Extracellular vesicle drug delivery system, preparation method thereof and application of extracellular vesicle drug delivery system in treatment of Alzheimer's disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an extracellular vesicle drug delivery system, a preparation method thereof and application of the extracellular vesicle drug delivery system in treatment of Alzheimer's disease. According to the invention, one or more of stilbene glucoside, emodin, kaempferol, resveratrol and quercetin are used for regulating and controlling the expression of miRNA (micro Ribonucleic Acid) related to the disease progress in the extracellular vesicles, and the result shows that one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b and miR-129-5p is remarkably up-regulated, and one or more of miR-342-3p, miR-16-5p and miR-125b-5p is remarkably down-regulated. The regulated extracellular vesicles can be used for treating Alzheimer's disease, relieving Tau protein abnormal phosphorylation, reducing amyloid protein beta deposition, inhibiting microglial cell activation, protecting neuronal cells and relieving memory deficits.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an extracellular vesicle drug delivery system, its preparation method, and its application in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD), a progressive neurodegenerative disease, has a complex pathological mechanism, posing significant challenges to clinical treatment. Currently used drugs, such as donepezil and memantine hydrochloride, can improve symptoms to some extent, but often cannot effectively reverse disease progression, and the long-term side effects limit their clinical application. Therefore, developing novel treatment strategies that can fundamentally intervene in the disease's pathology while possessing both safety and effectiveness has become a critical bottleneck that urgently needs to be overcome in this field.

[0003] In recent years, extracellular vesicles (EVs), as natural nanoscale information carriers, have demonstrated unique advantages in the treatment of central nervous system diseases such as Alzheimer's disease (AD). They can carry bioactive molecules such as proteins, lipids, and nucleic acids, participate in intercellular communication, and play important roles in various physiological and pathological processes. More importantly, some cell-derived vesicles possess natural brain-targeting properties, enabling them to penetrate biological barriers and directly deliver drug-active substances to target cells in the brain, thereby achieving highly effective therapeutic effects while reducing toxic side effects. However, using natural EVs directly as drug delivery carriers still has several drawbacks. First, their ability to penetrate the blood-brain barrier remains relatively limited, resulting in insufficient drug concentrations in the brain. Second, natural EVs have a short half-life in the blood, and after systemic administration, they are easily metabolized and cleared by organs such as the liver and kidneys, resulting in low accumulation at the lesion site and difficulty in achieving effective therapeutic concentrations. More importantly, the abundance of therapeutic bioactive substances contained in natural EVs, such as specific miRNAs that have therapeutic effects on AD, is often low and insufficient to produce significant therapeutic effects independently, which greatly limits their potential as an independent treatment. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art by upregulating the expression of miRNAs that negatively regulate disease progression in extracellular vesicles and downregulating the expression of miRNAs that positively regulate disease progression in extracellular vesicles, thereby endowing extracellular vesicles with stronger endogenous therapeutic activity and enabling extracellular vesicles to become a therapeutic system that can actively treat Alzheimer's disease, rather than a passive drug delivery carrier.

[0005] This invention provides the application of compounds in regulating the expression of extracellular vesicle miRNAs, wherein the regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p; The miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0006] Preferably, the extracellular vesicles are extracellular vesicles derived from stem cells.

[0007] 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.

[0008] This invention provides an extracellular vesicle drug delivery system, comprising extracellular vesicles and miRNAs related to disease progression loaded within the extracellular vesicles; The miRNAs associated with disease progression include miRNAs that negatively regulate disease progression and miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression are significantly upregulated in the extracellular vesicles; the miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p. The miRNAs that positively regulate disease progression are significantly downregulated in the extracellular vesicles; the miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p.

[0009] This invention provides a method for preparing the extracellular vesicle drug delivery system described above, comprising the following steps: The compound was co-cultured with stem cells, and the cell culture medium was collected. The cell culture medium was centrifuged for the first time, and the supernatant was collected to obtain the first centrifuged product; The first centrifuged product is subjected to a second centrifugation, and the supernatant is collected to obtain the second centrifuged product; The second centrifuged product was purified to obtain an extracellular vesicle drug delivery system; The centrifugal force of the first centrifugation is 1500~3000×g, and the time is 8~20 min; The centrifugal force for the second centrifugation is 8000~12000 ×g, and the time is 20~40 min; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0010] Preferably, the ratio of the compound to stem cells is 1~100000 nmol: 10 6 indivual; The co-culture method includes: after the stem cell density reaches 70-85% fusion, continue culturing for 12-24 hours.

[0011] Preferably, the co-culturing of the compound with stem cells includes one or more of the following: (1) Co-culture stilbene glycoside with bone marrow mesenchymal stem cells; the ratio of stilbene glycoside to bone marrow mesenchymal stem cells is 0.1~200 μmol: 10 6 indivual; (2) Co-culture emodin with umbilical cord mesenchymal stem cells; the ratio of emodin to umbilical cord mesenchymal stem cells is 1~100 μmol: 10 6 indivual; (3) Co-culture kaempferol with adipose-derived mesenchymal stem cells; the ratio of kaempferol to adipose-derived mesenchymal stem cells is 1~200 μmol: 10 6 indivual; (4) Co-culture resveratrol with skin mesenchymal stem cells; the ratio of resveratrol to skin mesenchymal stem cells is 0.1~200 μmol: 10 6 indivual; (5) Co-culture quercetin with placental mesenchymal stem cells; the ratio of quercetin to placental mesenchymal stem cells is 10~500 μmol: 10 6 indivual.

[0012] Preferably, the purification method includes one or more of ultracentrifugation, polymer precipitation, size exclusion chromatography, and ultrafiltration.

[0013] This invention provides the application of the compound or the extracellular vesicle drug delivery system described in the above technical solutions or the extracellular vesicle drug delivery system obtained by the preparation method described in the above technical solutions in the preparation of drugs for treating Alzheimer's disease; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0014] This invention provides a drug for treating Alzheimer's disease, wherein the active ingredient of the drug comprises the extracellular vesicle drug delivery system described in the above technical solution or the extracellular vesicle drug delivery system obtained by the preparation method described in the above technical solution.

[0015] Beneficial effects: This invention utilizes one or more of stilbene glycosides, emodin, kaempferol, resveratrol, and quercetin to specifically regulate the expression levels of miRNAs related to disease progression within extracellular vesicles. It significantly upregulates one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p, and significantly downregulates one or more of miR-342-3p, miR-16-5p, and miR-125b-5p, obtaining an extracellular vesicle drug delivery system with a "characteristic miRNA combination." This transforms extracellular vesicles from passive drug delivery carriers into an active therapeutic system for Alzheimer's disease. This invention possesses high determinism and reproducibility, solving industry problems such as complex sources, large batch-to-batch variations, and unstable activity of natural extracellular vesicles. It transforms an accidental biological phenomenon into a standardized industrial production process, providing a technological foundation for subsequent large-scale production and quality control. Furthermore, the extracellular vesicles in the extracellular vesicle drug delivery system of this invention retain their natural brain targeting tendency, phospholipid bilayer protection, and natural biocompatibility. The specific miRNAs enriched in them can enhance homing and retention at brain lesion sites, which is beneficial to improving the efficacy of Alzheimer's disease treatment, reducing abnormal phosphorylation of Tau protein, reducing amyloid β deposition, inhibiting microglia activation, protecting neurons, and alleviating memory deficits. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The miRNA expression of extracellular vesicles obtained from mesenchymal stem cells was regulated by different drugs; A was stilbene glycoside regulating bone marrow mesenchymal stem cells, B was emodin regulating adipose mesenchymal stem cells, C was kaempferol regulating skin mesenchymal stem cells, D was resveratrol regulating umbilical cord mesenchymal stem cells, and E was quercetin regulating placental mesenchymal stem cells; no significant difference was found in ns. p <0.05, p <0.01, p <0.001; Figure 2 To test the expression of Aβ1-42 in the hippocampus of mice in different treatment groups in Example 2; Figure 3 To test the CA1 region degenerative neuron index in mice of different treatment groups in Example 2; Figure 4 To test the expression of Aβ1-42 in the hippocampus of mice in different treatment groups in Example 3; Figure 5 To test the CA1 region degenerative neuron index in mice of different treatment groups in Example 3; Figure 6 To test the expression of Aβ1-42 in the hippocampus of mice in different treatment groups in Example 4; Figure 7 To test the CA1 region degenerative neuron index in mice of different treatment groups in Example 4; Figure 8 To test the expression of Aβ1-42 in the hippocampus of mice in different treatment groups in Example 5; Figure 9 To test the CA1 region degenerative neuron index in mice of different treatment groups in Example 5; Figure 10 To test the expression of Aβ1-42 in the hippocampus of mice in different treatment groups in Example 6; Figure 11 To test the degenerative neuron index in the CA1 region of mice in different treatment groups in Example 6; Figures 2-11 Compared with the model group, p <0.05, p <0.01, p <0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01, ### p <0.001. Detailed Implementation

[0018] This invention provides the application of compounds in regulating the expression of extracellular vesicle miRNAs, wherein the regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression; the miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p; the miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p; and the compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0019] In one embodiment, the upregulation of the miRNA that negatively regulates disease progression according to the present invention is at least 2-fold. In another embodiment, the downregulation of the miRNA that positively regulates disease progression according to the present invention is at least 50%.

[0020] In one embodiment, the extracellular vesicles described in this invention are stem cell-derived extracellular vesicles. In another embodiment, the stem cells described in this invention include one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose-derived mesenchymal stem cells, skin mesenchymal stem cells, and placental mesenchymal stem cells.

[0021] In one embodiment, the present invention utilizes stilbene glycosides to regulate the expression of extracellular vesicle miRNAs derived from bone marrow mesenchymal stem cells. The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include miR-let-7c-5p, miR-486-5p, and miR-129-5p; the miRNAs that positively regulate disease progression include miR-342-3p and miR-125b-5p.

[0022] In one embodiment, the present invention utilizes emodin to regulate the expression of extracellular vesicle miRNAs derived from umbilical cord mesenchymal stem cells. The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include miR-let-7c-5p, miR-486-5p, and miR-160b. The miRNAs that positively regulate disease progression include miR-342-3p and miR-125b-5p.

[0023] In one embodiment, the present invention utilizes kaempferol to regulate the expression of extracellular vesicle miRNAs derived from adipose-derived mesenchymal stem cells. The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include miR-486-5p, miR-132-3p, and miR-160b; the miRNAs that positively regulate disease progression include miR-16-5p and miR-125b-5p.

[0024] In one embodiment, the present invention utilizes resveratrol to regulate the expression of extracellular vesicle miRNAs derived from skin mesenchymal stem cells. The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include miR-let-7c-5p, miR-486-5p, and miR-132-3p; the miRNAs that positively regulate disease progression include miR-342-3p and miR-16-5p.

[0025] In one embodiment, the present invention utilizes quercetin to regulate the expression of extracellular vesicle miRNAs derived from placental mesenchymal stem cells. The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include miR-let-7c-5p, miR-132-3p, and miR-129-5p; the miRNAs that positively regulate disease progression include miR-342-3p, miR-16-5p, and miR-125b-5p.

[0026] This invention provides an extracellular vesicle drug delivery system, comprising extracellular vesicles and disease-related miRNAs loaded within the extracellular vesicles; the disease-related miRNAs include miRNAs that negatively regulate disease progression and miRNAs that positively regulate disease progression; the expression of the miRNAs that negatively regulate disease progression is significantly upregulated within the extracellular vesicles; the miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p; the expression of the miRNAs that positively regulate disease progression is significantly downregulated within the extracellular vesicles; the miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p.

[0027] In one embodiment, the expression of miR-let-7c-5p, miR-486-5p, and miR-129-5p in the extracellular vesicles of the present invention is significantly upregulated, while the expression of miR-342-3p and miR-125b-5p is significantly downregulated; or, the expression of miR-let-7c-5p, miR-486-5p, and miR-160b in the extracellular vesicles is significantly upregulated, while the expression of miR-342-3p and miR-125b-5p is significantly downregulated; or, the expression of miR-486-5p, miR-132-3p, and miR-160b in the extracellular vesicles is significantly downregulated. The expression of miR-60b was significantly upregulated, while the expression of miR-16-5p and miR-125b-5p was significantly downregulated in the extracellular vesicles; or, the expression of miR-let-7c-5p, miR-486-5p and miR-132-3p was significantly upregulated, while the expression of miR-342-3p and miR-16-5p was significantly downregulated in the extracellular vesicles; or, the expression of miR-let-7c-5p, miR-132-3p and miR-129-5p was significantly upregulated, while the expression of miR-342-3p, miR-16-5p and miR-125b-5p was significantly downregulated in the extracellular vesicles.

[0028] This invention provides a method for preparing the extracellular vesicle drug delivery system described above, comprising the following steps: The compound was co-cultured with stem cells, and the cell culture medium was collected. The cell culture medium was centrifuged for the first time, and the supernatant was collected to obtain the first centrifuged product; The first centrifuged product is subjected to a second centrifugation, and the supernatant is collected to obtain the second centrifuged product; The second centrifuged product was purified to obtain an extracellular vesicle drug delivery system; The centrifugal force of the first centrifugation is 1500~3000×g, and the time is 8~20 min; The centrifugal force for the second centrifugation is 8000~12000×g, and the time is 20~40 min; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0029] This invention involves co-culturing a compound with stem cells and collecting the cell culture medium. As one embodiment, the co-culturing method of this invention includes: after the stem cell density reaches 70-85% confluence, continuing to culture for 12-24 hours.

[0030] In one embodiment, the culture medium does not need to be changed during continued culturing according to the present invention. In another embodiment, the temperature for continued culturing according to the present invention is 37°C.

[0031] In one embodiment, the ratio of the compound and stem cells described in this invention is 1~100000 nmol: 10 6 One embodiment of the present invention includes one or more of the following: (1) co-culturing stilbene glycoside with bone marrow mesenchymal stem cells; (2) co-culturing emodin with umbilical cord mesenchymal stem cells; (3) co-culturing kaempferol with adipose mesenchymal stem cells; (4) co-culturing resveratrol with skin mesenchymal stem cells; (5) co-culturing quercetin with placental mesenchymal stem cells.

[0032] In one embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 0.1~200 μmol: 10 6 In another embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 0.2~50 μmol: 10 μmol. 6 One or 60~180 μmol: 10 6 In another embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 0.5~40 μmol: 10 μmol. 6 One or 80~160 μmol: 10 6 In another embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 1~30 μmol: 10 μmol. 6 One or 90~150 μmol: 10 6 In another embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 5-20 μmol: 10 μmol. 6 One or 100~120 μmol: 10 6 In another embodiment, the ratio of stilbene glycoside to bone marrow mesenchymal stem cells in this invention is 10~15 μmol: 10 μmol. 6 One or 110 μmol: 10 6 In one embodiment, the stilbene glycoside and bone marrow mesenchymal stem cells described in this invention are cultured for 24 hours.

[0033] In one embodiment, the ratio of emodin to umbilical cord mesenchymal stem cells in this invention is 1~100 μmol: 10 6 In another embodiment, the ratio of emodin to umbilical cord mesenchymal stem cells in this invention is 5-80 μmol: 10 μmol. 6 In another embodiment, the ratio of emodin to umbilical cord mesenchymal stem cells in this invention is 10~60 μmol: 10 μmol. 6In another embodiment, the ratio of emodin to umbilical cord mesenchymal stem cells in this invention is 20-50 μmol: 10. 6 In another embodiment, the ratio of emodin to umbilical cord mesenchymal stem cells in this invention is 30 μmol: 10 μmol. 6 In one embodiment, the emodin and umbilical cord mesenchymal stem cells described in this invention are cultured for 24 hours.

[0034] In one embodiment, the ratio of kaempferol to adipose-derived mesenchymal stem cells in this invention is 1-200 μmol: 10. 6 In another embodiment, the ratio of kaempferol to adipose-derived mesenchymal stem cells in this invention is 5-180 μmol: 10 μmol. 6 In another embodiment, the ratio of kaempferol to adipose-derived mesenchymal stem cells in this invention is 10~150 μmol: 10 μmol. 6 In another embodiment, the ratio of kaempferol to adipose-derived mesenchymal stem cells in this invention is 50-120 μmol: 10 μmol. 6 In another embodiment, the ratio of kaempferol to adipose-derived mesenchymal stem cells in this invention is 100 μmol: 10 μmol. 6 In one embodiment, the kaempferol and adipose-derived mesenchymal stem cells described in this invention are cultured for 12 hours.

[0035] In one embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 0.1~200 μmol: 10 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 0.5~180 μmol: 10 μmol. 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 1~160 μmol: 10 μmol. 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 5~150 μmol: 10 μmol. 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 10~120 μmol: 10 μmol. 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 50-100 μmol: 10 μmol. 6 In another embodiment, the ratio of resveratrol to skin mesenchymal stem cells in this invention is 60-80 μmol: 10 μmol. 6In one embodiment, the resveratrol and skin mesenchymal stem cells described in this invention are cultured for 12 hours.

[0036] In one embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 10~500 μmol: 10 6 In another embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 50-400 μmol: 10. 6 In another embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 80-300 μmol: 10 μmol. 6 In another embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 100-200 μmol: 10 μmol. 6 In another embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 120-180 μmol: 10 μmol. 6 In another embodiment, the ratio of quercetin to placental mesenchymal stem cells in this invention is 150-160 μmol: 10 μmol. 6 In one embodiment, the quercetin and placental mesenchymal stem cells described in this invention are cultured for 24 hours.

[0037] After obtaining the cell culture medium, the present invention performs a first centrifugation on the cell culture medium, collects the supernatant, and obtains a first centrifuged product.

[0038] The centrifugal force of the first centrifugation in this invention is 1500~3000×g; in one embodiment, the centrifugal force of the first centrifugation in this invention is 1600~2500×g; in another embodiment, the centrifugal force of the first centrifugation in this invention is 1800~2000×g. The centrifugation time of the first centrifugation in this invention is 8~20 min; in one embodiment, the centrifugation time of the first centrifugation in this invention is 10 min. The first centrifugation of the cell culture medium in this invention can remove residual intact cells and cell debris.

[0039] After obtaining the first centrifuged product, the present invention performs a second centrifugation on the first centrifuged product, collects the supernatant, and obtains the second centrifuged product.

[0040] The centrifugal force of the second centrifugation in this invention is 8000~12000×g; in one embodiment, the centrifugal force of the second centrifugation in this invention is 10000×g. The centrifugation time of the second centrifugation in this invention is 20~40 min; in one embodiment, the centrifugation time of the second centrifugation in this invention is 30 min. The second centrifugation of the first centrifuged product in this invention can remove large vesicles.

[0041] After obtaining the second centrifuged product, the present invention purifies the second centrifuged product to obtain an extracellular vesicle drug delivery system.

[0042] In one embodiment, the purification method of the present invention includes one or more of ultracentrifugation, polymer precipitation, size exclusion chromatography, and ultrafiltration. In one embodiment, the present invention uses ultracentrifugation to purify the second centrifuged product obtained after co-culturing stilbene glycosides with bone marrow mesenchymal stem cells. In one embodiment, the present invention uses ultracentrifugation to purify the second centrifuged product obtained after co-culturing emodin with umbilical cord mesenchymal stem cells. In one embodiment, the present invention uses polymer precipitation to purify the second centrifuged product obtained after co-culturing kaempferol with adipose-derived mesenchymal stem cells. In one embodiment, the present invention uses size exclusion chromatography to purify the second centrifuged product obtained after co-culturing resveratrol with skin mesenchymal stem cells. In one embodiment, the present invention uses ultrafiltration to purify the second centrifuged product obtained after co-culturing quercetin with placental mesenchymal stem cells. The specific steps of the ultracentrifugation, polymer precipitation, size exclusion chromatography, and ultrafiltration methods are not strictly required in the present invention; conventional operations in the art are sufficient.

[0043] In one embodiment, the present invention performs lyophilization on the extracellular vesicle drug delivery system to obtain a lyophilized agent of the extracellular vesicle drug delivery system. In one embodiment, the lyophilization process includes pre-freezing and freezing. In one embodiment, the pre-freezing temperature is -18 to -22°C; in another embodiment, the pre-freezing temperature is -20°C. In one embodiment, the pre-freezing time is 2 to 6 hours; in another embodiment, the pre-freezing time is 4 to 5 hours. In one embodiment, the freezing temperature is -82 to -78°C; in another embodiment, the freezing temperature is -80°C. In one embodiment, the freezing time is 23 to 25 hours; in another embodiment, the freezing time is 24 hours. In one embodiment, a lyophilization protectant is added to the lyophilization process, the lyophilization protectant including one or more of mannitol, sucrose, glucose, and trehalose. In one embodiment, the total weight-to-volume ratio of the lyophilization protectant and the extracellular vesicle drug delivery system of the present invention is 0.5% to 3%; in another embodiment, the total weight-to-volume ratio of the lyophilization protectant and the extracellular vesicle drug delivery system of the present invention is 1% to 2%. The present invention improves the therapeutic delivery effect of the lyophilized agent in the extracellular vesicle drug delivery system by limiting the parameters of the lyophilization process.

[0044] This invention provides the application of the compound or the extracellular vesicle drug delivery system described in the above-described technical solutions, or the extracellular vesicle drug delivery system obtained by the preparation method described in the above-described technical solutions, in the preparation of drugs for treating Alzheimer's disease; the compound includes one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

[0045] As one embodiment, the dosage form of the drug of the present invention includes one or more of the following: lyophilized preparation, injection, oral preparation, and topical preparation.

[0046] This invention provides a drug for treating Alzheimer's disease, wherein the active ingredient of the drug comprises the extracellular vesicle drug delivery system described in the above technical solution or the extracellular vesicle drug delivery system obtained by the preparation method described in the above technical solution.

[0047] In one embodiment, the drug of the present invention further includes pharmaceutically acceptable excipients. The present invention does not impose strict requirements on the type of excipients; they can be selected conventionally based on the drug's dosage form.

[0048] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an extracellular vesicle drug delivery system, its preparation method, and its application in the treatment of Alzheimer's disease. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1 A method for preparing extracellular vesicles of bone marrow mesenchymal stem cells, the specific steps of which are as follows: 1. Bone marrow mesenchymal stem cells (BMSCs) were seeded into a culture medium containing 10% fetal bovine serum (FBS) after extracellular vesicles were removed. Stilbene glycosides were added, and the cells were cultured until the BMSC density reached 80% confluence. The culture was then continued at 37°C for 12–24 h. The ratio of stilbene glycosides to BMSCs was 0.2 μmol:10. 6 indivual.

[0050] 2. After the culture is completed, collect the culture supernatant and centrifuge at 4℃ and 1500~3000×g for 10 min. Collect the supernatant, remove any remaining intact cells and cell debris to obtain the first centrifuged product. Centrifuge the first centrifuged product at 4℃ and 10000×g for 30 min, collect the supernatant, remove any remaining large vesicles, and obtain the second centrifuged product.

[0051] 3. Purification (Ultracentrifugation): The second centrifugation product was centrifuged at 4℃ and 100,000×g for 90 min. The precipitate was collected and resuspended in PBS to obtain a resuspension. The resuspension was centrifuged at 4℃ and 100,000×g for 90 min. The precipitate was collected and resuspended in 0.5 mL of PBS to obtain extracellular vesicles of bone marrow mesenchymal stem cells at a concentration of 5.5×10⁻⁶. 11 Particles / mL.

[0052] 4. Lyophilization: The extracellular vesicles resuspended in PBS were used as the lyophilization material. Mannitol was used as the cryoprotectant. Mannitol was added to the lyophilized material at a mass of 1% of the material's volume. The mixture was pre-frozen at -20 °C for 4 hours, then transferred to -80 °C for 24 hours. After pre-cooling in a freeze dryer for half an hour, the sample was rapidly transferred to the freeze dryer and lyophilized until all moisture was removed. The lyophilized product was then stored at -20 °C in a sealed container protected from light, yielding the lyophilized powder of the extracellular vesicle drug delivery system.

[0053] Comparative Example 1 Extracellular vesicles of bone marrow mesenchymal stem cells were prepared according to the method in Example 1, the only difference being that stilbene glycosides were not added for induction, resulting in blank extracellular vesicles of bone marrow mesenchymal stem cells.

[0054] Example 2 A method for preparing extracellular vesicles from adipose-derived mesenchymal stem cells, comprising the following specific steps: 1. Adipose-derived mesenchymal stem cells (ADMSCs) were seeded into a culture medium containing 10% fetal bovine serum (FBS), and emodin was added. The cells were cultured until the AMSC density reached 80% confluence, and then cultured at 37°C for 12–24 h. The ratio of emodin to AMSCs was 10 μmol: 10 μmol. 6 indivual.

[0055] 2. After the culture is completed, collect the culture supernatant and centrifuge at 4℃ and 1500~3000×g for 10 min. Collect the supernatant, remove any remaining intact cells and cell debris to obtain the first centrifuged product. Centrifuge the first centrifuged product at 4℃ and 10000×g for 30 min, collect the supernatant, remove any remaining large vesicles, and obtain the second centrifuged product.

[0056] 3. Purification (Ultracentrifugation): The second centrifugation product was centrifuged at 2℃ and 150,000×g for 60 min. The precipitate was collected and resuspended in PBS to obtain a resuspension. The resuspension was centrifuged at 2℃ and 150,000×g for 60 min. The precipitate was collected and resuspended in 1.0 mL of PBS to obtain extracellular vesicles of adipose-derived mesenchymal stem cells at a concentration of 4.8×10⁻⁶.11 Particles / mL.

[0057] 4. Lyophilization: The extracellular vesicles resuspended in PBS were used as the lyophilization material. Mannitol was added as a cryoprotectant to the lyophilized material at a mass of 3% of the total volume. The mixture was pre-frozen at -20 °C for 4 hours, then transferred to -80 °C for 24 hours. After pre-cooling in a freeze dryer for half an hour, the sample was rapidly transferred to the freeze dryer and lyophilized until all moisture was removed. The lyophilized product was then stored at -20 °C in a sealed container protected from light, yielding the lyophilized powder of the extracellular vesicle drug delivery system.

[0058] Comparative Example 2 Extracellular vesicles of adipose-derived mesenchymal stem cells were prepared according to the method in Example 2, the only difference being that no emodin was added for induction, resulting in blank extracellular vesicles of adipose-derived mesenchymal stem cells.

[0059] Example 3 A method for preparing extracellular vesicles of skin mesenchymal stem cells, the specific steps of which are as follows: 1. Skin mesenchymal stem cells were seeded into a culture medium containing 10% fetal bovine serum, and kaempferol was added. The cells were cultured until the skin mesenchymal stem cell density reached 80% confluence, and then cultured at 37 ℃ for 12–24 h. The ratio of kaempferol to skin mesenchymal stem cells was 10 μmol: 10 μmol. 6 indivual.

[0060] 2. After the culture is completed, collect the culture supernatant and centrifuge at 4℃ and 1500~3000×g for 10 min. Collect the supernatant, remove any remaining intact cells and cell debris to obtain the first centrifuged product. Centrifuge the first centrifuged product at 4℃ and 10000×g for 30 min, collect the supernatant, remove any remaining large vesicles, and obtain the second centrifuged product.

[0061] 3. Purification (Polymer Precipitation): Add PEG6000 solution to the second centrifuged product to a final PEG6000 concentration of 15%, then add NaCl to a final concentration of 0.3M. Mix well and incubate at 4°C for 12 h. Centrifuge at 10000×g for 60 min at 4°C, discard the supernatant, and collect the precipitate. Gently resuspend the precipitate in 500 μL PBS and centrifuge at 15000×g for 2 min at 4°C. Repeat three times, collecting the supernatant to obtain extracellular vesicles of skin mesenchymal stem cells at a concentration of 2.4×10⁻⁶. 11 Particles / mL.

[0062] 4. Lyophilization: The extracellular vesicles resuspended in PBS were used as the lyophilization material. Sucrose was used as the cryoprotectant. Sucrose was added to the lyophilized material at a mass of 1.5% of the material's volume. The mixture was pre-frozen at -20 °C for 4 h, then transferred to -80 °C for 24 h. After pre-cooling in a freeze dryer for half an hour, the sample was rapidly transferred to the freeze dryer and lyophilized until all moisture was removed. The lyophilized product was then stored at -20 °C in a sealed container protected from light, yielding the lyophilized powder of the extracellular vesicle drug delivery system.

[0063] Comparative Example 3 Skin mesenchymal stem cell extracellular vesicles were prepared according to the method of Example 3, the only difference being that kaempferol was not added for induction, resulting in blank skin mesenchymal stem cell extracellular vesicles.

[0064] Example 4 A method for preparing extracellular vesicles of umbilical cord mesenchymal stem cells, the specific steps of which are as follows: 1. Umbilical cord mesenchymal stem cells were seeded into a culture medium containing 10% fetal bovine serum, and resveratrol was added. The cells were cultured until the umbilical cord mesenchymal stem cell density reached 80% confluence, and then cultured at 37°C for 12–24 h. The ratio of resveratrol to umbilical cord mesenchymal stem cells was 0.5 μmol: 10 μmol / L. 6 indivual.

[0065] 2. After the culture is completed, collect the culture supernatant and centrifuge at 4℃ and 1500~3000×g for 10 min. Collect the supernatant, remove any remaining intact cells and cell debris to obtain the first centrifuged product. Centrifuge the first centrifuged product at 4℃ and 10000×g for 30 min, collect the supernatant, remove any remaining large vesicles, and obtain the second centrifuged product.

[0066] 3. Purification (size exclusion chromatography): A PBS-equilibrated column (Sepharose CL-2B column) was used, with an equilibration volume of 3 times the column volume. The second centrifuged product was loaded onto the column, with a loading volume less than or equal to 10% of the column volume. Elution was performed with PBS at a flow rate of 0.5 mL / min. Elution fractions were collected by volume, 1.0 mL per tube. Dynamic light scattering was used to identify the fractions in each tube. The extracellular vesicle enrichment fraction was combined to obtain extracellular vesicles of umbilical cord mesenchymal stem cells at a concentration of 8.4 × 10⁻⁶. 11 Particles / mL.

[0067] 4. Lyophilization: The extracellular vesicles resuspended in PBS were used as the lyophilization material. Glucose was added as a cryoprotectant, at a mass of 2% of the volume of the lyophilized material. The mixture was pre-frozen at -20 °C for 4 hours, then transferred to -80 °C for 24 hours. After pre-cooling in a freeze dryer for half an hour, the sample was rapidly transferred to the freeze dryer and lyophilized until all moisture was removed. The lyophilized product was then stored at -20 °C in a sealed container protected from light, yielding the lyophilized powder of the extracellular vesicle drug delivery system.

[0068] Comparative Example 4 Extracellular vesicles of umbilical cord mesenchymal stem cells were prepared according to the method in Example 4, the only difference being that resveratrol was not added for induction, resulting in blank extracellular vesicles of umbilical cord mesenchymal stem cells.

[0069] Example 5 A method for preparing extracellular vesicles of placental mesenchymal stem cells, the specific steps of which are as follows: 1. Placental mesenchymal stem cells were seeded into a culture medium containing 10% fetal bovine serum, and quercetin was added. The cells were cultured until the placental mesenchymal stem cell density reached 80% confluence, and then cultured at 37 °C for 12–24 h. The ratio of quercetin to placental mesenchymal stem cells was 50 μmol: 10 μmol. 6 indivual.

[0070] 2. After the culture is completed, collect the culture supernatant and centrifuge at 4℃ and 1500~3000×g for 10 min. Collect the supernatant, remove any remaining intact cells and cell debris to obtain the first centrifuged product. Centrifuge the first centrifuged product at 4℃ and 10000×g for 30 min, collect the supernatant, remove any remaining large vesicles, and obtain the second centrifuged product.

[0071] 3. Purification: The second centrifuged product was loaded into a 100 kDa ultrafiltration tube and centrifuged at 4°C and 4000×g for 30 min, concentrating to 0.5 mL to obtain the ultrafiltrate. PBS was added to the ultrafiltrate, and the mixture was centrifuged at 4°C and 4000×g for 30 min to remove small molecule impurities. The residue was resuspended in 1.0 mL of PBS to obtain extracellular vesicles of placental mesenchymal stem cells at a concentration of 9.3 × 10⁻⁶. 10 Particles / mL.

[0072] 4. Lyophilization: The extracellular vesicles resuspended in PBS were used as the lyophilization material. Trehalose was added as a cryoprotectant to the lyophilized material at a mass of 1.5% of the lyophilized material's volume. The mixture was pre-frozen at -20 °C for 4 h, then transferred to -80 °C for 24 h. After pre-cooling in a freeze dryer for half an hour, the sample was rapidly transferred to the freeze dryer and lyophilized until all moisture was removed. The lyophilized product was then stored at -20 °C in a sealed container protected from light, yielding the lyophilized powder of the extracellular vesicle drug delivery system.

[0073] Comparative Example 5 Placental mesenchymal stem cell extracellular vesicles were prepared according to the method in Example 5, the only difference being that quercetin was not added for induction, resulting in blank placental mesenchymal stem cell extracellular vesicles.

[0074] Test Example 1 Detection of extracellular vesicle miRNAs Samples of extracellular vesicles from Examples 1-5 and Comparative Examples 1-5 were collected, with three biological replicates for each group. Total RNA was extracted from the extracellular vesicles using column extraction. RNA concentration and purity were detected using NanoDrop. The expression of miRNAs in each group was analyzed using miRNA sequencing technology based on the Iliuma high-throughput sequencing platform. The screening criteria for differentially expressed miRNAs were: |log2(Fold Change)| ≥ 1.5 and p-value < 0.05. The results are as follows: Figure 1 As shown, compared with the untreated blank extracellular vesicles, each drug treatment group significantly altered the expression profile of miRNAs in stem cell extracellular vesicles (n=3, ...). p <0.05). Specifically, drug regulation upregulated several miRNAs with potential therapeutic effects on AD, including miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p. These miRNAs are known to be associated with reducing Tau protein phosphorylation, decreasing Aβ deposition, inhibiting microglia activation, protecting neuronal function, and alleviating memory deficits. Simultaneously, drug treatment downregulated miRNAs associated with AD disease progression, such as miR-342-3p, miR-16-5p, and miR-125b-5p. Specifically: stilbene glycoside treatment upregulated miR-let-7c-5p, miR-486-5p, and miR-129-5p in extracellular vesicles of bone marrow mesenchymal stem cells, and downregulated miR-342-3p and miR-125b-5p in extracellular vesicles of bone marrow mesenchymal stem cells. Figure 1(A). Emodin treatment upregulated miR-let-7c-5p, miR-486-5p, and miR-160b in extracellular vesicles of adipose-derived mesenchymal stem cells, and downregulated miR-342-3p and miR-125b-5p in extracellular vesicles of adipose-derived mesenchymal stem cells. Figure 1 (B). Kaempferol treatment upregulated miR-486-5p, miR-132-3p, and miR-160b in extracellular vesicles of skin mesenchymal stem cells, and downregulated miR-16-5p and miR-125b-5p in extracellular vesicles of skin mesenchymal stem cells. Figure 1 Resveratrol treatment upregulated miR-let-7c-5p, miR-486-5p, and miR-132-3p in extracellular vesicles of umbilical cord mesenchymal stem cells, and downregulated miR-342-3p and miR-16-5p in extracellular vesicles of umbilical cord mesenchymal stem cells. Figure 1 Quercetin treatment upregulated miR-let-7c-5p, miR-132-3p, and miR-129-5p in extracellular vesicles of placental mesenchymal stem cells, and downregulated miR-342-3p, miR-16-5p, and miR-125b-5p in extracellular vesicles of placental mesenchymal stem cells. Figure 1 (E).

[0075] Test Example 2 Extracellular vesicles from bone marrow mesenchymal stem cells for the treatment of Alzheimer's disease (AD) 1. Eight-week-old male C57BL / 6 mice were used to establish an AD model by stereotactic injection of streptozotocin (STZ) (3 mg / kg) into the lateral ventricle. A sham-operated group was set up as a control (n=8). Seven days after surgery, the AD mice were randomly divided into four groups (n=8): model group, positive drug group, blank extracellular vesicle group (blank-EVs), and TSG-EVs group, and treated as follows: Sham surgery group: 200 μL of normal saline was injected into the tail vein; Model group: 200 μL of normal saline was injected into the tail vein; Positive control group: donepezil administered by gavage at a dose of 1.5 mg / kg / day; The blank-EVs group: blank bone marrow mesenchymal stem cell extracellular vesicles obtained in Comparative Example 1 were injected via tail vein at a dose of 1.8 mg / kg / 2d. TSG-EVs group: Extracellular vesicles of bone marrow mesenchymal stem cells obtained in Example 1 were injected via tail vein at a dose of 1.8 mg / kg / 2 days.

[0076] 2. After 14 days of intervention, a new object recognition experiment was conducted, and behavioral tests were performed: adaptation period of 24 h, training period of 10 min, and testing period of 5 min. The time spent by mice exploring new objects and familiar objects was recorded, and the new object preference index (time spent exploring new objects / total time spent exploring objects) was calculated. The results are shown in Table 1.

[0077] Table 1 Results of the novel object experiment in each group of mice

[0078] Note: Compared to the model group, p <0.001; compared with the blank-EVs group, ## p <0.01.

[0079] As shown in Table 1, the novel object preference index of mice in the sham surgery group, positive drug group, blank-EVs group, and TSG-EVs group was significantly higher than that in the model group. p <0.001). Furthermore, the novel object preference index of mice in the TSG-EVs group was significantly higher than that in the blank extracellular vesicle-EVs group ( p The result is <0.01, indicating that the stilbene glycoside-regulated extracellular vesicles of bone marrow mesenchymal stem cells prepared in Example 1 can alleviate memory deficits in AD model mice and have better therapeutic effects.

[0080] 3. After the behavioral tests, mice were anesthetized and euthanized, and whole brain and blood samples were collected. A portion of hippocampal tissue was used for protein extraction, and the expression of p-Tau, total Tau, APP, and Iba1 proteins was detected by Western blotting, with GAPDH as an internal control. Another portion of hippocampal tissue was homogenized, and the Aβ1-42 content was measured using an ELISA kit. The remaining brain tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe the morphology and nuclear condensation of neurons in the CA1 region of the hippocampus. The degenerated neuron index (number of degenerated neurons / total number of neurons) in the CA1 region of the hippocampus was statistically analyzed to represent the degree of neurological damage in the brain tissue of each group of mice. The results are shown in Table 2 and [Table data missing]. Figures 2-3 As shown.

[0081] Table 2. Relative expression levels of target proteins in each group of mice (gray value of target protein / gray value of internal reference protein)

[0082] Note: Compared to the model group, p <0.05, p <0.01, p<0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01.

[0083] As shown in Table 2, TSG-EVs treatment significantly improved the expression levels of pathological molecular markers related to Alzheimer's disease. After TSG-EVs intervention, compared with the model group, the expression of p-Tau / t-Tau was significantly reduced (…). p <0.01, and its effect was significantly stronger than that of its corresponding unregulated extracellular vesicle blank-EVs ( p <0.05); TSG-EVs intervention also significantly downregulated abnormally elevated APP expression in the model group ( p <0.001, and its effect was significantly stronger than that of the blank-EVs group ( p <0.01); TSG-EVs can also significantly inhibit the overexpression of Iba1 in the model group ( p <0.01) Similarly, this effect was significantly stronger than that of the blank-EVs group ( p <0.05). The above results indicate that the stilbene glycoside-regulated extracellular vesicles of bone marrow mesenchymal stem cells prepared in Example 1 can synergistically exert multiple pharmacological activities, including inhibiting excessive phosphorylation of tau protein in the brain tissue of AD mouse models, reducing amyloid-β protein deposition, and alleviating microglial cell activation, thus providing a new and effective strategy for the treatment of Alzheimer's disease.

[0084] according to Figure 2 It can be seen that, compared with the model group, both the positive control drug and TSG-EVs intervention effectively reduced the concentration of Aβ1-42 in the hippocampus of mice. Furthermore, compared with the blank-EVs group, TSG-EVs intervention significantly reduced the concentration of Aβ1-42. p <0.05), indicating that the stilbene glycoside-regulated extracellular vesicles of bone marrow mesenchymal stem cells prepared in Example 1 can inhibit the deposition of Aβ1-42 in the brain tissue of an AD mouse model. According to Figure 3 It can be seen that the degenerated neuron index was significantly increased in the CA1 region of the hippocampus of the model group mice. Positive control drugs, blank-EVs, and TSG-EVs interventions all effectively improved neuronal morphology in the CA1 region of the small hippocampus and reduced the degenerated neuron index. Compared with blank-EVs, the proportion of degenerated neurons in the TSG-EVs group was significantly reduced ( p <0.001), and the neuronal morphology was better improved, indicating that the extracellular vesicles of bone marrow mesenchymal stem cells regulated by stilbene glycosides prepared in Example 1 can effectively protect the neuronal cell morphology in the CA1 region of the hippocampus in AD model mice.

[0085] Test Example 3 Extracellular vesicles from adipose-derived mesenchymal stem cells are used to treat Alzheimer's disease (AD). 1. Eight-week-old male C57BL / 6 mice were used to establish an AD model by stereotactic brain injection of streptozotocin (STZ) (3 mg / kg) into the lateral ventricle. A sham-operated group was set up as a control (n=8). Seven days after surgery, the AD mice were randomly divided into four groups (n=8): model group, positive drug group, blank extracellular vesicle group (blank-EVs), and EMO-EVs group, and treated as follows: Sham surgery group: 200 μL of normal saline was injected into the tail vein; Model group: 200 μL of normal saline was injected into the tail vein; Positive control group: donepezil administered by gavage at a dose of 1.5 mg / kg / day; blank-EVs group: blank adipose-derived mesenchymal stem cell extracellular vesicles obtained in Comparative Example 2 were injected via tail vein at a dose of 1.8 mg / kg / 2d; EMO-EVs group: Extracellular vesicles of adipose-derived mesenchymal stem cells obtained in Example 2 were injected via tail vein at a dose of 1.8 mg / kg / 2 days.

[0086] 2. The test was conducted according to the method in Test Example 2, and the results are shown in Tables 3-4 and 4. Figures 4-5 As shown.

[0087] Table 3 Results of the novel object experiment in each group of mice

[0088] Note: Compared to the model group, p <0.001; compared with the blank-EVs group, ### p <0.001.

[0089] Table 4. Relative expression levels of target proteins in each group of mice (gray value of target protein / gray value of internal reference protein)

[0090] Note: Compared to the model group, p <0.05, p <0.01, p <0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01.

[0091] As shown in Table 3, the novel object preference index of mice in the sham-operated group, positive drug group, and EMO-EVs group was significantly higher than that in the model group. p <0.01. Furthermore, the novel object preference index of mice in the EMO-EVs group was significantly higher than that of their corresponding unregulated blank extracellular vesicle-EVs group (…). p <0.001), indicating that the emodin-regulated extracellular vesicles of adipose-derived mesenchymal stem cells prepared in Example 2 can alleviate memory deficits in AD model mice and have better therapeutic effects. Table 4 shows that EMO-EVs treatment can significantly improve the expression levels of pathological molecular markers related to Alzheimer's disease models. After EMO-EVs intervention, compared with the model group, the expression of p-Tau / t-Tau was significantly reduced ( p <0.05%, and the effect was significantly stronger than that of its corresponding unregulated extracellular vesicle blank-EVs ( p <0.05); EMO-EVs intervention also significantly downregulated abnormally elevated APP expression in the model group ( p <0.001, and its effect was significantly stronger than that of the blank-EVs group ( p <0.01); TSG-EVs also significantly suppressed the overexpression of Iba1 in the model group (p<0.001). Similarly, this effect was significantly stronger than that of the blank-EVs group. p <0.01). The above results indicate that the emodin-regulated extracellular vesicles of adipose-derived mesenchymal stem cells prepared in Example 2 can synergistically exert multiple pharmacological activities, including inhibiting excessive phosphorylation of tau protein in the brain tissue of AD mouse models, reducing amyloid-β protein deposition, and alleviating microglial cell activation, thus providing a new and effective strategy for the treatment of Alzheimer's disease.

[0092] according to Figure 4 It can be seen that, compared with the model group, both the positive control drug and EMO-EVs intervention treatments effectively reduced the concentration of Aβ1-42 in the hippocampus of mice. Furthermore, compared with the blank-EVs group, TSG-EVs intervention significantly reduced the concentration of Aβ1-42. p <0.01), indicating that the emodin-regulated extracellular vesicles of adipose-derived mesenchymal stem cells prepared in Example 2 can inhibit the deposition of Aβ1-42 in the brain tissue of an AD mouse model. According to Figure 5 It can be seen that the degenerated neuron index was significantly increased in the CA1 region of the hippocampus of the model group mice. Both positive control drugs and EMO-EVs intervention effectively improved neuronal morphology in the CA1 region of the small hippocampus and reduced the degenerated neuron index. Compared with blank-EVs, the proportion of degenerated neurons in the EMO-EVs group was significantly reduced ( p<0.001), and the neuronal morphology was better improved, indicating that the extracellular vesicles of adipose-derived mesenchymal stem cells regulated by emodin prepared in Example 2 can effectively protect the neuronal cell morphology in the CA1 region of the hippocampus in AD model mice.

[0093] Test Example 4 Extracellular vesicles of skin mesenchymal stem cells for the treatment of Alzheimer's disease (AD) 1. Eight-week-old male C57BL / 6 mice were used to establish an Alzheimer's disease (AD) model by stereotactic injection of streptozotocin (STZ) (3 mg / kg) into the lateral ventricle. A sham-operated group was set up as a control (n=8). Seven days after surgery, the AD mice were randomly divided into four groups (n=8): model group, positive drug group, blank extracellular vesicle group (blank-EVs), and KAE-EVs group, and treated as follows: Sham surgery group: 200 μL of normal saline was injected into the tail vein; Model group: 200 μL of normal saline was injected into the tail vein; Positive control group: donepezil administered by gavage at a dose of 1.5 mg / kg / day; blank-EVs group: blank skin mesenchymal stem cell extracellular vesicles obtained in Comparative Example 3 were injected via tail vein at a dose of 1.8 mg / kg / 2d; KAE-EVs group: extracellular vesicles of skin mesenchymal stem cells obtained in Example 3 were injected via tail vein at a dose of 1.8 mg / kg / 2 days.

[0094] 2. The test was conducted according to the method in Test Example 2, and the results are shown in Tables 5-6. Figures 6-7 As shown.

[0095] Table 5 Results of the novel object experiment in each group of mice

[0096] Note: Compared to the model group, p <0.05, p <0.001; compared with the blank-EVs group, ### p <0.001.

[0097] Table 6. Relative expression levels of target proteins in each group of mice (gray value of target protein / gray value of internal reference protein)

[0098] Note: Compared to the model group, p <0.05, p <0.01, p <0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01.

[0099] As shown in Table 5, the novel object preference index of mice in the sham surgery group, positive drug group, blank-EVs group, and KAE-EVs group was significantly higher than that in the model group. p <0.05. Furthermore, the novel object preference index of mice in the KAE-EVs group was significantly higher than that of their corresponding unregulated blank extracellular vesicle (blank-EVs) group. p <0.001), indicating that the kaempferol-regulated extracellular vesicles of skin mesenchymal stem cells prepared in Example 3 can alleviate memory deficits in AD model mice and have better efficacy. Table 6 shows that KAE-EVs treatment can significantly improve the expression levels of pathological molecular markers related to Alzheimer's disease models. After KAE-EVs intervention, compared with the model group, the expression of p-Tau / t-Tau was significantly reduced ( p <0.01), and the effect was significantly stronger than that of its corresponding unregulated extracellular vesicle blank-EVs ( p <0.05); KAE-EVs intervention can also significantly downregulate the abnormally elevated APP expression in the model group ( p <0.05, and its effect was significantly stronger than that of the blank-EVs group ( p <0.05); KAE-EVs can also significantly inhibit the overexpression of Iba1 in the model group ( p <0.001) Similarly, this effect is significantly stronger than that of the blank-EVs group ( p <0.01). The above results indicate that the kaempferol-regulated extracellular vesicles of skin mesenchymal stem cells prepared in Example 3 can synergistically exert multiple pharmacological activities, including inhibiting excessive phosphorylation of tau protein in the brain tissue of AD mouse models, reducing amyloid-β protein deposition, and alleviating microglial cell activation, thus providing a new and effective strategy for the treatment of Alzheimer's disease.

[0100] according to Figure 6 It can be seen that, compared with the model group, the positive control drug, blank-EVs, and KAE-EVs interventions all effectively reduced the concentration of Aβ1-42 in the hippocampus of mice. Furthermore, compared with the blank-EVs group, KAE-EVs intervention significantly reduced the concentration of Aβ1-42. p<0.01), indicating that the kaempferol-regulated extracellular vesicles of skin mesenchymal stem cells prepared in Example 3 can inhibit the deposition of Aβ1-42 in the brain tissue of an AD mouse model. According to Figure 7 It can be seen that the degenerated neuron index was significantly increased in the CA1 region of the hippocampus of the model group mice. Positive control drugs, blank-EVs, and KAE-EVs interventions all effectively improved neuronal morphology in the CA1 region of the small hippocampus and reduced the degenerated neuron index. Compared with blank-EVs, the proportion of degenerated neurons in the KAE-EVs group was significantly reduced ( p <0.01), and the neuronal morphology was better improved, indicating that the extracellular vesicles of skin mesenchymal stem cells regulated by kaempferol prepared in Example 3 can effectively protect the neuronal cell morphology in the CA1 region of the hippocampus in AD model mice.

[0101] Test Example 5 Umbilical cord mesenchymal stem cell extracellular vesicle therapy for Alzheimer's disease (AD) 1. Eight-week-old male C57BL / 6 mice were used to establish an AD model by stereotactic injection of streptozotocin (STZ) (3 mg / kg) into the lateral ventricle. A sham-operated group was set up as a control (n=8). Seven days after surgery, the AD mice were randomly divided into four groups (n=8): model group, positive drug group, blank extracellular vesicle group (blank-EVs), and RES-EVs group, and treated as follows: Sham surgery group: 200 μL of normal saline was injected into the tail vein; Model group: 200 μL of normal saline was injected into the tail vein; Positive control group: donepezil administered by gavage at a dose of 1.5 mg / kg / day; blank-EVs group: blank umbilical cord mesenchymal stem cell extracellular vesicles obtained in Comparative Example 4 were injected via tail vein at a dose of 1.8 mg / kg / 2d; RES-EVs group: extracellular vesicles of umbilical cord mesenchymal stem cells obtained in Example 4 were injected via tail vein at a dose of 1.8 mg / kg / 2d.

[0102] 2. The test was conducted according to the method described in Test Example 2, and the results are shown in Tables 7-8. Figures 8-9 As shown.

[0103] Table 7 Results of the novel object experiment in each group of mice

[0104] Note: Compared to the model group, p <0.05, p <0.001; compared with the blank-EVs group, ###p <0.001.

[0105] Table 8. Relative expression levels of target proteins in each group of mice (gray value of target protein / gray value of internal reference protein)

[0106] Note: Compared to the model group, p <0.05, p <0.01, p <0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01.

[0107] As shown in Table 7, the novel object preference index of mice in the sham surgery group, positive drug group, and RES-EVs group was significantly higher than that in the model group. p <0.001). Furthermore, the novel object preference index of mice in the RES-EVs group was significantly higher than that of their corresponding unregulated blank extracellular vesicle-EVs group (<0.001). p <0.001), indicating that the resveratrol-regulated extracellular vesicles of umbilical cord mesenchymal stem cells prepared in Example 4 can alleviate memory deficits in AD model mice and have better efficacy. Table 8 shows that RES-EVs treatment can significantly improve the expression levels of pathological molecular markers related to Alzheimer's disease models. After RES-EVs intervention, compared with the model group, the expression of p-Tau / t-Tau was significantly reduced ( p <0.001), and the effect was significantly stronger than that of its corresponding unregulated extracellular vesicle blank-EVs ( p <0.05); RES-EVs intervention also significantly downregulated abnormally elevated APP expression in the model group ( p <0.01, and its effect was significantly stronger than that of the blank-EVs group ( p <0.01); RES-EVs can also significantly inhibit the overexpression of Iba1 in the model group ( p <0.001) Similarly, this effect is significantly stronger than that of the blank-EVs group ( p <0.001). The above results indicate that the resveratrol-regulated extracellular vesicles of umbilical cord mesenchymal stem cells prepared in Example 4 can synergistically exert multiple pharmacological activities, including inhibiting excessive phosphorylation of tau protein in the brain tissue of AD mouse models, reducing amyloid-β protein deposition, and alleviating microglial cell activation, thus providing a new and effective strategy for the treatment of Alzheimer's disease.

[0108] according to Figure 8 It can be seen that, compared with the model group, both the positive control drug and RES-EVs intervention treatment can effectively reduce the concentration of Aβ1-42 in the hippocampus of mice. Furthermore, compared with the blank-EVs group, RES-EVs intervention can significantly reduce the concentration of Aβ1-42. p <0.05), indicating that the resveratrol-regulated extracellular vesicles of umbilical cord mesenchymal stem cells prepared in Example 4 can inhibit the deposition of Aβ1-42 in the brain tissue of an AD mouse model. According to Figure 9 It can be seen that the degenerated neuron index was significantly increased in the CA1 region of the hippocampus of the model group mice. Both the positive control drug and RES-EVs intervention can effectively improve the neuronal morphology in the CA1 region of the small hippocampus and reduce the degenerated neuron index. Compared with blank-EVs, the proportion of degenerated neurons in the RES-EVs group was significantly reduced (p<0.01), and the neuronal morphology was better improved, indicating that the resveratrol-regulated extracellular vesicles of umbilical cord mesenchymal stem cells prepared in Example 4 can effectively protect the neuronal cell morphology in the CA1 region of the hippocampus in AD model mice.

[0109] Test Example 6 Placental mesenchymal stem cell extracellular vesicle therapy for Alzheimer's disease (AD) 1. Eight-week-old male C57BL / 6 mice were used to establish an Alzheimer's disease (AD) model by stereotactic injection of streptozotocin (STZ) (3 mg / kg) into the lateral ventricle. A sham-operated group was set up as a control (n=8). Seven days after surgery, the AD mice were randomly divided into four groups (n=8): model group, positive drug group, blank extracellular vesicle group (blank-EVs), and QUE-EVs group, and treated as follows: Sham surgery group: 200 μL of normal saline was injected into the tail vein; Model group: 200 μL of normal saline was injected into the tail vein; Positive control group: donepezil administered by gavage at a dose of 1.5 mg / kg / day; The blank-EVs group was injected via tail vein with extracellular vesicles of blank placental mesenchymal stem cells obtained in Comparative Example 5 at a dose of 1.8 mg / kg / 2d. QUE-EVs group: extracellular vesicles of placental mesenchymal stem cells obtained in Example 5 were injected via tail vein at a dose of 1.8 mg / kg / 2 days.

[0110] 2. The test was conducted according to the method in Test Example 2, and the results are shown in Tables 9-10 and... Figures 10-11 As shown.

[0111] Table 9 Results of the novel object experiment in each group of mice

[0112] Note: Compared to the model group, p <0.05, p <0.001; compared with the blank-EVs group, ### p <0.001.

[0113] Table 10. Relative expression levels of target proteins in each group of mice (gray value of target protein / gray value of internal reference protein)

[0114] Note: Compared to the model group, p <0.05, p <0.01, p <0.001; compared with the blank-EVs group, # p <0.05, ## p <0.01.

[0115] As shown in Table 9, the novel object preference index of mice in the sham-operated group, the positive drug group, and the QUE-EVs group was significantly higher than that in the model group. p <0.01. Furthermore, the novel object preference index of the QUE-EVs group mice was significantly higher than that of their corresponding unregulated blank extracellular vesicle (blank-EVs) group (…). p <0.001), indicating that the quercetin-regulated extracellular vesicles of placental mesenchymal stem cells prepared in Example 5 can alleviate memory deficits in AD model mice and have better efficacy. Table 10 shows that QUE-EVs treatment can significantly improve the expression levels of pathological molecular markers related to Alzheimer's disease. After QUE-EVs intervention, compared with the model group, the expression of p-Tau / t-Tau was significantly reduced ( p <0.01, and the effect was significantly stronger than that of its corresponding unregulated extracellular vesicle blank-EVs (p<0.05); QUE-EVs intervention can also significantly downregulate the abnormally elevated APP expression in the model group ( p <0.01, and its effect was significantly stronger than that of the blank-EVs group ( p <0.05); QUE-EVs also significantly suppressed the overexpression of Iba1 in the model group ( p <0.001) Similarly, this effect is significantly stronger than that of the blank-EVs group ( p<0.01). The above results indicate that the quercetin-regulated extracellular vesicles of placental mesenchymal stem cells prepared in Example 5 can synergistically exert multiple pharmacological activities, including inhibiting excessive phosphorylation of tau protein in the brain tissue of AD mouse models, reducing amyloid-β protein deposition, and alleviating microglial cell activation, thus providing a new and effective strategy for the treatment of Alzheimer's disease.

[0116] according to Figure 10 It can be seen that, compared with the model group, the positive control drug, blank-EVs, and QUE-EVs interventions all effectively reduced the concentration of Aβ1-42 in the hippocampus of mice. Furthermore, compared with the blank-EVs group, QUE-EVs intervention significantly reduced the concentration of Aβ1-42. p <0.05), indicating that the quercetin-regulated extracellular vesicles of placental mesenchymal stem cells prepared in Example 5 can inhibit the deposition of Aβ1-42 in the brain tissue of an AD mouse model. According to Figure 11 It can be seen that the degenerated neuron index was significantly increased in the CA1 region of the hippocampus of the model group mice. Both positive control drugs and QUE-EVs intervention effectively improved neuronal morphology in the CA1 region of the small hippocampus and reduced the degenerated neuron index. Compared with blank-EVs, the proportion of degenerated neurons in the QUE-EVs group was significantly reduced ( p <0.05), and the neuronal morphology was better improved, indicating that the extracellular vesicles of placental mesenchymal stem cells regulated by quercetin prepared in Example 5 can effectively protect the neuronal cell morphology in the CA1 region of the hippocampus in AD model mice.

[0117] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the compound in regulating extracellular vesicle miRNA expression, characterized in that, The regulation includes upregulating the expression of miRNAs that negatively regulate disease progression and downregulating the expression of miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p; The miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

2. The application according to claim 1, characterized in that, The extracellular vesicles are stem cell-derived extracellular vesicles.

3. The application according to claim 2, characterized in that, The stem cells include one or more of the following: bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells, and placental mesenchymal stem cells.

4. An extracellular vesicle drug delivery system, characterized in that, This includes extracellular vesicles and miRNAs related to disease progression loaded within the extracellular vesicles; The miRNAs associated with disease progression include miRNAs that negatively regulate disease progression and miRNAs that positively regulate disease progression. The miRNAs that negatively regulate disease progression are significantly upregulated in the extracellular vesicles; the miRNAs that negatively regulate disease progression include one or more of miR-let-7c-5p, miR-486-5p, miR-132-3p, miR-160b, and miR-129-5p. The miRNAs that positively regulate disease progression are significantly downregulated in the extracellular vesicles; the miRNAs that positively regulate disease progression include one or more of miR-342-3p, miR-16-5p, and miR-125b-5p.

5. The method for preparing the extracellular vesicle drug delivery system according to claim 4, characterized in that, Includes the following steps: The compound was co-cultured with stem cells, and the cell culture medium was collected. The cell culture medium was centrifuged for the first time, and the supernatant was collected to obtain the first centrifuged product; The first centrifuged product is subjected to a second centrifugation, and the supernatant is collected to obtain the second centrifuged product; The second centrifuged product was purified to obtain an extracellular vesicle drug delivery system; The centrifugal force of the first centrifugation is 1500~3000×g, and the time is 8~20 min; The centrifugal force for the second centrifugation is 8000~12000×g, and the time is 20~40 min; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

6. The preparation method according to claim 5, characterized in that, The ratio of the compound to stem cells is 1~100000 nmol: 10 6 indivual; The co-culture method includes: after the stem cell density reaches 70-85% fusion, continue culturing for 12-24 hours.

7. The preparation method according to claim 5, characterized in that, The co-culture of compounds with stem cells includes one or more of the following: (1) Co-culture stilbene glycoside with bone marrow mesenchymal stem cells; the ratio of stilbene glycoside to bone marrow mesenchymal stem cells is 0.1~200 μmol: 10 6 indivual; (2) Co-culture emodin with umbilical cord mesenchymal stem cells; the ratio of emodin to umbilical cord mesenchymal stem cells is 1~100 μmol: 10 6 indivual; (3) Co-culture kaempferol with adipose-derived mesenchymal stem cells; the ratio of kaempferol to adipose-derived mesenchymal stem cells is 1~200 μmol: 10 6 indivual; (4) Co-culture resveratrol with skin mesenchymal stem cells; the ratio of resveratrol to skin mesenchymal stem cells is 0.1~200 μmol: 10 6 indivual; (5) Co-culture quercetin with placental mesenchymal stem cells; the ratio of quercetin to placental mesenchymal stem cells is 10~500 μmol: 10 6 indivual.

8. The preparation method according to claim 5, characterized in that, The purification method includes one or more of ultracentrifugation, polymer precipitation, size exclusion chromatography, and ultrafiltration.

9. The use of the compound or the extracellular vesicle drug delivery system according to claim 4 or the extracellular vesicle drug delivery system obtained by the preparation method according to any one of claims 5 to 8 in the preparation of a drug for treating Alzheimer's disease; The compounds include one or more of stilbene glycoside, emodin, kaempferol, resveratrol, and quercetin.

10. A drug for treating Alzheimer's disease, characterized in that, The active ingredient of the drug includes the extracellular vesicle drug delivery system of claim 4 or the extracellular vesicle drug delivery system obtained by the preparation method of any one of claims 5 to 8.

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