivery system according to claim 2, characterized in that, In step (2), hollow mesoporous silica nanoparticles are first modified with 3-aminopropyltriethoxysilane to form amino-modified hollow mesoporous silica nanoparticles, which are then dispersed in DMSO. 3,3'-(propane-2,2-diylbis(thioalkyldiyl))dipropionic acid, N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are ultrasonically mixed in DMSO. The two solutions are then mixed and triethanolamine is added as a catalyst. After stirring at room temperature for 24 hours, HMSN-TK with ketethiolated bonds on the surface is obtained after washing.

4. The method for preparing the nano-drug delivery system according to claim 3, characterized in that, The ratio of hollow mesoporous silica nanoparticles, 3-aminopropyltriethoxysilane, 3,3'-(propane-2,2-diylbis(thioalkyldiyl))dipropionic acid, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and triethanolamine used was 50 mg:50 μL:32 mg:22.4 mg:36 mg:10 μL.

5. The method for preparing the nano-drug delivery system according to claim 2, characterized in that, In step (3), HMSN-TK is dispersed in anhydrous ethanol, and then quercetin is added at a mass ratio of 1:1 to HMSN-TK. After stirring at 4°C in the dark for 8 hours, the precipitate is collected by centrifugation and washed with PBS to obtain drug-loaded Q@HMSN-TK.

6. The method for preparing the nano-drug delivery system according to claim 2, characterized in that, The mass ratio of Q@HMSN-TK to bacterial outer membrane vesicles used in step (4) is 1:2; the co-incubation is carried out at 4°C with shaking for 1 hour.

7. The method for preparing the nano-drug delivery system according to claim 2, characterized in that, In step (5), AMD3100 is first synthesized into the targeting molecule DSPE-PEG-AMD3100 through the Michael addition reaction of maleimide-thiol. Then, it is mixed with Q@HMSN-O in PBS at a mass ratio of 1:50 and incubated at 4°C for 2 hours to obtain the nano-drug delivery system Q@HMSN-O@A.

8. The application of the nano-drug delivery system as described in claim 1 in the preparation of drugs for treating ischemic stroke, characterized in that, The quercetin loaded in the nano-drug delivery system can treat ischemic stroke through a cascade mechanism of "neutrophil loading - CXCR4 active targeting - ROS-responsive drug release".

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