Artemisia apiacea extracellular vesicle and preparation method thereof, pharmaceutical composition and medicine for treating cerebral apoplexy, and application of Artemisia apiacea extracellular vesicle in preparation of medicine for treating cerebral apoplexy

Abstract

The invention discloses a preparation method of artemisia apiacea extracellular vesicles, which comprises the following steps: step 1, pre-treating artemisia apiacea, carrying out primary centrifugal treatment, removing large plant tissues and cell debris, carrying out secondary centrifugal treatment, filtering by a needle filter, and collecting; step 2, transferring the filtered suspension to a sucrose density gradient solution, then carrying out third centrifugal treatment, collecting the solution with the concentration of 30% in the layer, and carrying out fourth centrifugal treatment to obtain artemisia apiacea extracellular vesicles; the invention further discloses an artemisia apiacea extracellular vesicle, a pharmaceutical composition for treating cerebral apoplexy, a medicine and application of the artemisia apiacea extracellular vesicle in preparation of the medicine for treating cerebral apoplexy. The naturally-sourced vesicle delivery system provided by the invention is expected to have higher safety and lower toxic and side effects, and the enrichment concentration of the therapeutic component at the focus part of cerebral apoplexy is improved, so that the bioavailability and the treatment efficiency of the medicine are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of stroke drug preparation, specifically to an artemisinin extracellular vesicle and its preparation method, a stroke treatment drug composition, a drug, and its application in the preparation of stroke treatment drugs. Background Technology

[0002] Stroke is an acute cerebrovascular disease with high incidence, high disability rate, and high mortality. Its pathological mechanisms are complex, mainly including post-ischemic inflammatory response, oxidative stress, blood-brain barrier disruption, and neuronal apoptosis. Currently, clinical treatments (such as thrombolysis and thrombectomy) can effectively achieve vascular recanalization, but they have strict treatment time windows and cannot effectively address secondary damage caused by reperfusion, resulting in most survivors still facing severe neurological deficits and sequelae. Therefore, developing novel treatment strategies that can effectively penetrate the blood-brain barrier, regulate the post-stroke pathological process at multiple targets, and promote neurological repair is an urgent need and a major challenge in current medical research.

[0003] Artemisinin and its derivative artesunate, derived from the traditional Chinese medicine Artemisia annua L., have been proven to possess a variety of pharmacological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects, demonstrating great potential for the treatment of stroke. However, its inherent problems, such as poor solubility, low bioavailability, rapid metabolism in vivo, and difficulty in effectively accumulating in the affected brain, severely limit its clinical translation and application.

[0004] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles naturally secreted by cells. As natural endogenous drug delivery carriers, they possess unique advantages such as low immunogenicity, high biocompatibility, excellent targeting, and easy crossing of biological barriers (such as the blood-brain barrier). Furthermore, the active substances carried within the vesicles, such as proteins, nucleic acids, and lipids, can also play important therapeutic roles.

[0005] The Chinese patent on Artemisia annua exosomes, their extraction method, and their use in antitumor therapy specifically describes the Artemisia annua exosomes extracted from Artemisia annua. These exosomes exhibit excellent antitumor effects, significantly inhibiting tumor growth, and their tumor-inhibiting effect is significantly superior to that of artemisinin, a common extract from Artemisia annua. However, this patent suffers from insufficient purity of the exosomes. Furthermore, the patent does not investigate their impact on stroke. Therefore, issues regarding exosome purity and stroke treatment urgently need to be addressed. Summary of the Invention

[0006] This invention designs and develops an artemisinin extracellular vesicle ADNV, and the purpose of this invention is to provide a natural endogenous drug delivery carrier.

[0007] This invention designs and develops a method for preparing extracellular vesicles of Artemisia annua. The purpose of this invention is to solve the purity problem that exists in the preparation process of extracellular vesicles of Artemisia annua.

[0008] This invention designs and develops an application of artemisinin extracellular vesicles in the preparation of drugs for treating stroke. The purpose of this invention is to utilize the natural targeting properties of extracellular vesicles to solve the problems of low delivery efficiency and limited therapeutic effect of artemisinin active ingredients in the prior art, enhance its ability to cross the blood-brain barrier, and thus significantly improve the bioavailability and therapeutic efficiency of the drug.

[0009] This invention also includes the design and development of a drug for treating stroke.

[0010] The present invention also designed and developed a pharmaceutical composition for treating stroke.

[0011] The technical solution provided by this invention is as follows:

[0012] A method for preparing extracellular vesicles of Artemisia annua, comprising the following steps:

[0013] Step 1: After pretreating Artemisia annua, perform the first centrifugation to remove large plant tissues and cell debris, then perform the second centrifugation. Add phosphate buffered saline to the sample containing the supernatant, filter it through a needle filter, and collect it.

[0014] Step 2: After transferring the filtered suspension to a sucrose density gradient solution, perform a third centrifugation. Collect the solution in the 30% concentration layer and perform a fourth centrifugation. Add phosphate buffered saline to the sample containing the discarded supernatant to obtain the Artemisia annua extracellular vesicles.

[0015] Preferably, in step one, the pretreatment process includes: washing the fresh artemisia three times with purified water, then placing the plant in a phosphate buffer solution and chopping it at high speed for 5 minutes.

[0016] Preferably, in step one, the conditions for the first centrifugation are: centrifugation at 200×g for 10 minutes, centrifugation at 2000×g for 20 minutes, and centrifugation at 10000×g for 30 minutes in sequence.

[0017] Preferably, in step one, the conditions for the second centrifugation are centrifugation at 150,000 × g for 2 hours at a temperature of 4°C.

[0018] Preferably, in step two, the conditions for the third and fourth centrifugation treatments are both centrifugation at 150,000 × g for 2 hours at a temperature of 4°C.

[0019] Preferably, in step two, the concentrations of the sucrose density gradient solution are 15%, 30%, 45%, and 60%, respectively.

[0020] An artemisia annua extracellular vesicle, prepared using the aforementioned method for preparing artemisia annua extracellular vesicles;

[0021] Qualitative analysis of the extracellular vesicles of Artemisia annua revealed the following cations: di(2-ethylhexyl) phthalate, quercetin, caprolactam, galactose, chlorogenic acid, tagatose, D-proline, nobiletin, kaempferol, and cryptochlorogenic acid; and

[0022] Qualitative analysis of the extracellular vesicles of Artemisia annua revealed anions including: oleic acid, trans-11-octadecenoic acid, linoleic acid, cis-9-palmitic acid, myristic acid, nonanoic acid, sucrose, trehalose, α-linolenic acid, and undecanoic acid.

[0023] A pharmaceutical composition for treating stroke, comprising the artemisinin extracellular vesicles.

[0024] A drug for treating stroke, comprising the aforementioned artemisinin extracellular vesicles.

[0025] An application of artemisinin extracellular vesicles in the preparation of a drug for treating stroke, using the aforementioned artemisinin extracellular vesicles.

[0026] The beneficial effects of this invention are as follows:

[0027] 1. This invention provides a novel, efficient, and stable drug delivery system by providing a method for preparing Artemisia annua extracellular vesicles and the prepared Artemisia annua extracellular vesicles: using extracellular vesicles derived from Artemisia annua cells as natural nanocarriers, which themselves are derived from medicinal plants and have inherent biocompatibility and synergy with the active ingredients of Artemisia annua, it is expected to efficiently load and deliver therapeutic substances (such as flavonoids, polysaccharides, and their derivatives) in Artemisia annua;

[0028] 2. This invention utilizes the ability of artemisinin extracellular vesicles to enhance the targeting and bioavailability of therapeutic drugs. By leveraging the natural targeting properties of extracellular vesicles, the concentration of therapeutic components at the lesion site of stroke is increased, thereby significantly improving the bioavailability and therapeutic efficiency of the drug.

[0029] 3. This invention provides the application of artemisinin extracellular vesicles in the preparation of stroke drugs. Compared with synthetic nanocarriers and direct use of artemisinin extracts, the natural vesicle delivery system provided by this invention is expected to have higher safety and lower toxicity, providing stroke patients with a novel and more promising biological treatment option. Attached Figure Description

[0030] Figure 1a This is a flowchart illustrating the separation process of the ADNVs preparation method described in this invention.

[0031] Figure 1b This is a TEM image of the ADNVs described in this invention.

[0032] Figure 1c This is a schematic diagram illustrating the detection of ADNVs particle size and concentration using NTA as described in this invention.

[0033] Figure 1d This is a schematic diagram illustrating the potential detection of ADNVs via NTA according to the present invention.

[0034] Figure 1e This is a schematic diagram of protein separation of ADNVs described in this invention.

[0035] Figure 1f This is a schematic diagram of the TIC icon peaks for the top 10 anions selected based on their scores, as described in this invention.

[0036] Figure 1g This is a schematic diagram of the TIC icon peaks for the top 10 cations selected based on their scoring values ​​according to the present invention.

[0037] Figure 2a The invention describes the detection of fluorescence signals in the brain, lungs, liver, kidneys, and spleen of mice injected with DiR-labeled ADNVs via the tail vein.

[0038] Figure 2b This is a graph showing the changes in fluorescence intensity in the brain, lung, liver, kidney, and spleen at different time points after intravenous injection of DiR-labeled ADNVs into mice as described in this invention.

[0039] Figure 2c This is a pathological diagram showing the co-localization of ADNVs with astrocytes (GFAP), neurons (NeuN), and microglia (Iba-1) as described in the invention.

[0040] Figure 3a The survival curve diagram described in the invention.

[0041] Figure 3b This is a graph showing the experimental results of the neurological function score (mNSS) described in this invention.

[0042] Figure 3c The diagram shows the experimental results of the wire hanging test described in this invention.

[0043] Figure 3d This is a diagram showing the experimental results of the cornering method described in this invention.

[0044] Figure 3e This is a schematic diagram illustrating the observation of brain tissue damage after HE staining as described in this invention.

[0045] Figure 3f This is a schematic diagram illustrating the observation of brain tissue damage after Nissl staining as described in this invention.

[0046] Figure 4a This is a schematic diagram illustrating the immunofluorescence staining for observing the tight junction ZO-1 protein in brain tissue as described in this invention.

[0047] Figure 4b This is a schematic diagram illustrating the immunofluorescence staining for observing the Occludin protein in tight junctions of brain tissue, as described in this invention.

[0048] Figure 4c This is a schematic diagram illustrating the immunofluorescence staining for observing Claudin-5 protein in tight junctions of brain tissue as described in this invention. Detailed Implementation

[0049] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0050] This invention provides a method for preparing extracellular vesicles of Artemisia annua, comprising the following steps:

[0051] Step 1, Pretreatment: Wash the fresh Artemisia annua three times with purified water in a beaker, then put the plant into phosphate-buffered saline (PBS) and chop it at high speed for 5 minutes.

[0052] Step 2, Extraction: The obtained juice was centrifuged sequentially at 200×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes to remove large plant tissues and cell debris. The plant supernatant was centrifuged in an ultracentrifuge for 2 hours (4℃, 150000×g), the supernatant was discarded, PBS was added to the sample tube to resuspend the particles, and the mixture was filtered through a 0.45μm needle filter before collection.

[0053] Step 3, Purification: Transfer the filtered suspension to a gradient of sucrose solutions (15%, 30%, 45%, and 60%) and centrifuge for 2 hours (4°C, 150,000 × g). Collect the solution from the 30% layer. Centrifuge again for 2 hours (4°C, 150,000 × g), discard the supernatant, and resuspend the particles in PBS in a sample tube. Artemisia annua extracellular vesicles (ADNVs) can be used fresh or stored at -80°C until reuse.

[0054] This invention also provides an extracellular vesicle of Artemisia annua. The prepared extracellular vesicles of Artemisia annua are characterized by electron microscopy, concentration and particle size, and potential. The components of the extracellular vesicles of Artemisia annua are identified, including quantitative protein analysis, SDS-PAGE analysis, and identification of small molecule compounds. At the same time, the distribution and absorption of the extracellular vesicles of Artemisia annua in vivo are determined and analyzed.

[0055] In another embodiment, the electron microscopy characterization process includes: diluting Artemisia annua extracellular vesicle samples to an appropriate fold for particle size analysis. A copper mesh is placed on filter paper, and 10 μL of exosome suspension is added to the mesh. After standing for 10 minutes, one drop of PBS solution is added. Once the liquid has slightly dried, 4% paraformaldehyde solution is added, and the mixture is allowed to air dry for 3 minutes. The exosomes are then observed under an electron microscope, and their morphology and size are recorded by photograph.

[0056] In another embodiment, the concentration and particle size characterization process includes: diluting Artemisia annua extracellular vesicle samples with electrolyte buffer, followed by detecting single-particle size distribution and absolute concentration using a nanopore electrical impedance analyzer. First, 200 μL of the diluted sample is injected into the sample slot of the nanopore chip, which has been pre-cleaned with washing solution. An equal volume of buffer is injected into the control slot, and the sample is allowed to stand for 5 minutes. Then, a constant voltage of 1.2V is applied, and resistance pulse signals are continuously acquired for 120 seconds at a sampling frequency of 10 kHz. The pulse amplitude threshold is set to 0.5-5V. Finally, concentration and particle size data are collected.

[0057] In another embodiment, the potential characterization included: diluting the artemisinin extracellular vesicle samples with an electrolyte buffer, followed by single-particle surface charge detection using a nanopore electrical impedance analyzer. First, 200 μL of the diluted sample was injected into the sample slot of the nanopore chip, which had been pre-cleaned with a cleaning solution. An equal volume of buffer was injected into the control slot, and the samples were allowed to stand for 5 min. Then, a gradient electric field (0-15 V / cm, step size 3 V / cm) was applied, and data was collected for 60 seconds at each field strength. The signal filtering threshold was set to 100-1000 μs. Finally, the potential data were collected.

[0058] In another embodiment, the protein quantification analysis process includes: mixing reagent A and reagent B (50:1) from the BCA kit to prepare the BCA working solution; taking an appropriate amount of 20 μL of the pre-amplified BSA protein standard solution (2 mg / mL, 40 μL) and diluting it 2-fold with ultrapure water to obtain 6 concentrations; adding 10 μL of Artemisia annua extracellular vesicle sample and each concentration of BSA standard protein to a 96-well microplate, adding 200 μL of BCA working solution to each well, shaking thoroughly, and incubating at 37°C for 30 min. The absorbance value of each sample at 562 nm is measured using a microplate reader, and a standard curve is plotted to calculate the protein concentration of the sample.

[0059] In another embodiment, the SDS-PAGE analysis process includes: separating proteins from Artemisia annua extracellular vesicle samples using sodium dodecyl sulfate polyacrylamide gel electrophoresis. ADNVs (BCA protein quantification is 10 μg protein) are spotted onto prepared 5% stacking gels and 10% separating gels, electrophoretically separated at 120V for 60 min, followed by colloidal Coomassie brilliant blue staining, and the positions of the protein bands are observed and photographed.

[0060] In another embodiment, the small molecule compound identification process includes: sample pretreatment: after lyophilization, two homogenizing beads are added, followed by 500 μL of extraction buffer (methanol:acetonitrile:water (2:2:1, v / v / v)), containing an isotope-labeled internal standard. The sample is vortexed for 30 s; homogenized in a homogenizer (35 Hz, 240 s), then transferred to an ice-water bath and sonicated for 5 min; this sonication step is repeated three times; the sample is placed in a -40℃ freezer for 30 min; centrifuged at 4℃, 12000 rpm for 15 min, and the supernatant is collected; the supernatant is placed in a -40℃ freezer for 10 min; the supernatant is centrifuged at 4℃, 12000 rpm for 15 min; the supernatant is filtered through a 0.22 μm microporous membrane, diluted 100 times, and added to a vial for analysis. The target compounds were separated chromatographically using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UHPLC) with a Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). Phase A of the HPLC was aqueous containing 0.01% acetic acid, and Phase B was isopropanol:acetonitrile (1:1, v / v). The sample pan temperature was 4 °C, and the injection volume was 2 μL. An Orbitrap Exploris 120 mass spectrometer was used for primary and secondary mass spectrometry data acquisition under the control of Xcalibur (version 4.4, Thermo) software. The raw data were converted to mzXML format using ProteoWizard software, and metabolite identification was performed using a collaboratively developed R package. The databases used were Biotree TCM (V 1.0) and BT-HERB (V 1.0). Visualization analysis was then performed using a self-developed R package.

[0061] In another embodiment, the in vivo distribution of artemisinin extracellular vesicles was analyzed, including the following steps:

[0062] Step 1: DiR labeling of ADNVs: ADNVs were labeled with DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide) according to the manufacturer's instructions. 500 μL of ADNVs (2.0 mg / mL) was placed in a 15 mL centrifuge tube, and 10 mL of DiR dye working solution was added. The mixture was thoroughly mixed by pipetting and incubated at 37°C in the dark for 20 min. The tube was then centrifuged at 120,000 g at 4°C for 2 h. The supernatant was discarded, and the ADNVs were resuspended in PBS to wash them. The precipitate was resuspended in 1.25 mL of PBS to obtain DiR-ADNVs. After washing twice in PBS, the DiR-labeled ADNVs were ready for experimental use and stored at 4°C until further notice.

[0063] Step 2: Determining the tissue distribution of ADNVs: C57BL / 6J mice (n=3) were injected intravenously with DiR-ADNVs (8 mg / kg), and the tissue distribution of ADNVs was observed. Rats were sacrificed at 0, 1, 4, 6, and 24 hours post-injection, and major organs such as brain, liver, spleen, lung, and kidney were collected. The intensity of DiR-labeled ADNVs in the isolated organs was measured using an in vivo imaging system (IVIS Spectrum, Caliper, USA) (excitation filter 750 and emission filter 800). The brain was preserved in 4% paraformaldehyde solution for subsequent tissue sectioning.

[0064] In another embodiment, the in vivo uptake of artemisinin extracellular vesicles was analyzed using the following steps: Mice were euthanized with 2.5% v / v Avertin and perfused intracardiacly with ice-cold PBS. Fresh brain tissue was isolated and fixed in PLP buffer (0.075 M lysine, 0.37 M sodium phosphate, 2% formaldehyde, and 0.01 M NaIO4). After dehydration with 30% sucrose solution, the tissue was embedded in a discarded mold containing optimal cutting temperature (OCT) medium. The samples were frozen overnight at -80°C. Next, the OCT-mounted tissue was transferred to a freezer at -20°C and then cut into 8 μm thick sections using a cryosurgical knife. The sections were thawed and mounted on charged microscope slides, dried on a heating block at 37°C, and blocked with 5% BSA solution containing 0.3% Triton X-100. Diluted primary antibodies (anti-GFAP, affinity, DF6040; anti-NeuN, Wuhan Sanying, 66836-1-ig; anti-lba1, Wuhan Sanying, cl488-81728) were applied and incubated overnight at 4°C. After rinsing twice in PBS, the slides were stained in the dark for 1 hour with secondary antibodies conjugated to fluorescent dyes (Wuhan Sanying, SA00013-2). DAPI staining was performed before covering the slides with anti-fading mounting buffer. Images were visualized using a Nikon A1R-A1 confocal microscope equipped with a digital image analysis system (CSIM130, Beijing Century Sunny Technology Co., Ltd.).

[0065] This invention also provides an application of artemisinin extracellular vesicles in the preparation of drugs for treating stroke.

[0066] This invention uses animal experiments, hematoxylin-eosin staining experiments, Nissler staining experiments, and immunofluorescence staining experiments to determine the application of artemisinin extracellular vesicles in the preparation of drugs for treating stroke.

[0067] The specific process of animal model preparation includes:

[0068] Male C57BL / 6J mice, weighing 19-23g, were used in the experiment. Twelve hours prior to surgery, the mice were fasted but allowed free access to water. After isoflurane gas anesthesia, the neck skin was disinfected, and a longitudinal incision was made along the midline. The neck muscles were bluntly dissected to fully expose the right common carotid artery (CCA), and the accompanying vagus nerve was thoroughly dissected. The bifurcation between the external carotid artery (ECA) and internal carotid artery (ICA) was located distally and separated using forceps. The proximal ends of the ECA and CCA were ligated with surgical sutures, and the ICA was temporarily clamped with an arterial clamp. A suture was prepared distal to the CCA. A small incision was made at the CCA, 3mm from the bifurcation of the ECA and ICA. A suture plug was inserted, the arterial clamp was released, and the suture plug was advanced through the ICA to the origin of the middle cerebral artery. The suture plug was inserted to a length of approximately 12mm. The suture at the CCA was tied tightly to prevent bleeding. The incision was sutured and disinfected with povidone-iodine. Throughout the occlusion phase, the animal's body temperature was maintained at 37±0.5℃ using a heating pad. After 60 minutes of ischemia, the mice were re-anesthetized, and the suture plug was slowly removed to restore blood flow and achieve reperfusion. Sham-operated mice underwent the same procedure, but without the insertion of the suture plug into the ICA.

[0069] 1. Drug preparation, experimental grouping, and administration: Artemisinin (ART) was dissolved sequentially in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% ddH2O. First, it was dissolved in DMSO to prepare a 5.56 mg / mL stock solution. After dissolving until clear, the remaining solubilizers were added sequentially to prepare a 0.556 mg / mL working solution, prepared fresh each time.

[0070] All mice were randomly divided into 6 groups: sham operation group (Sham), model group (tMCAO), tMCAO + exosome low-dose group (ADNVs / L) 2 mg / kg, tMCAO + exosome medium-dose group (ADNVs / M) 4 mg / kg, tMCAO + exosome high-dose group (ADNVs / H) 8 mg / kg and tMCAO + artesunate group (ART) 150 mg / kg.

[0071] Mice were given exosomes (via tail vein injection) after recovery from surgery. Mice in the Sham group and tMCAO group were given the same volume of physiological saline.

[0072] 2. Neurological deficit scoring: Neurological function was assessed in mice 24, 48, and 72 hours after tMCAO reperfusion. The mNSS (modified neurological score) covered four aspects: motor, sensory, reflex, and balance, with a maximum total score of 18 points, while 0 points indicated normal neurological function. As shown in Table 1, higher scores indicated more severe neurological dysfunction.

[0073] Table 1 Neurological deficit scores

[0074]

[0075]

[0076] 3. Survival analysis: Data were recorded daily for 3 days, and the survival rate of mice was analyzed using Kaplan-Meier curves.

[0077] Wire Hang Test: The wire hang test is used to assess the upper limb muscle strength of mice. It records the time a mouse can grip a wire with its forelimbs and remain suspended without falling. The mouse is placed on a 9-inch wire 50cm above the ground, with a protective pad placed below to prevent injury in case of a fall. The time it takes to fall from the wire is recorded and scored, with 60 seconds being the longest possible time. A score of 0 is given for falling instantly without attempting to maintain balance; 1 point is given for attempting to maintain balance but falling within 20 seconds; 2 points are given for attempting to maintain balance but falling within 20-40 seconds; 3 points are given for attempting to maintain balance but falling within 40-60 seconds; and 4 points are given for maintaining balance for more than 60 seconds.

[0078] Cornering Test: The cornering test measures sensorimotor impairment and quantifies a mouse's turning preference (right or left) when approaching a 30-degree angle. Mice without brain ischemia-reperfusion injury turn left / right at similar frequencies, but animals with brain ischemia-reperfusion injury tend to turn to the side of the injury (right). A 30-degree angle is constructed using two 30 cm × 20 cm × 1 cm pieces of cardboard for the cornering test. The mouse is allowed to enter the 30-degree angle; to leave the angle, the mouse can turn left or right, which is recorded. The test is repeated 10 times, with at least 30 seconds between each test (note: ventral folding or horizontal turning is excluded; the mouse is not immediately picked up after each turn, so they do not perceive their dominant turning response), and the percentage of right turns is calculated. Note: The animal's turning choice is recorded; turns without standing are not recorded.

[0079] Hematoxylin-eosin staining: Preparation of paraffin sections of brain tissue: Paraffin sections were cut into 5µm thick sections using a paraffin microtome and baked in a slide oven (60℃) for 2 hours; Dewaxing to water sequence: Immersed in xylene I and II for 10 min each, then in 100% ethanol I and II for 5 min each, followed by immersion in 95% ethanol I and 95% ethanol II for 5 min each, 85% ethanol for 5 min each, and 75% ethanol for 5 min each, and finally rinsed with running water for 5 min; Hematoxylin staining: Immersed in hematoxylin staining solution for 3 min to stain the cell nuclei purple, and rinsed with running water for 5 min; Differentiation: Differentiated with 0.1% hydrochloric acid-ethanol for 10 s, and rinsed with running water for 3 min; Bluing: The sections were placed in PBS solution to re-blue for 3 min, and rinsed with running water for 2 min; Eosin staining: Stained with eosin staining solution for 1 min, and rinsed with running water for 2 min; Dehydration and clearing: Cleared in anhydrous ethanol for 1-2 s, and immediately placed in xylene; Mounting: Mounted with neutral resin.

[0080] Nissell staining: Preparation of paraffin sections of brain tissue: Cut paraffin into 5µm thick sections using a paraffin microtome, and bake the sections in a slide oven (60℃) for 2 hours; Dewaxing to water sequence: Immerse in xylene I and II for 10 min each, immerse in 100% ethanol I and II for 5 min each, then immerse in 95% ethanol I and 95% ethanol II for 5 min each, 85% ethanol for 5 min each, and 75% ethanol for 5 min each, and finally rinse with running water for 5 min; Staining: Place the sections in a 60°C incubator and immerse in Nissell staining solution for 40 min, then rinse with running water for 1 min; Differentiation: Rapidly differentiate with 95% ethanol for 10 s; Dehydration and clearing: Clear the sections in anhydrous ethanol for 1-2 s, then immediately immerse in xylene; Mounting: Mount the sections with neutral resin.

[0081] Immunofluorescence staining: Section warming: Remove frozen sections from the -20°C freezer and place them at room temperature for 15 min; Rinsing: Immerse tissue sections in PBS solution and wash on a shaker for 5 min at 100 rpm; Blocking: Block sections with BSA containing 10% goat serum for 45 min, then spin dry the blocking serum; Primary antibody incubation: Cover the entire section with ZO-1 antibody, Occludin, and Claudin-5 antibody, place in a humidified chamber, and incubate overnight at 4°C. All antibodies were diluted 1:200 with primary antibody dilution buffer. Washing: Tissue sections were washed three times with PBS for 10 minutes each time, then the surface moisture was removed. Secondary antibody incubation: Fluorescent secondary antibody was added under light-protected conditions, and the sections were incubated in a humidified chamber for 1 hour. The antibody was diluted 1:300 with secondary antibody dilution buffer. Washing: Tissue sections were washed three times with PBS for 10 minutes each time, then the surface moisture was removed. Mounting: Sections were mounted with mounting solution containing fluorescence quencher (including DAPI). Protein expression was observed using a digital slide scanner. Brain tissue sections were observed at 400× magnification. Analysis: Three non-overlapping regions were randomly selected from each section, and the average fluorescence intensity of tight junction proteins was analyzed using ImageJ software.

[0082] Example

[0083] This invention provides a method for preparing extracellular vesicles of Artemisia annua, comprising the following steps:

[0084] Step 1, Pretreatment: Wash the fresh Artemisia annua three times with purified water in a beaker, then put the plant into phosphate-buffered saline (PBS) and chop it at high speed for 5 minutes.

[0085] Step 2, Extraction: The obtained juice was centrifuged sequentially at 200×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes to remove large plant tissues and cell debris. The plant supernatant was centrifuged in an ultracentrifuge for 2 hours (4℃, 150000×g), the supernatant was discarded, PBS was added to the sample tube to resuspend the particles, and the mixture was filtered through a 0.45μm needle filter before collection.

[0086] Step 3, Purification: Transfer the filtered suspension to a gradient of sucrose solutions (15%, 30%, 45%, and 60%) and centrifuge for 2 hours (4°C, 150,000 × g). Collect the solution from the 30% layer. Centrifuge again for 2 hours (4°C, 150,000 × g), discard the supernatant, and resuspend the particles in PBS in a sample tube. Artemisia annua extracellular vesicles (ADNVs) can be used fresh or stored at -80°C until reuse.

[0087] In this embodiment, the precipitate obtained by ultracentrifugation was weighed and the yield was calculated. Approximately 5 mg of ADNVs could be separated from every 150 g of Artemisia annua.

[0088] Test case

[0089] like Figure 1a As shown, Artemisia annua cells were isolated as vesicles (ADNVs) from the juice of fresh Artemisia annua leaves using a combination of differential centrifugation and ultracentrifugation.

[0090] like Figure 1b As shown, the structure of extracellular vesicles in Artemisia annua can be observed using a transmission electron microscope.

[0091] like Figure 1c , Figure 1d As shown, the particles are concentrated at approximately 148.9 nm, with a concentration of 9.3E+11 (particles / mL), and the particle size distribution exhibits a unimodal normal distribution. The detection potential for ADNVs is -18.40 mV.

[0092] like Figure 1e As shown, ADNVs proteins were separated on a 10% separating gel by SDS-PAGE gel electrophoresis. ADNVs proteins are widely distributed. Coomassie brilliant blue staining showed that ADNVs protein bands existed in the range of 15kDa-180kDa, but they were mainly concentrated in the range of 35kDa-70kDa.

[0093] Qualitative analysis of small molecule compounds within ADNVs was performed using HPLC. Raw data were converted to mzXML format using ProteoWizard software, and metabolite identification was performed using MetDNA2 (https: / / github.com / ZhuMetLab / MetDNA2) with Biotree TCM (V 1.0) and BT-HERB (V 1.0) databases. Visualization analysis was then performed using R (ggplot2). Figure 1f , 1gAs shown, based on the scoring values, the top 10 substances were selected for TIC icon peak examples. We detected the following cations: (di(2-ethylhexyl) phthalate DEHP, quercetin, caprolactam, galactose, chlorogenic acid, tagatose, D-proline, nobiletin, kaempferol, cryptochlorogenic acid), and the following anions: (oleic acid, trans-vaccenic acid, linoleic acid, cis-9-palmitoleic acid, myristic acid, pelargonic acid, sucrose, trehalose, alpha-linolenic acid, undecanoic acid).

[0094] like Figure 2a , Figure 2b As shown, major organs of mice, including the brain, liver, spleen, lungs, and kidneys, were subjected to IVIS imaging. DiR-labeled ADNVs were injected into the tail vein of the mice. One hour later, strong fluorescence signals were detected in the brain and liver. After 4 hours, the fluorescence signals in the organs other than the liver gradually weakened. After 24 hours, almost no fluorescence signals were detected in the spleen, kidneys, and lungs. A slight signal was present in the brain, but the fluorescence signal in the liver did not show a strong weakening trend.

[0095] like Figure 2c As shown, after intravenous injection in mice, brain sections and fluorescence staining were observed. It was found that ADNVs could co-localize with astrocytes (GFAP), neurons (NeuN), and microglia (Iba-1). The three cell types showed green fluorescence, while ADNVs showed red fluorescence. This indicates that they can be taken up by astrocytes, neurons, and microglia.

[0096] like Figures 3b-3d As shown, the results of the string hanging test and corner turning test indicate that mice experiencing ischemic brain injury also show significant impairment in limb sensation, balance, and motor function. On day 3 after tMCAO, these neurological and behavioral deficits were effectively improved in both the ADNVs / H and ART groups (p<0.05), but the effects were not significant in the ADNVs / L and ADNVs / M groups. Figure 3aAs shown, the survival curve results also indicate that the ADNVs / H group and the ART group can significantly improve the survival rate of tMCAO mice.

[0097] like Figure 3e , Figure 3f As shown, further observations using HE and Nissl staining revealed that the tMCAO group mice had larger cerebral infarction areas, extensive vacuolar changes in brain tissue, and severe nuclear pyknosis. The reduced number of Nissl bodies indicated significant neuronal cell necrosis. This phenomenon was significantly alleviated after intervention in the ADNVs / H group and the ART group. Therefore, high-dose ADNVs have an ameliorative effect on ischemic brain injury in mice.

[0098] like Figures 4a-4c As shown, the damage to the blood-brain barrier in each group of mice was detected. Immunofluorescence staining was used to observe the expression of tight junction proteins in brain tissue, and further analysis of the blood-brain barrier damage was conducted. ZO-1, Occludin, and Claudin-5 proteins were mainly expressed on the cell membrane of brain microvascular endothelial cells. In the Sham group, the fluorescence signal was strong and the positive expression area was large, indicating that the tight junction structure was intact. In the tMCAO group, the fluorescence signal intensity of ZO-1, Occludin, and Claudin-5 proteins in brain tissue was weakened (P<0.05), and the expression area was reduced. Compared with the tMCAO group, the positive expression area of ​​ZO-1, Occludin, and Claudin-5 proteins in the ADNVs / H group and the ART group tended to recover, and the fluorescence intensity increased (P<0.05), suggesting that the damage to the blood-brain barrier was improved.

[0099] The above experiments show that artemisinin exosomes can be successfully isolated using ultracentrifugation and have been shown to effectively reduce brain damage in stroke treatment.

[0100] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing extracellular vesicles of Artemisia annua, characterized in that, Includes the following steps: Step 1: After pretreating Artemisia annua, perform the first centrifugation to remove large plant tissues and cell debris, then perform the second centrifugation. Add phosphate buffered saline to the sample containing the supernatant, filter it through a needle filter, and collect it. Step 2: After transferring the filtered suspension to a sucrose density gradient solution, perform a third centrifugation. Collect the solution in the 30% concentration layer and perform a fourth centrifugation. Add phosphate buffered saline to the sample containing the discarded supernatant to obtain the Artemisia annua extracellular vesicles.

2. The method for preparing artemisia annua extracellular vesicles as described in claim 1, characterized in that, In step one, the pretreatment process includes: washing the fresh artemisia three times with purified water, then placing the plant in a phosphate buffer solution and chopping it at high speed for 5 minutes.

3. The method for preparing artemisia annua extracellular vesicles as described in claim 1 or 2, characterized in that, In step one, the conditions for the first centrifugation are: centrifugation at 200×g for 10 minutes, centrifugation at 2000×g for 20 minutes, and centrifugation at 10000×g for 30 minutes in sequence.

4. The method for preparing artemisia annua extracellular vesicles as described in claim 3, characterized in that, In step one, the conditions for the second centrifugation treatment are centrifugation at 150,000 × g for 2 hours at a temperature of 4°C.

5. The method for preparing artemisia annua extracellular vesicles as described in claim 3, characterized in that, In step two, the conditions for the third and fourth centrifugation treatments are both centrifugation at 150,000 × g for 2 hours at a temperature of 4°C.

6. The method for preparing artemisia annua extracellular vesicles as described in claim 5, characterized in that, In step two, the concentrations of the sucrose density gradient solution are 15%, 30%, 45%, and 60%, respectively.

7. An extracellular vesicle of Artemisia annua, characterized in that, It was prepared using the method for preparing Artemisia annua extracellular vesicles as described in any one of claims 1-6; Qualitative analysis of the extracellular vesicles of Artemisia annua revealed the following cations: di(2-ethylhexyl) phthalate, quercetin, caprolactam, galactose, chlorogenic acid, tagatose, D-proline, nobiletin, kaempferol, and cryptochlorogenic acid; and Qualitative analysis of the extracellular vesicles of Artemisia annua revealed anions including: oleic acid, trans-11-octadecenoic acid, linoleic acid, cis-9-palmitic acid, myristic acid, nonanoic acid, sucrose, trehalose, α-linolenic acid, and undecanoic acid.

8. A pharmaceutical composition for treating stroke, characterized in that, Including the artemisia extracellular vesicles as described in claim 7.

9. A drug for treating stroke, characterized in that, Including the artemisia extracellular vesicles as described in claim 7.

10. The application of artemisinin extracellular vesicles in the preparation of drugs for treating stroke, characterized in that, Using the artemisia extracellular vesicles as described in claim 7.

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