Brain-targeted delivery carrier as well as preparation method and application thereof

Abstract

The invention provides a brain-targeted delivery carrier as well as a preparation method and application thereof, and belongs to the technical field of biology and medicine. The brain-targeted delivery carrier provided by the invention is prepared from extracellular vesicles and cell membranes of immune cells through a stable hybridization method; the extracellular vesicles are loaded with active pharmaceutical ingredients and / or bioactive substances for treating diseases; the treatment diseases comprise Alzheimer's disease or brain glioma. The brain-targeted delivery carrier provided by the invention has the advantages of small particle size and improved stability, and the brain-targeted delivery carrier provided by the invention has the capability of penetrating an in-vivo biological barrier, and can directly deliver pharmacodynamic substances in extracellular vesicles to target cells in the brain, so that the pharmacodynamic effect is improved, the toxic and side effects are reduced, and an efficient treatment effect is played.

Description

Technical Field

[0001] The present invention belongs to the field of biology and medicine technology, and specifically relates to a brain-targeted delivery carrier and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (EVs) are a type of membrane vesicles secreted by various cells. At present, they are divided into three categories: exosomes, microvesicles, and apoptotic bodies, but the existing separation methods cannot completely separate these three types of EVs. Extracellular vesicles can usually be divided into small extracellular vesicles (<200nm, nanoscale) and large extracellular vesicles (>200nm) according to their particle size. EVs carry bioactive molecules such as proteins, lipids, and nucleic acids, can participate in intercellular communication, and play an important role in a variety of physiological and pathological processes. Although some cell-derived vesicles have natural brain-targeting properties, there are still problems that need to be solved as drug delivery carriers. First, their ability to penetrate the blood-brain barrier is relatively limited; second, extracellular vesicles have a short blood half-life, and after systemic administration, they are easily metabolized and eliminated in the blood, liver, kidneys, etc., resulting in less distribution in the targeted sites. Therefore, how to improve the ability of EVs to cross the blood-brain barrier and prolong the blood half-life of EVs so that they can act on targeted parts of the brain completely, efficiently and safely is an urgent problem to be solved in this field. Summary of the invention

[0003] In view of this, one of the objects of the present invention is to provide a brain-targeted delivery vector having the advantages of small particle size, good stability, easy penetration of the blood-brain barrier, improved efficacy and reduced toxic and side effects.

[0004] The second object of the present invention is to provide a method for preparing the brain-targeted delivery vector.

[0005] The third object of the present invention is to provide the use of the brain-targeted delivery vector in the preparation of products for treating Alzheimer's disease or brain glioma.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a brain-targeted delivery carrier, which is obtained by hybridizing an extracellular vesicle with a cell membrane of an immune cell in the presence of a stabilizer; the extracellular vesicle is loaded with active pharmaceutical ingredients and / or bioactive substances for treating a disease; the disease includes Alzheimer's disease or brain glioma; the stabilizer includes one of mercaptoethanol, dithiothreitol, dithioerythritol, tri-(2-formylethyl)phosphine hydrochloride and reduced glutathione.

[0008] Preferably, the immune cells include macrophages, T lymphocytes, B lymphocytes or neutrophils; and the extracellular vesicles are derived from one or more of stem cells, nerve cells and glial cells.

[0009] Preferably, the stem cells include one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells and placental mesenchymal stem cells; the neural cells include one or more of human neuroblastoma cell lines, cortical neural cells, hippocampal neuronal cells and neural stem cells; the glial cells include one or more of astrocytes, oligodendrocytes and microglia.

[0010] Preferably, when the disease is Alzheimer's disease, the active pharmaceutical ingredients include one or more of stilbene glycosides, rhein, rhein, rhein methyl ether, rhein-1,6-dimethyl ether, icariin, curcumin, gambogic acid, artemisinin, dihydroartemisinin, artesunate, matrine, berberine, sinomenine, angelica coumarin, quercetin, glycyrrhizic acid, liquiritigenin, liquiritigenin, isoliquiritigenin, peony glucoside, platycoside, resveratrol and houttuynia cordata, or derivatives of the active pharmaceutical ingredients.

[0011] Preferably, when the disease is brain glioma, the active pharmaceutical ingredient includes one or more of temozolomide, methylcarbazine, lomustine, vinblastine, vincristine, carmustine, irinotecan, etoposide, cisplatin, carboplatin, methotrexate, docetaxel, paclitaxel, camptothecin, doxorubicin, cyclophosphamide, epirubicin, hydroxycamptothecin, gemcitabine, pentostatin and gambogic acid, or derivatives of the active pharmaceutical ingredient.

[0012] Preferably, the bioactive substance includes one or more of microRNA, lncRNA, protein and lipid.

[0013] The present invention also provides a method for preparing the above-mentioned brain-targeted delivery vector, comprising the following steps: adding 0.1 to 100 mM of a stabilizer to the cell membrane of immune cells, incubating at 37° C. for 0.5 to 4 hours, and centrifuging to obtain a cell membrane precipitate; uniformly mixing the cell membrane precipitate with extracellular vesicles at a protein mass ratio of 0.5 to 5:1 to obtain a brain-targeted delivery vector; the extracellular vesicles are loaded with active pharmaceutical ingredients and / or biologically active substances for treating diseases.

[0014] Preferably, the preparation of the cell membrane of the immune cells comprises the following steps: culturing the immune cells for 12 to 72 hours, centrifuging to obtain the cell pellet, and resuspending the cells to a cell density of 0.5 to 2.5×10 7 / mL, under ice bath conditions, ultrasonically disrupt the cells, mix the disrupted cell suspension with sucrose solution to make the concentration of sucrose solution 0.1-0.5M, and collect the cell membrane precipitate by centrifugation.

[0015] Preferably, the methods for loading extracellular vesicles with active pharmaceutical ingredients and / or biologically active substances for treating diseases include the following two methods: one is to co-culture the active pharmaceutical ingredients with cells, and then obtain the extracellular vesicles loaded with active pharmaceutical ingredients and / or biologically active substances by differential centrifugation; the other is to obtain the extracellular vesicles by differential centrifugation, mix them with the active pharmaceutical ingredients, and treat them by ultrasound, high-pressure homogenization or microporous membrane filtration to incubate the extracellular vesicles loaded with active pharmaceutical ingredients and / or biologically active substances.

[0016] The present invention also provides the use of the above-mentioned brain-targeted delivery carrier in the preparation of a product for treating Alzheimer's disease or brain glioma.

[0017] Beneficial effects of the present invention:

[0018] The brain-targeted delivery vector provided by the present invention has the advantages of small particle size and improved stability. In addition, the brain-targeted delivery vector provided by the present invention has the ability to penetrate biological barriers in the body and can directly deliver the pharmacological substances in the extracellular vesicles to the target cells in the brain, thereby improving the efficacy of the drug, reducing toxic side effects, and exerting a highly effective therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 for the uptake of brain-targeted delivery vehicles by microglia;

[0020] Figure 2 The uptake of brain-targeted delivery vectors by human glioma cells U-87MG;

[0021] Figure 3 is the tumor weight of different groups after administration, *p<0.05, **p<0.01 represent significant differences; compared with each administration group, ## p<0.01. DETAILED DESCRIPTION

[0022] The present invention provides a brain-targeted delivery carrier, which is obtained by hybridizing an extracellular vesicle with a cell membrane of an immune cell in the presence of a stabilizer; the extracellular vesicle is loaded with active pharmaceutical ingredients and / or bioactive substances for treating a disease; the disease includes Alzheimer's disease or brain glioma; the stabilizer includes one of mercaptoethanol, dithiothreitol, dithioerythritol, tri-(2-formylethyl)phosphine hydrochloride and reduced glutathione.

[0023] The present invention uses cells that efficiently secrete extracellular vesicles as parent cells, and adopts the method of co-incubating the active pharmaceutical ingredient with the cell or loading the active pharmaceutical ingredient after the drug to realize the loading of the extracellular vesicle on the drug, and obtains the extracellular vesicle loaded with the active pharmaceutical ingredient and / or the biologically active substance, and the obtained extracellular vesicle contains a phospholipid bilayer, which protects the loaded active pharmaceutical ingredient and / or the biologically active substance from being degraded in the systemic circulation. And the method of hybridizing the cell membrane with the extracellular vesicle across the blood-brain barrier is used to improve the targeted distribution in the brain, and the method of adding a stabilizer is adopted to reduce the particle size of the carrier and improve the stability of the carrier. The brain-targeted delivery carrier provided by the present invention has the following characteristics: ① It has good plasma stability; ② It has the advantages of brain-targeted modification, easy to penetrate the blood-brain barrier, and targeted accumulation at the lesion site; ③ The biologically active substances inside the extracellular vesicles or the loaded active pharmaceutical ingredients efficiently exert the disease treatment effect. The present invention enhances the function of drug substances being internalized by target cells by means of ligand-receptor targeting, enables the drug substances to penetrate biological barriers in the body, and directly delivers the drug substances in extracellular vesicles to the brain and target cells, thereby exerting a highly effective therapeutic effect.

[0024] In the present invention, the immune cells preferably include macrophages, T lymphocytes, B lymphocytes or neutrophils; the extracellular vesicles preferably originate from one or more of stem cells, neural cells and glial cells. In the present invention, the stem cells preferably include one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells and placental mesenchymal stem cells; the neural cells preferably include one or more of human neuroblastoma cell lines, cortical neural cells, hippocampal neuronal cells and neural stem cells; the glial cells preferably include one or more of astrocytes, oligodendrocytes and microglia.

[0025] In the present invention, when the disease is Alzheimer's disease, the active pharmaceutical ingredients preferably include one or more of stilbene glycosides, rhein, rhein, rhein methyl ether, rhein-1,6-dimethyl ether, icariin, curcumin, gambogic acid, artemisinin, dihydroartemisinin, artesunate, matrine, berberine, sinomenine, angelica coumarin, quercetin, glycyrrhizic acid, liquiritigenin, liquiritigenin, isoliquiritigenin, peony glucoside, platycoside, resveratrol and houttuynia cordata, or derivatives of the active pharmaceutical ingredients. When the disease is glioma, the active pharmaceutical ingredient preferably includes one or more of temozolomide, methylcarbazine, lomustine, vinblastine, vincristine, carmustine, irinotecan, etoposide, cisplatin, carboplatin, methotrexate, docetaxel, paclitaxel, camptothecin, doxorubicin, cyclophosphamide, epirubicin, hydroxycamptothecin, gemcitabine, pentostatin and gambogic acid or a derivative of the active pharmaceutical ingredient. In the present invention, the bioactive substance refers to the bioactive substance inside the extracellular vesicle, that is, the bioactive substance derived from the parent cell, preferably including one or more of microRNA, lncRNA, protein and lipid. In the present invention, the extracellular vesicle is preferably a nanoscale extracellular vesicle.

[0026] The present invention also provides a method for preparing the above-mentioned brain-targeted delivery vector, comprising the following steps: adding 0.1 to 100 mM of a stabilizer to the cell membrane of immune cells, incubating at 37° C. for 0.5 to 4 hours, and centrifuging to obtain a cell membrane precipitate; mixing the cell membrane precipitate with extracellular vesicles at a protein ratio of 0.5 to 5:1 to obtain a brain-targeted delivery vector; the extracellular vesicles are loaded with active pharmaceutical ingredients and / or biologically active substances for treating diseases.

[0027] In the present invention, the preparation of the cell membrane of the immune cells preferably comprises the following steps: culturing the immune cells for 12 to 72 hours, centrifuging to obtain the cell pellet, and resuspending the cells to a cell density of 0.5 to 2.5×10 7 / mL, under ice bath conditions, ultrasonically disrupt the cells, mix the disrupted cell suspension with a sucrose solution to make the concentration of the sucrose solution 0.1-0.5M, and centrifuge to collect the cell membrane precipitate. The present invention does not specifically limit the specific method of culturing immune cells, and the conventional method of culturing immune cells can be used. In the present invention, the time for culturing immune cells is preferably 20-50h. After the culture is completed, it is preferably necessary to use trypsin digestion. After the culture is completed, centrifugation is performed, and the centrifugation conditions are preferably 1000×g room temperature centrifugation for 5min to obtain a cell precipitate, and the buffer used for resuspending the cells preferably includes PBS buffer and 1×TM buffer containing 1% PMSF. In the present invention, the power of the ultrasonic disruption is preferably 50-200W, more preferably 70-150W, the time of the ultrasonic disruption is preferably 1-5min, more preferably 2-3min, and the ultrasonic disruption preferably needs to be circulated 3-6 times. The disrupted cell suspension is mixed with a sucrose solution to make the concentration of the sucrose solution preferably 0.2-0.4 M. After centrifugation to collect the cell membrane precipitate, it is preferably washed, and the washing is preferably washed with ice-cold 1×TM buffer containing 0.1-0.5 M sucrose, and centrifuged again to obtain the cell membrane precipitate.

[0028] In the present invention, the method for preparing the extracellular vesicles preferably includes the following steps: culturing parent cells for 12 to 72 hours to obtain a first cell culture solution; replacing the culture medium of the first cell culture solution with a complete culture medium without extracellular vesicles containing or not containing drugs, and continuing to culture the cells for 12 to 72 hours to obtain a second cell culture solution; centrifuging the second cell culture solution to obtain a supernatant; subjecting the supernatant to differential centrifugation, collecting the precipitate to obtain extracellular vesicles, and the extracellular vesicles are nanoscale extracellular vesicles. In the present invention, the extracellular vesicle-free culture medium is a complete culture medium that removes extracellular vesicles and contains 10% fetal bovine serum. In the present invention, the parent cells preferably include one or more of stem cells, neural cells and glial cells. In the present invention, the temperature for culturing the parent cells is preferably 37°C. The culture time of the parent cells in the present invention is preferably 20 to 48 hours. The extracellular vesicles prepared in the prior art are often distributed in the liver in the human body, and other lesions in the human body are less targeted. The present invention uses stem cells, nerve cells or glial cells to prepare extracellular vesicles. There are many types of parent cells, high loading capacity for therapeutic drugs or bioactive substances, and prolonged drug circulation time. It can target multiple lesions in the brain, thereby improving the therapeutic effect of the pharmacological substance. In the present invention, after obtaining the second cell culture fluid, the second cell culture fluid is centrifuged to obtain a supernatant and precipitated cells. The present invention does not specifically limit the parameters of the centrifugation, and conventional methods can be used. In an embodiment of the present invention, the centrifugation condition is 300×g centrifugation for 10 minutes. The supernatant is obtained, and the present invention performs differential centrifugation on the supernatant to collect the precipitate, i.e., small extracellular microvesicles. The temperature of the differential centrifugation of the present invention is preferably 2 to 6°C, more preferably 4°C. The differential centrifugation method of the present invention preferably comprises: subjecting the supernatant to a first centrifugation supernatant, subjecting the first centrifugation supernatant to a second centrifugation to obtain a second centrifugation supernatant, subjecting the second centrifugation supernatant to a third centrifugation to obtain a third centrifugation precipitate, subjecting the third centrifugation precipitate to a fourth centrifugation after resuspending the third centrifugation precipitate to obtain a fourth centrifugation precipitate, and resuspending the fourth centrifugation precipitate to obtain small extracellular microvesicles in the functionalized delivery system of extracellular vesicles. The centrifugal force of the first centrifugation of the present invention is preferably 1500-3000×g, and the first centrifugation time is preferably 10min; the first centrifugation of the present invention can remove dead cells in the cell supernatant. The centrifugal force of the second centrifugation of the present invention is preferably 10000×g, and the second centrifugation time is preferably 30min; the second centrifugation of the present invention facilitates the removal of cell debris in the cell supernatant. The centrifugal force of the third centrifugation of the present invention is preferably 100000-200000×g, and the third centrifugation time is preferably 60-90min. The centrifugal force of the fourth centrifugation of the present invention is preferably 100,000 to 200,000×g, and the time of the fourth centrifugation is preferably 60 to 90 min; the purpose of the fourth centrifugation of the present invention is to obtain extracellular vesicle precipitation.The present invention resuspends the fourth centrifugal precipitate to obtain the nanoscale extracellular vesicles in the brain-targeted delivery vector. The resuspending of the present invention preferably uses PBS solution.

[0029] In the present invention, the method for loading nanoscale extracellular vesicles with active pharmaceutical ingredients and / or bioactive substances for treating diseases preferably includes the following steps: co-culturing the active pharmaceutical ingredients with cells, and then performing differential centrifugation to obtain nanoscale extracellular vesicles loaded with active pharmaceutical ingredients and / or bioactive substances; or after obtaining the nanoscale extracellular vesicles by differential centrifugation, mixing with the active pharmaceutical ingredients, treating with ultrasound, high-pressure homogenization or microporous membrane filtration, and incubating to obtain nanoscale extracellular vesicles loaded with active pharmaceutical ingredients and / or bioactive substances.

[0030] In the present invention, after the extracellular vesicles and the cell membranes of immune cells are mixed to obtain a mixture, it is preferred that the mixture is subjected to one or more of ultrasonic treatment, filtration treatment, vortex treatment and high-pressure homogenization treatment; more preferably, ultrasonic treatment, filtration treatment and vortex treatment. The present invention does not specifically limit the parameters of the ultrasonic treatment, filtration treatment, vortex treatment and high-pressure homogenization treatment, as long as the mixing is uniform.

[0031] In the present invention, after the extracellular vesicles and the cell membranes of immune cells are hybridized, they are preferably freeze-dried to obtain a brain-targeted delivery vector. The freeze-drying method is preferably: 0.5% to 3% mannitol is added to the hybridized sample liquid as a freeze-drying protectant, pre-frozen at -20°C for 4 hours, and transferred to -80°C for freezing for 24 hours. After the freeze dryer is pre-cooled for half an hour, the sample is transferred to a freeze dryer and freeze-dried until there is no moisture, taken out, and stored in a sealed container at 4°C away from light.

[0032] The particle size of the brain-targeted delivery carrier obtained by the preparation method of the present invention is 74 to 131 nm, and the Zeta potential is -36.3 to -27.1 mV.

[0033] The present invention also provides the use of the above-mentioned brain-targeted delivery carrier in the preparation of a product for treating Alzheimer's disease or brain glioma. In the present invention, the product preferably includes a drug. When the product is a drug, the dosage form of the drug preferably includes one or more of an injection, an oral agent, and an external dosage form. The injection of the present invention preferably includes one or more of an intravascular injection, an intramuscular injection, and an in situ injection; the oral agent preferably includes an oral liquid and / or a tablet; the external dosage form preferably includes one or more of a solution, a powder, and an ointment.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1

[0038] Preparation of extracellular vesicles released from bone marrow mesenchymal stem cells and loading of bioactive substances and stilbene glycosides

[0039] DMEM / F12 medium containing 10% fetal bovine serum by mass concentration is recorded as complete medium; the composition of DMEM / F12 medium is DMEM medium and F12 medium mixed in a volume ratio of 1:1.

[0040] (1) Rat bone marrow mesenchymal stem cells with good growth status were inoculated into a cell culture flask at a cell number of 2×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0041] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 72 h in an incubator at 37° C. and 5% by volume of carbon dioxide, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell and cell debris precipitate (the cell and cell debris precipitate is referred to as a cell precipitate, the same below).

[0042] (3) The supernatant obtained in step (2) is centrifuged at 1500×g for 10 min at 4°C, the supernatant is taken, and centrifuged at 10,000×g for 30 min. The supernatant is further taken and centrifuged at 200,000×g for 90 min. The supernatant is removed and the precipitate is resuspended with PBS, and centrifuged again at 200,000×g for 90 min. Finally, the precipitate is resuspended with PBS to obtain extracellular vesicles secreted by bone marrow mesenchymal stem cells, which contain bioactive substances derived from bone marrow mesenchymal stem cells.

[0043] (4) Adding a diphenylethylene glycoside aqueous solution to the extracellular vesicles obtained in step (3) at a ratio of 1 mM diphenylethylene glycoside aqueous solution: 0.5 mg / mL extracellular vesicles, ultrasonicating at 100 W for 15 min under ice bath conditions, and incubating at 37° C. for 1 h to obtain extracellular vesicles loaded with bioactive substances and diphenylethylene glycoside.

[0044] According to sequencing results, the bioactive substances are microRNAs in extracellular vesicles that have therapeutic effects on Alzheimer's disease (AD), including miR-let-7c, miR-122, miR-26a, miR-22 and miR-146b.

[0045] Example 2

[0046] Preparation of extracellular vesicles released from umbilical cord mesenchymal stem cells and loading of bioactive substances and icariin

[0047] DMEM / F12 medium containing 10% fetal bovine serum by mass concentration is recorded as complete medium; the composition of DMEM / F12 medium is DMEM medium and F12 medium mixed in a volume ratio of 1:1.

[0048] (1) Mouse umbilical cord mesenchymal stem cells with good growth status were inoculated into a cell culture flask at a cell number of 1×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0049] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 60 h in an incubator at 37° C. and 5% carbon dioxide by volume, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0050] (3) The same as step (3) of Example 1, obtaining extracellular vesicles secreted by umbilical cord mesenchymal stem cells, which contain bioactive substances derived from umbilical cord mesenchymal stem cells.

[0051] (4) Adding icariin solution to the extracellular vesicles obtained in step (3) at a ratio of 2 mM icariin solution: 0.8 mg / mL extracellular vesicles, vortexing at 2000 rpm for 2 min, and then subjecting the mixture to 3 cycles of high-pressure homogenization at 500 bar, and incubating at 37° C. for 0.5 h to obtain extracellular vesicles loaded with bioactive substances and icariin.

[0052] According to sequencing results, the bioactive substances are microRNAs in extracellular vesicles that have therapeutic effects on AD, including miR-16, miR-23a, miR-103a, miR-221 and miR-423.

[0053] Example 3

[0054] Preparation of extracellular vesicles released from adipose-derived mesenchymal stem cells and loading of bioactive substances and matrine

[0055] DMEM / F12 medium containing 10% fetal bovine serum by mass concentration is recorded as complete medium; the composition of DMEM / F12 medium is DMEM medium and F12 medium mixed in a volume ratio of 1:1.

[0056] (1) Adipose-derived mesenchymal stem cells with good growth status were inoculated into a cell culture flask with a cell number of 5×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0057] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 48 h in an incubator at 37° C. and 5% carbon dioxide by volume, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0058] (3) The same as step (3) of Example 1, obtaining extracellular vesicles secreted by adipose-derived mesenchymal stem cells.

[0059] (4) Adding matrine solution to the extracellular vesicles obtained in step (3) at a ratio of 1.5 mM matrine solution: 0.6 mg / mL extracellular vesicles, filtering repeatedly three times with a 0.45 μm microporous filter membrane, and incubating at 37° C. for 2 h to obtain extracellular vesicles loaded with matrine.

[0060] The bioactive substances were analyzed by mass spectrometry-based proteomics method for proteins in extracellular vesicles. The analysis results showed that IL-8 and VEGF were upregulated and may be proteins related to the treatment of AD.

[0061] Example 4

[0062] Preparation of extracellular vesicles released from human neuroblastoma cell line SH-SY5Y and loading of astragalus polysaccharide

[0063] MEM medium containing 10% fetal bovine serum was recorded as complete medium.

[0064] (1) SH-SY5Y cells with good growth status were inoculated into a cell culture flask with a cell number of 2×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 48 h to obtain a first cell culture solution.

[0065] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and then an astragalus polysaccharide solution was added at a concentration of 800 μM. The culture was continued for 72 h in an incubator at 37°C and a carbon dioxide volume percentage of 5%, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0066] (3) The same as step (3) of Example 1, to obtain extracellular vesicles secreted by SH-SY5Y cells, which contained astragalus polysaccharides.

[0067] Example 5

[0068] Preparation of extracellular vesicles released from rat hippocampal neurons and loading of temozolomide

[0069] DMEM high-glucose medium containing 10% fetal bovine serum was recorded as complete medium.

[0070] (1) Well-growing rat hippocampal neuronal cells H19-7 were inoculated into a cell culture flask at a cell number of 5×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0071] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 48 h in an incubator at 37° C. and 5% carbon dioxide by volume, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0072] (3) The same as step (3) of Example 1, obtaining extracellular vesicles secreted by neuronal cells.

[0073] (4) adding temozolomide solution to the extracellular vesicles obtained in step (3) at a ratio of 20 mM temozolomide solution: 0.5 mg / mL extracellular vesicles, ultrasonicating at 100 W for 15 min under ice bath conditions, and incubating at 37° C. for 0.5 h to obtain extracellular vesicles loaded with temozolomide.

[0074] Example 6

[0075] Preparation of extracellular vesicles released from astrocytes and loading with vincristine

[0076] DMEM high-glucose medium containing 10% fetal bovine serum was recorded as complete medium.

[0077] (1) Human astrocytes in good growth condition were inoculated into a cell culture flask at a cell number of 5×10 6, add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0078] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 48 h in an incubator at 37° C. and 5% carbon dioxide by volume, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0079] (3) The same as step (3) of Example 1, to obtain extracellular vesicles secreted by astrocytes.

[0080] (4) Add vincristine solution to the extracellular vesicles obtained in step (3) at a ratio of 20 mM vincristine solution: 0.5 mg / mL extracellular vesicles, vortex at 1000 rpm for 5 min, and then perform 3 cycles of high-pressure homogenization at 500 bar. Incubate at 37° C. for 1 h to obtain extracellular vesicles loaded with vincristine.

[0081] Example 7

[0082] Preparation of extracellular vesicles released from microglia and loading of paclitaxel

[0083] DMEM high-glucose medium containing 10% fetal bovine serum was recorded as complete medium.

[0084] (1) Mouse microglial cells BV2 with good growth status were inoculated into a cell culture flask with a cell number of 5×10 6 , add 2 mL of complete culture medium, and culture at 37° C. in an incubator with a carbon dioxide volume percentage of 5% for 24 h to obtain a first cell culture solution.

[0085] (2) The culture medium in the first cell culture medium was replaced with a complete culture medium that removed extracellular vesicles, and the culture was continued for 72 h in an incubator at 37° C. and 5% carbon dioxide by volume, and the cell culture medium was collected. The cell culture medium was centrifuged at 300×g for 10 min to obtain a supernatant and a cell pellet.

[0086] (3) The same as step (3) of Example 1, obtaining extracellular vesicles secreted by microglia.

[0087] (4) Adding paclitaxel solution to the extracellular vesicles obtained in step (3) at a ratio of 10 mM paclitaxel solution: 0.5 mg / mL extracellular vesicles, filtering repeatedly 5 times with a 0.45 μm microporous filter membrane, and incubating at 37° C. for 2 h to obtain paclitaxel-loaded extracellular vesicles.

[0088] Example 8

[0089] Preparation of macrophage-derived cell membranes

[0090] (1) RAW264.7 mouse macrophages were cultured in DMEM high-glucose medium containing 10% fetal bovine serum for 48 h, digested with trypsin, and prepared into a cell suspension. The cells were centrifuged at 1000×g for 5 min at room temperature to obtain a cell pellet, the supernatant was removed, and the cell pellet was resuspended in PBS. The cells were centrifuged again at 1000×g for 5 min at room temperature, and the cells were resuspended in 1×TM buffer containing 1% PMSF. The cell density was adjusted to 2×10 7 Pieces / mL.

[0091] (2) Under ice bath conditions, use an ultrasonic cell crusher to crush cells at a power of 100 W for 5 minutes and repeat 4 times. The crushed cell suspension is mixed with a 1M sucrose solution to make the sucrose solution concentration 0.25M, and the mixed solution is centrifuged at 4°C and 2000×g for 10 minutes. Take the supernatant and continue to centrifuge at 4°C and 10000×g for 30 minutes to collect the cell membrane precipitate. The cell membrane precipitate is washed with ice-cold 1×TM buffer containing 0.25M sucrose, and centrifuged again at 4°C and 10000×g for 30 minutes to obtain macrophage-derived cell membranes.

[0092] Example 9

[0093] Preparation of cell membranes from T lymphocytes

[0094] (1) H9 human T lymphocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum for 48 h. The cells were centrifuged at 1000×g for 5 min at room temperature to obtain a cell pellet, the supernatant was removed, and the cell pellet was resuspended in PBS. The cells were centrifuged again at 1000×g for 5 min at room temperature, and the cells were resuspended in 1×TM buffer containing 1% PMSF. The cell density was adjusted to 2.5×10 7 Pieces / mL.

[0095] (2) Under ice bath conditions, use an ultrasonic cell crusher to crush cells at a power of 150W for 3 minutes and repeat 3 times. The crushed cell suspension is mixed with a 1M sucrose solution to make the sucrose solution concentration 0.5M, and the mixed solution is centrifuged at 4°C and 2000×g for 10 minutes. Take the supernatant and continue to centrifuge at 4°C and 10000×g for 30 minutes to collect the cell membrane precipitate. The cell membrane precipitate is washed with ice-cold 1×TM buffer containing 0.5M sucrose, and centrifuged again at 4°C and 10000×g for 30 minutes to obtain T lymphocyte-derived cell membranes.

[0096] Example 10

[0097] Preparation of cell membranes from B lymphocytes

[0098] (1) Human peripheral blood B lymphocytes were cultured in RPMI-1640 medium containing 10% fetal bovine serum for 72 h. The cells were centrifuged at 1000 × g for 5 min at room temperature to obtain a cell pellet, the supernatant was removed, and the cell pellet was resuspended in PBS. The cells were centrifuged again at 1000 × g for 5 min at room temperature, and the cells were resuspended in 1× TM buffer containing 1% PMSF. The cell density was adjusted to 1×10 7 Pieces / mL.

[0099] (2) Under ice bath conditions, use an ultrasonic cell crusher to crush cells at a power of 75W for 5 minutes and repeat 3 times. The crushed cell suspension is mixed with a 1M sucrose solution to make the sucrose solution concentration 0.25M, and the mixed solution is centrifuged at 4°C and 2000×g for 10 minutes. Take the supernatant and continue to centrifuge at 4°C and 10000×g for 30 minutes to collect the cell membrane precipitate. The cell membrane precipitate is washed with ice-cold 1×TM buffer containing 0.25M sucrose, and centrifuged again at 4°C and 10000×g for 30 minutes to obtain the cell membrane derived from B lymphocytes.

[0100] Embodiment 11

[0101] Preparation of Neutrophil-derived Cell Membranes

[0102] (1) Mouse neutrophils were cultured in DMEM high-glucose medium containing 10% fetal bovine serum for 24 h. The cells were centrifuged at 1000×g for 5 min at room temperature to obtain a cell pellet, the supernatant was removed, and the cell pellet was resuspended in PBS. The cells were centrifuged again at 1000×g for 5 min at room temperature, and the cells were resuspended in 1×TM buffer containing 1% PMSF. The cell density was adjusted to 2.5×10 7 Pieces / mL.

[0103] (2) Under ice bath conditions, use an ultrasonic cell crusher to crush cells at a power of 1500W for 3 minutes and repeat 5 times. The crushed cell suspension is mixed with a 1M sucrose solution to make the sucrose solution concentration 0.5M, and the mixed solution is centrifuged at 4°C and 2000×g for 10 minutes. Take the supernatant and continue to centrifuge at 4°C and 10000×g for 30 minutes to collect the cell membrane precipitate. The cell membrane precipitate is washed with ice-cold 1×TM buffer containing 0.5M sucrose, and centrifuged again at 4°C and 10000×g for 30 minutes to obtain the cell membrane derived from neutrophils.

[0104] Example 12

[0105] Preparation of brain-targeted delivery vectors by hybridization of extracellular vesicles and cell membranes derived from immune cells

[0106] Add PBS containing 0.1-100mM stabilizer to the cell membrane of immune cells, incubate at 37°C for 0.5-4h, centrifuge at 4°C and 10000×g for 30min to obtain cell membrane precipitate, remove the supernatant, and resuspend the cell membrane precipitate with PBS. The cell membrane precipitate is immediately mixed with extracellular vesicles according to different total protein ratios, and after mixing, a carrier after hybridization of extracellular vesicles and immune cell cell membranes is obtained. The stabilizer is one of mercaptoethanol, dithiothreitol, dithioerythritol, tri-(2-formylethyl)phosphine hydrochloride, and reduced glutathione; the protein ratio is between (0.5:1) and (5:1). The preparation conditions of different brain-targeted delivery carriers are shown in Table 1:

[0107] Table 1 Preparation of different brain-targeted delivery vectors

[0108]

[0109] Embodiment 13

[0110] Example 12 Preparation and characterization of the lyophilized product of the brain-targeted delivery vector obtained

[0111] 1. Freeze-drying process

[0112] The suspension of the extracellular vesicle hybrid cell membrane obtained in Example 12 and the suspension obtained without adding a stabilizer (the group without adding a stabilizer and each group in Example 12 follow the single control principle) were taken as the lyophilized material, mannitol was used as a lyophilization protectant, mannitol was added to the lyophilized material, the mass of mannitol added was 1% of the volume of the lyophilized material, pre-frozen at -20°C for 4h, and transferred to -80°C for 24h. After the freeze dryer was pre-cooled for half an hour, the sample was quickly transferred to a freeze dryer for freeze drying until there was no moisture, the freeze-dried product was taken out, and sealed and stored in the dark at 4°C, which was the freeze-dried brain-targeted delivery carrier.

[0113] 2. Determination of particle size

[0114] The lyophilized products of the brain-targeted delivery vectors obtained in step 1 were taken separately, and ultrapure water was added to dissolve them. The particle size and Zeta potential of the samples were determined using a Zetasizer (Nano ZS90, Malvern Instruments Ltd., UK) dynamic light scattering method (633 nm, 25°C, 4 mW He-Ne). The results are shown in Table 2 (n=3, indicating that three parallel experiments were performed).

[0115] Table 2 Particle size and potential (mean ± SD)

[0116]

[0117] As shown in Table 2, compared with the brain-targeted delivery vector prepared without adding stabilizer, the particle size of the brain-targeted delivery vector prepared with adding stabilizer was significantly reduced (p<0.05), and the zeta potential was also decreased.

[0118] Embodiment 14

[0119] Study on the Plasma Stability of the Freeze-dried Brain Targeted Delivery Vector Obtained in Example 13

[0120] The lyophilized products of the brain-targeted delivery vector obtained in Example 13 were taken separately, dissolved in ultrapure water, and the delivery system was labeled with the fluorescent dye PKH67, and then mixed with an equal volume of rat plasma, and the final concentration of PKH67 was 200 ng / mL. After that, it was incubated in a 37°C water bath for 48 h, centrifuged (3000 rpm, 5 min), and 100 μL of the supernatant was taken into an EP tube, heated at 90°C until the liquid evaporated completely, and an EP tube precipitate was obtained. An equal volume of methanol was added to dissolve the precipitate in the EP tube, and then filtered through a 0.22 μm microporous membrane, and the amount of PKH67 M was determined by an ELISA instrument. t , set up three parallel experiments; after the PKH67 labeled delivery vector was mixed with an equal volume of rat plasma, the amount of PKH67 without water bath incubation was measured by an ELISA instrument, recorded as M0 (drug amount at time 0), and set up three parallel experiments. t / M0×100% was used to evaluate the plasma stability. The result data are shown in Table 3 (n=3).

[0121] Table 3 Stability of different groups in rat plasma (mean ± SD)

[0122]

[0123] As shown in Table 3, after incubation with rat plasma for 48 hours, the leakage rate of the brain-targeted delivery carrier prepared by adding a stabilizer was significantly reduced compared with the hybrid carrier prepared without adding a stabilizer. The hybrid carrier without adding a stabilizer has a rat plasma stability of 75% to 85%, and the hybrid carrier with adding a stabilizer has a rat plasma stability of 87% to 95%. Since the various enzymes present in plasma can have a significant effect on the stability of the carrier, it will cause the loaded pharmacological substances to leak, and the brain-targeted delivery carrier with stabilizers prepared by the present invention has enhanced stability, which is conducive to the delivery of loaded pharmacological substances to target tissues.

[0124] Embodiment 15

[0125] Evaluation of targeted uptake of freeze-dried brain-targeted delivery vectors

[0126] 1. Uptake of targeted delivery vectors by microglia in the brain

[0127] After the microglial cells BV2 were cultured to 80% confluence, the culture medium was discarded and washed 3 times with PBS. The brain-targeted delivery vector labeled with fluorescent dye PKH26 (red fluorescence) was added and cultured for 4 hours. The culture medium was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature and away from light for 30 minutes, and washed 3 times with PBS. DAPI was stained in the dark for 10 minutes (blue fluorescence), washed 3 times with PBS and a little PBS was retained. The uptake of the brain-targeted delivery system by microglia was observed using a laser confocal microscope (TCS SP5, Leica).

[0128] The results are as follows Figure 1 As shown, compared with the hybrid carrier prepared without adding stabilizer, the brain-targeted delivery carrier prepared with adding stabilizer has stronger intracellular fluorescence intensity, indicating that the microglia in the brain have a stronger ability to take up the brain-targeted delivery carrier prepared by the present invention.

[0129] 2. Uptake of targeted delivery vectors by glioma cells

[0130] After the human brain glioma cells U-87MG were cultured to 80% confluence, the culture medium was discarded and washed 3 times with PBS. The brain-targeted delivery vector labeled with fluorescent dye PKH67 (green fluorescence) was added and cultured for 4 hours. The culture medium was discarded, washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature and protected from light for 30 minutes, and washed 3 times with PBS. The uptake of brain-targeted delivery vector by brain glioma cells U-87MG was observed using an inverted microscope (TI-DH, Nikon).

[0131] The results are as follows Figure 2 As shown, compared with the hybrid carrier prepared without adding a stabilizer, the brain-targeted delivery carrier prepared with a stabilizer has a stronger intracellular fluorescence intensity, indicating that glioma cells have a stronger ability to take up the brain-targeted delivery carrier prepared by the present invention.

[0132] Example 16

[0133] Therapeutic effects on Alzheimer's disease

[0134] 1. The therapeutic effect of simultaneously loading bioactive substances and small molecule drugs

[0135] Using Aβ 1-42The Alzheimer's disease (AD) model was prepared by intracerebral localized injection, and the model mice were randomly divided into groups, with 10 mice in each group. The dosing groups were: sham group, AD model group, positive drug group (memantine hydrochloride, oral gavage dose 3.33 mg / kg / d), Example 12-1 without stabilizer group (stilbene glycoside injection dose 1.2 mg / kg / 2d per mouse, abbreviated as N-EV-1), Example 12-1 with stabilizer group (stilbene injection dose 1.2 mg / kg / 2d per mouse, abbreviated as EV-1), Example 12-2 without stabilizer group (icariin injection dose 15 mg / kg / 2d per mouse, abbreviated as N-EV-2), Example 12-2 with stabilizer group (icariin injection dose 15 mg / kg / 2d per mouse, abbreviated as EV-2), Example 12-3 without stabilizer group (matrine injection dose Each mouse received 10 mg / kg / 2d, abbreviated as N-EV-3), Example 12-3 plus stabilizer group (matrine injection amount of each mouse 10 mg / kg / 2d, abbreviated as EV-3), Example 12-4 without stabilizer group (stilbene glycoside injection amount of each mouse 1.2 mg / kg / 2d, abbreviated as N-EV-4), Example 12-1 plus stabilizer group (stilbene glycoside injection amount of each mouse 1.2 mg / kg / 2d, abbreviated as EV-4), Example 12-5 without stabilizer group (astragalus polysaccharide injection amount of each mouse 1.8 mg / kg / 2d, abbreviated as N-EV-5), Example 12-5 plus stabilizer group (stilbene injection amount of each mouse 1.2 mg / kg / 2d, abbreviated as EV-5). Each mouse in the sham group and AD model group was gavaged with 0.2 mL of sterile PBS every day, memantine hydrochloride was administered by gavage, and the carriers in each group in Example 12 were administered by intravenous injection.

[0136] After 14 days of drug administration, the spatial behavioral memory ability of mice in each group was tested by novel object recognition test. The novel object preference index was calculated as: the time spent exploring the novel object / the total time spent exploring the novel and familiar objects, and statistical analysis was performed. The results are shown in Table 4. The data in Table 4 are all mean±SD.

[0137] Table 4 Experimental results of new object recognition in each group in Example 12

[0138]

[0139] In Table 4, compared with the AD group, **p<0.01, ***p<0.001; compared with the corresponding carrier group without stabilizer, #p<0.05. As shown in Table 4, the new object preference index of the sham group, positive drug group, N-EV-1 group, EV-1 group, N-EV-2 group, EV-2 group, N-EV-3 group, EV-3 group, N-EV-4 group, EV-4 group, N-EV-5 group, and EV-5 group was significantly higher than that of the AD group (p<0.01 or p<0.001). In addition, the preference index of the EV-1 group, EV-2 group, EV-3 group, EV-4 group, and EV-5 group was significantly higher than that of the corresponding delivery carrier prepared without stabilizer (p<0.05), indicating that the brain-targeted delivery carrier obtained in Example 12 can enhance the memory ability of AD model mice and has better therapeutic effect.

[0140] 2. The therapeutic effect of loading only active substances

[0141] There are four groups of brain-targeted delivery vehicles that only carry biologically active substances:

[0142] The difference between the first group and Example 12-1 is that the extracellular vesicles were prepared without loading diphenylethylene glycosides, and the rest were the same as Example 12-1, and were recorded as B-EV-1;

[0143] The difference between the second group and Example 12-2 is that icariin is not loaded during the preparation of the extracellular vesicles, and the rest is the same as Example 12-2, and is denoted as B-EV-2;

[0144] The difference between the third group and Example 12-3 is that the extracellular vesicles were not loaded with matrine during preparation, and the rest were the same as Example 12-3, and were recorded as B-EV-3;

[0145] The fourth group differs from Example 12-4 in that diphenylethylene glycoside is not loaded during the preparation of the extracellular vesicles, and the rest is the same as Example 12-4, and is denoted as B-EV-4;

[0146] Using Aβ 1-42The Alzheimer's disease (AD) model was prepared by intracerebral localized injection method, and the model mice were randomly divided into groups, with 10 mice in each group. The drug administration groups were: sham group, AD model group, positive drug group (memantine hydrochloride, intragastric dose 3.33 mg / kg / d), B-EV-1 group loaded with bioactive substances only (injection dose 5 mg EVs protein / kg per mouse), B-EV-2 group loaded with bioactive substances only (injection dose 5 mg EVs protein / kg per mouse), B-EV-3 group loaded with bioactive substances only (injection dose 5 mg EVs protein / kg per mouse), and B-EV-4 group loaded with bioactive substances only (injection dose 5 mg EVs protein / kg per mouse). Each mouse in the sham group and AD model group was intravenously injected with 0.2 mL of sterile PBS every day, memantine hydrochloride was administered by intragastric administration, and each group loaded with bioactive substances was administered by intravenous injection.

[0147] After 14 days of drug administration, the spatial behavioral memory ability of mice in each group was tested by novel object recognition test. The novel object preference index was calculated as: the time spent exploring the novel object / the total time spent exploring the novel and familiar objects, and statistical analysis was performed. The results are shown in Table 5. The data in Table 5 are all mean±SD.

[0148] Table 5 Experimental results of new object recognition in each group in Example 12

[0149]

[0150] In Table 5, compared with the AD group, *p<0.05, **p<0.01, ***p<0.001. As shown in Table 5, the novel object preference index of the B-EV-1 group, B-EV-2 group, B-EV-3 group, and B-EV-4 group was significantly higher than that of the AD group (p<0.05 or p<0.01), indicating that the brain-targeted delivery carrier prepared by the present invention, when not loaded with small molecule drugs, the bioactive substances loaded in the extracellular vesicles also have the effect of treating AD, can enhance the memory ability of AD model mice, and has a good therapeutic effect.

[0151] Embodiment 17

[0152] Evaluation of anti-glioma efficacy and brain distribution

[0153] Human brain glioma U-87MG cells in the logarithmic growth phase were digested with trypsin, and then digested with complete medium. The cells were centrifuged, the supernatant was discarded, and serum-free DMEM medium was added to make a cell suspension. The suspension was placed in a 2 mL EP tube and the cell concentration was adjusted to 5 × 10 55×10 cells / 5μL, wrap the EP tube with cotton and place it on ice for later use. Wipe the skin of the nude mouse's abdomen and back with a 75% alcohol cotton ball, draw 100μL of the cell suspension with a disposable sterile syringe, and inoculate it on the right side of the nude mouse's abdomen and back. The number of cells injected is 5×10 6 The growth status, diet, time of tumor onset, growth trend of subcutaneous tumors, and survival of nude mice after subcutaneous tumors were observed every day.

[0154] On the 11th day after inoculation, the model mice were randomly divided into groups, with 5 mice in each group. The dosing groups were: model group, positive drug group (temozolomide, oral gavage dose 20 mg / kg / d), Example 12-6 without stabilizer group (temozolomide injection dose 20 mg / kg / 2d per mouse, abbreviated as N-EV-6), Example 12-6 with stabilizer group (temozolomide injection dose 20 mg / kg / 2d per mouse, abbreviated as EV-6), Example 12-7 without stabilizer group (vincristine injection dose 10 mg / kg / 2d per mouse, abbreviated as N-EV-7), Example 12-7 with stabilizer group (vincristine injection dose 10 mg / kg / 2d per mouse, abbreviated as N-EV-7). mg / kg / 2d, abbreviated as EV-7), Example 12-8 without stabilizer group (temozolomide injection amount of 20 mg / kg / 2d per mouse, abbreviated as N-EV-8), Example 12-8 with stabilizer group (temozolomide injection amount of 20 mg / kg / 2d per mouse, abbreviated as EV-8), Example 12-9 without stabilizer group (paclitaxel injection amount of 10 mg / kg / 2d per mouse, abbreviated as N-EV-9), Example 12-9 with stabilizer group (paclitaxel injection amount of 10 mg / kg / 2d per mouse, abbreviated as EV-9). The model group was gavaged with 0.2 mL of sterile PBS per day, temozolomide was gavage-administered, and the carriers in each group in Example 12 were administered intravenously. After the administration, the tumor was removed and the tumor weight was weighed for comparison.

[0155] like Figure 3 As shown, the tumor weights of the positive drug group, N-EV-6 group, EV-6 group, N-EV-7 group, EV-7 group, N-EV-8 group, EV-8 group, N-EV-9 group, and EV-9 group were significantly lower than those of the model group (p<0.01). In addition, the tumor weights of the EV-6 group, EV-7 group, EV-8 group, and EV-9 group were significantly lower than those of the corresponding delivery vectors prepared without adding stabilizers (p<0.05 or p<0.01), indicating that the brain-targeted delivery vector of the present invention can significantly inhibit the growth of brain gliomas and has better therapeutic effects.

[0156] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A brain-targeted delivery vector, characterized in that: The extracellular vesicles are hybridized with the cell membrane of immune cells in the presence of a stabilizer; the extracellular vesicles are loaded with active pharmaceutical ingredients and / or biologically active substances for treating diseases; the diseases include Alzheimer's disease or brain glioma; the stabilizer includes one of mercaptoethanol, dithiothreitol, dithioerythritol, tri-(2-formylethyl)phosphine hydrochloride and reduced glutathione.

2. The brain-targeted delivery vector according to claim 1, characterized in that: The immune cells include macrophages, T lymphocytes, B lymphocytes or neutrophils; the nanoscale extracellular vesicles are derived from one or more of stem cells, nerve cells and glial cells.

3. The brain-targeted delivery vector according to claim 2, characterized in that: The stem cells include one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells and placental mesenchymal stem cells; the neural cells include one or more of human neuroblastoma cell lines, cortical neural cells, hippocampal neuronal cells and neural stem cells; the glial cells include one or more of astrocytes, oligodendrocytes and microglia.

4. The brain-targeted delivery vector according to claim 1, characterized in that: When the disease is Alzheimer's disease, the active pharmaceutical ingredients include one or more of diphenylethylene glycosides, rhein, rhein, rhein methyl ether, rhein-1,6-dimethyl ether, icariin, curcumin, gambogic acid, artemisinin, dihydroartemisinin, artesunate, matrine, berberine, sinomenine, angelica coumarin, quercetin, glycyrrhizic acid, liquiritigenin, liquiritigenin, isoliquiritigenin, peony glucoside, platycoside, resveratrol and houttuynia cordata, or derivatives of the active pharmaceutical ingredients.

5. The brain-targeted delivery vector according to claim 1, characterized in that: When the disease is brain glioma, the active pharmaceutical ingredients include one or more of temozolomide, methylcarbazine, lomustine, vinblastine, vincristine, carmustine, irinotecan, etoposide, cisplatin, carboplatin, methotrexate, docetaxel, paclitaxel, camptothecin, doxorubicin, cyclophosphamide, epirubicin, hydroxycamptothecin, gemcitabine, pentostatin and gambogic acid, or derivatives of the active pharmaceutical ingredients.

6. The brain-targeted delivery vector according to claim 1, characterized in that: The bioactive substances include one or more of microRNA, lncRNA, protein and lipid.

7. The method for preparing the brain-targeted delivery vector according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: adding 0.1 to 100 mM of a stabilizer to the cell membrane of immune cells, incubating at 37°C for 0.5 to 4 hours, and centrifuging to obtain a cell membrane precipitate; uniformly mixing the cell membrane precipitate with extracellular vesicles at a protein mass ratio of 0.5 to 5:1 to obtain a brain-targeted delivery carrier; the extracellular vesicles are loaded with active pharmaceutical ingredients and / or bioactive substances for treating diseases.

8. The preparation method according to claim 7, characterized in that: The preparation of the cell membrane of the immune cells comprises the following steps: culturing the immune cells for 12 to 72 hours, centrifuging to obtain the cell pellet, and resuspending the cells to a cell density of 0.5 to 2.5×10 7 / mL, under ice bath conditions, ultrasonically disrupt the cells, mix the disrupted cell suspension with sucrose solution to make the concentration of sucrose solution 0.1-0.5M, and collect the cell membrane precipitate by centrifugation.

9. The preparation method according to claim 7, characterized in that: The method for loading extracellular vesicles with active pharmaceutical ingredients and / or bioactive substances for treating diseases comprises the following steps: co-culturing the active pharmaceutical ingredients with cells, and then performing differential centrifugation to obtain the extracellular vesicles loaded with the active pharmaceutical ingredients and / or bioactive substances; or after obtaining the extracellular vesicles by differential centrifugation, mixing with the active pharmaceutical ingredients, treating with ultrasound, high-pressure homogenization or microporous membrane filtration, and incubating to obtain the extracellular vesicles loaded with the active pharmaceutical ingredients and / or bioactive substances.

10. Use of the brain-targeted delivery vector according to any one of claims 1 to 6 in the preparation of a product for treating Alzheimer's disease or brain glioma.

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