Exosome composition for resisting neurodegeneration as well as preparation method and application thereof

Abstract

The invention discloses an anti-neurodegeneration exosome composition as well as a preparation method and application thereof. The anti-neurodegeneration exosome composition is prepared from the following components in parts by mass: 5 to 25 parts of human exfoliated deciduous tooth stem cell exosome, 25 to 85 parts of plant-derived exosome-like nano-vesicles, 12 to 10 parts of ginsenoside Rg, 2 to 10 parts of curcumin and 10 to 20 parts of auxiliary materials. The anti-neurodegeneration exosome composition contains a large amount of cell factors, growth factors, miRNA, mRNAs and other components, is easy to fuse with cell membranes of intestinal epithelial cells, can selectively deliver bioactive substances to recipient cells through a blood-brain barrier, carries out information transmission among different cells, regulates signal transduction among the cells, and has a good anti-neurodegeneration effect. The effects of resisting oxidation, resisting inflammation, resisting aging and the like are achieved.

Description

Technical Field

[0001] The present invention relates to the application of engineering technology in biological tissues, and particularly relates to an exosome composition for anti-neural degeneration, a preparation method thereof, and an application thereof. Background Art

[0002] With the aging of the population, the number of people suffering from neurodegenerative diseases is increasing rapidly. Neurodegenerative diseases are a class of chronic progressive nervous system diseases characterized by neuronal degeneration or apoptosis. The main characteristics of these diseases are brain atrophy, neuronal degeneration and damage, resulting in the loss of function, which is usually irreversible, and current treatments cannot prevent the development of the diseases.

[0003] In recent years, stem cell therapy has been a promising treatment method for tissue regeneration and repair in many central nervous system or neurodegenerative diseases. Stem cells from human exfoliated deciduous teeth (SHED) are mesenchymal stem cells derived from the neural crest, with a tendency for neural differentiation, and their potential for regenerating neurons is superior to that of mesenchymal stem cells from other sources. SHED is an earlier embryonic-derived cell in development, with stronger proliferation levels, population doubling numbers, colony formation abilities, and multi-directional differentiation abilities. Deciduous teeth are an easier way to obtain seed cells, and multiple SHED stem cell banks have been established abroad. However, the direct use of stem cells has the disadvantages of inconvenient storage and transportation and immune rejection. Moreover, stem cells are prone to differentiating into other stromal cells, which may promote tumor cell metastasis and stimulate epithelial-mesenchymal cell transformation, posing a safety hazard.

[0004] Exosomes are extracellular vesicle structures produced by the invagination of the endosomal membrane, which encapsulate a large number of substances such as proteins, lipids, various RNAs, and DNA, with a diameter of about 30-150 nm and a density of 1.13-1.19 g / mL. Exosomes can act on neighboring cells through autocrine and paracrine methods, or be transmitted to distant cells through the blood circulation system, participating in the material exchange and information communication between cells. Exosome therapy avoids the risks of low survival rate, immune rejection, and malignant transformation in cell transplantation, and the administration method is relatively simple, stable, and controllable, suitable for large-scale clinical production. In vitro studies have found that Exo produced by SHED (SHED-Exo) can prevent the death of 80% of dopamine neurons in a Parkinson's disease model. Therefore, SHED-Exo combines the characteristics of low immunogenicity, a nano-carrier, and strong neuronal targeting ability, and has great potential in preventing and treating neurodegenerative diseases.

[0005] Plant exosome-like nanovesicles (PELNVs) derived from plants are also rich in various bioactive components such as lipids, proteins, and RNAs, and have significant regulatory effects in aspects such as immunomodulation, anti-inflammatory and anti-infection, and regenerative medicine. In addition, exosomes derived from plants such as edible fruits, vegetables, and traditional Chinese medicines have the advantages of low immunogenicity, small size, and strong tissue penetrability, and can maintain good physicochemical stability at different pH values and temperatures, and can regulate the homeostatic balance between host immunity and the gut microbiota (Dad HA, Gu TW, Zhu AQ, et al. Plant Exosome-like Nanovesicles: Emerging Therapeutics and Drug Delivery Nanoplatforms. Mol Ther. 2021 Jan 6; 29(1): 13-31.). Plant exosomes can be used as an efficient and safe carrier to effectively deliver bioactive substances into cells, mediate cell-to-cell communication, reduce adverse reactions and side effects, achieve more precise targeted therapy, and have broader application prospects.

[0006] Studies have shown that yam-derived exosome-like nanovesicles (Y-ELNs) have great potential in anti-aging of the body (Hwang JH, Park YS, Kim HS, et al. Yam-derived exosome-like nanovesicles stimulate osteoblast formation and prevent osteoporosis in mice. J Control Release. 2023 Mar;355:184-198.). Exosome-like vesicles extracted from blueberries (blueberry exosome-like nanoparticles, B-ELNs) have significant antioxidant stress effects (De Robertis M, Sarra A, D'Oria V, et al. Blueberry-Derived Exosome-Like Nanoparticles Counter the Response to TNF-α-Induced Change on Gene Expression in EA.hy926 Cells. Biomolecules. 2020 May 10;10(5):742.). Wolfberry is considered to have anti-aging effects in the Chinese Pharmacopoeia (Chang RCC, Ho YS, Yu MS, et al. Medicinal and nutraceutical uses of wolfberry in preventing neurodegeneration in Alzheimer’s disease. Recent Advances on Nutrition and the Prevention of Alzheimer’s disease, 2010:169-185.). Fresh wolfberry fruits are rich in up to 18 amino acids and trace elements beneficial to human health, and are food materials with both nutritional value and health care effects that are homologous in medicine and food. Currently, extracellular vesicles derived from wolfberry (GQ-ELNs) have not been reported.

[0007] However, at present, the combined utilization and development of plant exosome-like nanovesicles and exosomes from mammalian-derived stem cells are still relatively scarce, and the research and development of functional products based on exosome compositions against neurodegeneration are of great significance. Summary of the Invention

[0008] In view of this, the present invention provides an exosome composition for anti - neurodegeneration, and its preparation method and application. The stem cell exosome composition prepared by the present invention contains a large number of components such as cytokines, growth factors, miRNAs, mRNAs, etc. It is easy to fuse with the cell membrane of intestinal epithelial cells and can also cross the blood - brain barrier, selectively delivering biologically active substances to recipient cells, transmitting information between different cells, regulating inter - cellular signal transduction, having antioxidant, anti - inflammatory, anti - aging effects, etc., which can significantly improve the comprehensive intervention effect on neurodegenerative diseases, and realizing precise targeting and long - acting treatment of different dosage forms by using excipient modification and sequential release strategies.

[0009] The technical solution of the present invention is realized as follows:

[0010] In the first aspect of the present invention, there is provided an exosome composition for anti - neurodegeneration, comprising human exfoliated deciduous teeth stem cell exosomes, plant - derived exosome - like nanovesicles, ginsenoside Rg1, curcumin, and excipients.

[0011] Ginsenoside Rg1 has effects such as neuroprotection, promoting the proliferation of neural stem cells, anti - inflammation, and antioxidant, especially in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Curcumin can promote neurogenesis, anti - inflammation and antioxidant, and inhibit the aggregation of Aβ and tau proteins. However, the inherent defects of curcumin and Rg1 limit their drug development and application. For example, the oral bioavailability of free ginsenoside Rg1 is low (<5%); curcumin has low solubility (<1 μg / mL), strong photosensitivity, low blood - brain barrier penetration rate, and short half - life (<1 hour). The unique advantage of exosomes as an efficient carrier can overcome the inherent defects of curcumin and Rg1. Encapsulating curcumin in the lipid bilayer of SHED - Exo can improve the stability and half - life of curcumin, and exosome delivery can increase the content of ginsenoside Rg1 and curcumin in the brain.

[0012] This exosome composition can form a multi - dimensional treatment network of "neural regeneration (SHED - Exo, ginsenoside Rg1)+antioxidation (GQ - ELN)+metabolism regulation (B - ELN)+anti - inflammation (Y - ELN)+anti - pathological protein aggregation (curcumin)", significantly improving the comprehensive intervention effect on neurodegenerative diseases. Overcoming the inherent defects of ginsenoside Rg1 and curcumin through the exosome delivery system, and realizing precise targeting and long - acting treatment of different dosage forms by using excipient modification and sequential release strategies.

[0013] On the basis of the above technical solutions, further, it comprises the following components in parts by weight:

[0014]

[0015]

[0016] Among them, the unit portion is 10^8 human exfoliated deciduous tooth stem cell exosomes or plant-derived exosome-like nanovesicles, and the unit of the mass portion is g.

[0017] 1 unit portion is 10^8 human exfoliated deciduous tooth stem cell exosomes or plant-derived exosome-like nanovesicles.

[0018] 1 mass portion is 1 g of ginsenoside Rg1 or curcumin or excipient.

[0019] On the basis of the above technical solution, further, the plant-derived exosome-like nanovesicles include the following components in parts by number: 10-50 unit portions of yam exosome-like nanovesicles, 5-15 unit portions of blueberry exosome-like nanovesicles, and 10-20 unit portions of wolfberry exosome-like nanovesicles.

[0020] On the basis of the above technical solution, further, it includes the following components in parts by number: 7 mass portions of the human exfoliated deciduous tooth stem cell exosomes, 30 mass portions of the plant-derived exosome-like nanovesicles, 5 mass portions of ginsenoside Rg1, 7 mass portions of curcumin, and 10 mass portions of excipient.

[0021] On the basis of the above technical solution, further, the excipient includes a solubilizer and a lyoprotectant, and the mass ratio of the solubilizer to the lyoprotectant is (1-5):(5-9).

[0022] The solubilizer includes hydroxypropyl cyclodextrin;

[0023] The lyoprotectant includes any one of trehalose and sodium hyaluronate.

[0024] Hydroxypropyl cyclodextrin (HP-β-CD) can increase the solubility of the hydrophobic component curcumin and form an inclusion complex, and particles similar in size to exosomes can be formed by filtration extrusion; the lyoprotectant can maintain the integrity of exosomes and the stability of drugs during lyophilization and storage.

[0025] In the second aspect of the present invention, a method for preparing the above exosome composition for anti-neural degeneration is provided, including the following steps:

[0026] Mix the human exfoliated deciduous tooth stem cell exosomes, plant-derived exosome-like nanovesicles, ginsenoside Rg1, curcumin, and excipient evenly to prepare the exosome composition for anti-neural degeneration.

[0027] On the basis of the above technical solution, further, the method for preparing the human exfoliated deciduous tooth stem cell exosomes is: culturing, digesting, and subculturing human exfoliated deciduous tooth stem cells, collecting the supernatant of the human exfoliated deciduous tooth stem cell culture solution, and centrifuging to obtain the human exfoliated deciduous tooth stem cell exosomes.

[0028] On the basis of the above technical solutions, further, human deciduous tooth stem cells with passage numbers of 3 to 6 are collected.

[0029] In the third aspect of the present invention, there is provided an application of the above exosome composition against neurodegeneration, which is applied to the preparation of health products or drugs with the effect of promoting nerve regeneration, and health products or drugs for preventing and / or treating neurodegenerative diseases such as age-related physical aging, Alzheimer's disease, Parkinson's syndrome, etc.

[0030] The exosome composition prepared by the present invention can maintain neuronal activity, inhibit the effect of the neurotoxin MPP+ on the activity of neurons SH-SY5Y, and prevent neuronal aging.

[0031] It has the following beneficial effects compared with the prior art:

[0032] The stem cell exosome composition of the present invention contains a large number of components such as cytokines, growth factors, miRNAs, mRNAs, etc. It is easy to fuse with the cell membranes of adjacent cells, selectively deliver biologically active substances to recipient cells, conduct information transmission between different cells, regulate signal transduction between cells, and has effects such as antioxidant, anti-inflammatory, and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0034] Figure 1 It is the growth morphology diagram of primary and passaged deciduous tooth stem cells;

[0035] Figure 2 It is the detection result of various exosome-like nanovesicles and deciduous tooth stem cell exosomes by transmission electron microscopy;

[0036] Figure 3 It is the result of protein immunoblotting to detect the marker proteins of deciduous tooth stem cell exosomes;

[0037] Figure 4 It is the detection result of plant-derived exosome-like nanovesicles and deciduous tooth stem cell exosomes by nanoparticle tracking analyzer;

[0038] Figure 5 It is the detection result of CCK8 reagent to analyze the protection of neuronal activity by plant-derived exosome-like nanovesicles, deciduous tooth stem cell exosomes and exosome composition;

[0039] Figure 6 To detect the protective effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on neuronal activity for senescence detection reagents;

[0040] Figure 7 To detect the effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on the recovery of motor function in a MPTP-induced Parkinson's mouse model by pole climbing test;

[0041] Figure 8 To detect the effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on the recovery of motor function in a MPTP-induced Parkinson's mouse model by balance beam test;

[0042] Figure 9 To detect the effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on the recovery of motor function in a MPTP-induced Parkinson's mouse model by forelimb suspension test;

[0043] Figure 10 To detect the results of Nissl staining, immunohistochemistry, and Western blot of brain tissues in a mouse model;

[0044] Figure 11 To analyze the effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on the intestinal flora abundance in a MPTP-induced Parkinson's mouse model by 16S rRNA sequencing of intestinal microbiota, at the phylum, class, order, and family taxonomic levels respectively;

[0045] Figure 12 To analyze the effect of plant-derived exosome-like nanovesicles, exfoliated deciduous tooth stem cell exosomes, and exosome compositions on the intestinal flora abundance in a MPTP-induced Parkinson's mouse model by 16S rRNA sequencing of intestinal microbiota, at the genus and species taxonomic levels respectively;

[0046] Figure 13 To analyze the bar chart of the LDA value distribution based on ASV for the 16S rRNA sequencing results of intestinal microbiota;

[0047] Figure 14 To perform OPLS-DA model validation and T-test differential analysis of differential bacterial genera for the 16S rRNA sequencing results of intestinal microbiota;

[0048] Figure 15 To perform T-test differential analysis of Tax4Fun2 functional annotation based on ASV for the 16S rRNA sequencing results of intestinal microbiota;

[0049] Figure 16Results of case detection, immunohistochemical detection, and Western blot detection of colon tissue in a mouse model. Detailed implementation manners

[0050] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0051] Example 1 Preparation and identification of exosomes from exfoliated deciduous teeth stem cells

[0052] 1. Collection of exfoliated deciduous teeth stem cells

[0053] The collection method includes the following steps carried out in sequence:

[0054] Exfoliated deciduous teeth are taken from children who seek medical treatment at the Department of Stomatology of the First People's Hospital Affiliated to Hubei University of Medicine in Xiangyang. The teeth are complete, without caries, pulp disease, periapical disease, and periodontal disease. The age is 6 - 12 years old, gender is not limited, and there are no infectious diseases such as hepatitis and tuberculosis.

[0055] Before tooth exfoliation, the tooth is disinfected with iodophor III. After complete extraction, the surface of the deciduous tooth is disinfected again. It is placed in a culture medium centrifuge tube containing 2 - fold triple antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B, all purchased from Invitrogen, USA), and taken to the laboratory as soon as possible.

[0056] 2. Isolation of exfoliated deciduous teeth stem cells

[0057] The operation is carried out in a laminar flow hood. The exfoliated deciduous teeth are placed in a sterile 35 - mm culture dish, and rinsed repeatedly 3 times with PBS containing 2 - fold triple antibiotics. The dental pulp is removed as completely as possible using an endodontic broach, and the dental pulp is placed in an EP tube. 0.5 mL of 3 mg / mL type I collagenase and 0.5 mL of 4 mg / mL neutral protease (Dispase) are added respectively. The dental pulp tissue is minced with ophthalmic scissors and digested in a 37°C carbon dioxide incubator for 30 minutes, gently shaken every 10 minutes until there are no obvious tissue blocks floating.

[0058] An equal volume of human mesenchymal stem cell - free serum medium (Procell, CM - SC01) is added, mixed well, centrifuged at 800 r / min for 5 min, and the supernatant is discarded to obtain the digested and dissociated dental pulp cells.

[0059] The above - mentioned dental pulp cells are resuspended with stem cell culture medium, counted after repeated pipetting and mixing, and the remaining cells are transferred to a 6 - well plate and cultured in an incubator at 37°C with a CO2 saturation of 5%.

[0060] The above cells were cultured until they adhered to the wall, and the culture medium was changed by half every 3 days. The growth status of the cells was observed under an inverted microscope. After the cells reached 80% confluence, they were passaged at a ratio of 1:3. The cells used for passage were inoculated into a cell culture flask with air holes, thereby obtaining stem cells from exfoliated deciduous teeth, as Figure 1 shown.

[0061] 3. Cryopreserve the stem cells from exfoliated deciduous teeth, that is, establish a stem cell bank of exfoliated deciduous teeth

[0062] When the cell confluence reached 80%, cryopreservation was carried out, which specifically included the following steps:

[0063] Take out the culture dish from the incubator, discard the original culture medium, and rinse twice with PBS; add an appropriate volume of digestive solution containing 0.05% trypsin and 0.004% EDTA, gently shake the culture dish to make the digestive solution cover all cell surfaces; digest at 37°C for 2 min, and observe the cell morphology under the microscope; when the cell gaps become larger, the cell shapes become round, and a few cells fall off, add human mesenchymal stem cell serum-free medium at a volume ratio of 1:1 to terminate digestion.

[0064] Gently pipette the culture medium in a clockwise direction at the bottom of the culture flask to detach the cells from the wall. While pipetting the cells to detach them from the wall, collect the detached cells. Collect the detached cells into a 15 mL centrifuge tube. After most of the cells have fallen off, add PBS and pipette at the bottom of the flask, and then collect into the same centrifuge tube. Place the centrifuge tube containing the cells into a centrifuge, with the centrifuge speed at 1000 r / min and centrifuge for 5 min, and discard the supernatant; add a cell cryopreservation solution containing 0.9 mL of culture medium and 0.1 mL of DMSO to the centrifuge tube, mix well, and gently disperse the cell clumps, then transfer them into a 1.5 mL cryopreservation tube and seal it with a sealing film. Make good marks, place the cryopreservation tube in a programmable cooling box, put it into a -80°C refrigerator, and transfer it to a dedicated liquid nitrogen tank for stem cells after 24 h.

[0065] 4. Extraction of exosomes from stem cells of exfoliated deciduous teeth

[0066] After resuscitating or passaging the stem cells from exfoliated deciduous teeth in step 3, when the cells grew to 80% confluence, collect the culture supernatant, continuously collect about 500 mL of cell culture medium, and aliquot it into 50 mL centrifuge tubes;

[0067] The centrifuge was precooled at 4°C; first, the 50 mL centrifuge tube containing the culture supernatant of deciduous dental stem cells was leveled and placed in the precooled centrifuge, and the cells were removed by centrifugation at 4°C for 10 min with a centrifugal force of C=300 g (where C represents the centrifugal force); then, the supernatant was transferred to a new 50 mL centrifuge tube, and then centrifuged at 2000 g and 4°C for 20 min to remove cell debris; then, the supernatant was transferred to a new 50 mL centrifuge tube, and then centrifuged at 10000 g and 4°C for 30 min to remove large vesicles; finally, the supernatant was carefully transferred to an ultracentrifuge tube, and centrifuged at 4°C, 110000 g, for 90 min, the supernatant was discarded, the bottom precipitate was resuspended with PBS, and then centrifuged at 4°C, 110000 g, for 90 min; the supernatant was carefully discarded, and the bottom precipitate was resuspended with an appropriate amount of PBS to obtain the exosomes of the deciduous dental stem cells.

[0068] 5. Identification of exosomes from deciduous tooth stem cells

[0069] Observation using a transmission electron microscope includes the following steps:

[0070] The extracted exosomes of deciduous stem cells were resuspended with PBS and dropped onto a sample-carrying copper mesh with a pore size of 2 nm. The sample was allowed to stand at room temperature for 2 minutes. The liquid was blotted from the side of the mesh with filter paper. The exosomes of deciduous stem cells were negatively stained with 3% phosphotungstic acid solution at room temperature for 5 minutes. The negative staining liquid was blotted with filter paper, dried at room temperature, and observed and photographed under an electron microscope. Under an electron microscope, the exosomes have very obvious membrane boundaries and are saucer or cup-shaped structures of varying sizes of 30 to 100 nm.

[0071] Electron microscope image reference Figure 2 As shown. Figure 2 It can be seen that the prepared exosomes present vesicle structures of different sizes, with diameters ranging from 30-100 nm, scale = 100 nm.

[0072] 6. Western blotting detection of exosomes from deciduous tooth stem cells

[0073] The extracted exosomes from deciduous tooth stem cells were resuspended in PBS, lysed with RIPA lysis buffer, and the total protein was extracted. The protein concentration was determined by BCA method to ensure that the total protein loading amount was 30 μg / well. The expression of positive markers CD63 and HSP70 of exosomes in the samples, as well as the expression of negative marker GM130, were detected by immunoblotting. Figure 3 As shown in the figure, GM130 is a Golgi matrix protein that can be detected in total cell proteins and should be negative in exosomes, while CD63 and HSP70 are exosome marker proteins and should be positive in exosomes. Figure 3It can be seen that GM130 was not detected in the exosomes of shed deciduous teeth stem cells, while GM130 was positive in shed deciduous teeth stem cells. CD63 and HSP70 were positive in the exosome proteins of shed deciduous teeth stem cells. Therefore, Western blotting detection confirmed the successful extraction of exosomes from shed deciduous teeth stem cells.

[0074] Example 2 Preparation and Identification of Exosome-like Nanoparticles Derived from Plant Cells

[0075] The preparation of exosome-like nanoparticles derived from plant cells includes the following steps:

[0076] 1. Weigh 50 g of the edible parts of washed Chinese yam / blueberry / wolfberry, mix and chop or mash them preliminarily, then add 8 times the amount of sterile PBS, and homogenize at 50 r / min for 15 min in a wall breaker (Joyoung Z8-V82) until completely broken.

[0077] 2. Filter and collect the Chinese yam / blueberry / wolfberry juice with a 200-μm nylon mesh into a 50-mL centrifuge tube, centrifuge at 4°C for 30 min, then transfer the supernatant to a new 50-mL centrifuge tube and centrifuge at 2000 g at 4°C for 10 min; then transfer the supernatant to a new 50-mL centrifuge tube and centrifuge at 6000 g at 4°C for 20 min.

[0078] 3. Filter the supernatant obtained in step 2 with a 0.22-μm filter membrane to remove large-particle-size substances.

[0079] 4. Carefully transfer the supernatant obtained in step 3 to an ultracentrifuge tube and centrifuge at 4°C, 110000 g for 90 min.

[0080] 5. Discard the supernatant, resuspend the bottom precipitate with PBS, and then purify it with a sucrose cushion. The specific operation is as follows: Carefully load the above resuspended PBS liquid into an ultracentrifuge tube with a 3-4 mL 30% deuterated water sucrose cushion at the bottom of the tube, and the liquid should be filled to 2-3 mm from the centrifuge tube mouth. Centrifuge at 4°C, 110000 g for 90 min.

[0081] 6. Remove as much supernatant above the sucrose cushion as possible, and dilute the bottom sucrose layer with an appropriate amount of DPBS. Transfer the DPBS-diluted sucrose layer solution into a new ultracentrifuge tube and centrifuge at 4°C, 110000 g for 90 min.

[0082] 7. Carefully remove the supernatant, and the bottom precipitate is the plant-derived exosome-like nanovesicles (PDLNVs).

[0083] 8. Identification of plant-derived exosome-like nanovesicles: nanoparticle tracking analysis (NTA). Resuspend the extracted plant-derived exosome-like nanovesicles with PBS, and determine the protein concentration by BCA method to ensure that the total protein concentration is ≥ 0.2 μg / μL. Take 20 μL and add it to a new 1.5 mL EP tube, then add 980 μL of DPBS to dilute 50 times. Select the required measurement method, adjust the measurement parameter to the dilution factor, and draw the particle size and concentration distribution diagram.

[0084] Prepare and identify yam-derived exosome-like nanovesicles (Y-ELNs), blueberry-derived exosome-like nanovesicles (B-ENVs), and Gouqizi-derived exosome-like nanovesicles (GQ-ENVs) according to steps 1 - 8.

[0085] The identification of exosome-like nanoparticles derived from plant cells includes the following steps:

[0086] Use the nanoparticle tracking analyzer (model: Zetaview-PMX120-Z) of Particle Metrix Company in Germany to detect the particle size distribution and particle concentration of yam-derived exosome-like nanovesicles (Y-ELNs), Gouqizi-derived exosome-like nanovesicles (GQ-ENVs), blueberry-derived exosome-like nanovesicles (B-ENVs), and stem cell-derived exosomes from shed deciduous teeth (SHED-Exos) extracted by the above method.

[0087] As Figure 4 shown, the particle size ranges of Y-ELN, GQ-ELN, B-ELN, and SHED-Exo are all between 50 - 150 nm, which is in line with the size range of exosomes.

[0088] The morphological characteristics of yam-derived exosome-like nanovesicles (Y-ELNs), Gouqizi-derived exosome-like nanovesicles (GQ-ENVs), blueberry-derived exosome-like nanovesicles (B-ENVs), and stem cell-derived exosomes from exfoliated deciduous teeth (SHED-Exos) extracted by the above method were detected using a transmission electron microscope (model: IHT7700) from Hitachi, Japan. The prepared suspension sample was aspirated with a pipette and dropped onto a copper grid with a supporting film. According to the sample concentration in the suspension, after immediately or leaving it for several minutes, the excess liquid was aspirated from the edge of the droplet with filter paper, and then the staining solution could be dropped, and stained for 3 - 5 min. Then the staining solution was aspirated with filter paper, dried, and observed under a transmission electron microscope.

[0089] As Figure 2 shown, Y-ELN, GQ-ELN, B-ELN, and SHED-Exo all have a typical lipid bilayer membrane structure. The morphologies of Y-ELN, GQ-ELN, and B-ELN are round, with a diameter of about 100 nm, which conforms to the characteristics of plant exosomes; the morphology of SHED-Exo is a saucer or cup-like structure, with a diameter of about 100 nm, which conforms to the characteristics of mammalian exosomes.

[0090] Example 3 Preparation of an exosome composition against neurodegeneration

[0091] This example provides a method for preparing an exosome composition against neurodegeneration. By parts, 15 parts of SHED-Exo, 18 parts of Y-ELN, 18 parts of GQ-ELN, 18 parts of B-ELN, 8 g of ginsenoside Rg1, 8 g of curcumin, 3 g of HP-β-CD, and 8 g of trehalose were taken, and after mixing evenly, the exosome (ELNs-Mix) composition against neurodegeneration was obtained.

[0092] Example 4 Neuron protection experiment

[0093] The SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and ELNs-Mix prepared in the above examples were applied to the neuron protection experiment.

[0094] The SH-SY5Y nerve cells are close to mature human neurons found in the body. SH-SY5Y cells are widely used in experimental studies of neurodegenerative diseases, especially in Parkinson's disease, Alzheimer's disease, etc. Moreover, SH-SY5Y cells express tyrosine hydroxylase, dopamine 2B2 hydroxylase, and dopamine transporter, which are unique to catecholaminergic neurons. Therefore, SH-SY5Y cells induced by 1-methyl-4-phenylpyridine (MPP+) are widely used in the study of the pathogenesis of PD.

[0095] It includes the following steps:

[0096] 1. Digest the SH-SY5Y cells in the logarithmic growth phase and inoculate them into 3 96-well plates (5000 cells / well), and culture overnight;

[0097] 2. In the first 96-well plate, treat the cells with MPP+ and various exosome-like nanovesicles (ELNs) and exosome composition (ELNs-Mix) simultaneously for 24 hours;

[0098] 3. In the second 96-well plate, pre-protect the cells with various ELNs and ELNs-Mix for 24 hours and then add MPP+ to treat for 24 hours; as a pre-protection experiment.

[0099] 4. In the third 96-well plate, treat the cells with MPP+ for 24 hours first, and then add various ELNs and ELNs-Mix to intervene for 24 hours; as a therapeutic experiment.

[0100] 5. The SH-SY5Y cells in steps 2-4 are finally treated with CCK-8 reagent, and the absorbance value is detected with an enzyme-labeled instrument at a wavelength of 450 nm, and the cell viability is calculated. The results are as Figure 5 shown.

[0101] It can be Figure 5 seen that ELNs-Mix has a promoting effect on cell viability, MPP+ causes a decrease in cell viability, but the cell viability can be improved when ELNs-Mix is used for pre-protection or added simultaneously with MPP+. After treating the cells with MPP+ for 24 hours first, stem cell-derived extracellular vesicles from exfoliated deciduous teeth (SHED-Exo) and ELNs-Mix can also improve cell viability. In addition, the pre-protection effect of Y-ELN and GQ-ELN on SH-SY5Y cells is basically the same as that of SHED-Exo on SH-SY5Y cells, but the pre-protection effect of ELNs-Mix is better than that of GQ-ELN. The results of the therapeutic experiment show that the therapeutic effect of ELNs-Mix is better than that of SHED-Exo.

[0102] Example 5 Cell Senescence Experiment

[0103] As people age, the nervous system gradually becomes fragile and unstable. This natural aging process may lead to nerve cell death, loss of synaptic connections, and decline in neuron function. Many neurological diseases can also cause nerve aging. Aging leads to the loss of neuron morphology and function, thus becoming the most important risk factor for neurodegenerative diseases.

[0104] SA-β-Gal activity and oxidative stress (ROS) levels are common detection indicators often used to evaluate cell aging.

[0105] Apply the SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and ELNs-Mix prepared in the above examples to the cell aging experiment, including the following steps:

[0106] 1. Digest and count the SH-SY5Y cells in the logarithmic growth phase and inoculate them into a 12-well plate (5 * 10^5 cells / well), and culture overnight;

[0107] 2. Add various ELNs and ELNs-Mix to the wells in step 1, pre-protect the cells for 24 hours first, and then add MPP+ and treat for 24 hours;

[0108] 3. Treat the cells in step 2 with an SA-β-Gal activity detection kit, take pictures with a microscope, and evaluate the cell aging level according to the number of β-Gal positive cells. The results are as Figure 6 shown.

[0109] It can be Figure 6 seen that there are some β-Gal positive cells in the untreated SH-SY5Y cells (Ctrl group), fewer β-Gal positive cells in the group only using ELNs-Mix, significantly more β-Gal positive cells in the MPP+ group, and the number of β-Gal positive cells in the cells pre-protected by each group of ELNs and ELNs-Mix is significantly less than that in the MPP+ group.

[0110] Example 6 Motor Ability Experiment

[0111] The incidence of Parkinson's disease (PD) in people over 65 years old in China is 1% - 2%, which is the second most common neurodegenerative disease after Alzheimer's disease. Currently, there are about more than 3 million PD patients in the country, and the disease shows a trend of getting younger. MPTP is the only known dopaminergic neurotoxin that can cause clinical manifestations similar to Parkinson's disease in humans and monkeys.

[0112] The pole climbing test, hanging test, and balance beam test are commonly used indicators to evaluate the motor ability, posture, and coordination of the model as well as its motor skills, and are also methods to evaluate the success rate and drug efficacy of the model.

[0113] The SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and ELNs-Mix prepared in the above embodiments were applied to the motor ability experiment, including the following steps:

[0114] 1. Seventy 10-week-old male C57BL / 6J mice with a body weight of (30 ± 5) g were adaptively fed for 1 week and then divided into a control group, an MPTP group, and treatment groups including an MPTP+SHED-Exo group, an MPTP+Y-ELNs group, an MPTP+B-ELNs group, an MPTP+GQ-ELNs group, and an MPTP+ELNs-Mix group according to the random number table method, with 10 mice in each group.

[0115] The model group was intraperitoneally injected with MPTP (30 mg / kg) for 7 days, and the control group was intraperitoneally injected with an equal dose of PBS. The treatment groups were given SHED-Exo, Y-ELNs, B-ELNs, GQ-ELNs, and ELNs-Mix for treatment on the second day of modeling, once every two days for 15 consecutive times;

[0116] 2. The experimental mice in step 1 were pre-trained before drug administration. During the pre-training and the formal experiment, each mouse was detected 3 times repeatedly, with a certain time interval between each detection, and the average value of the three experimental results was calculated; the motor function detection included steps 3-5:

[0117] 3. Pole Test: A wooden pole with a diameter of 1 cm and a length of 50 cm was self-made, with a softwood ball with a diameter of 2.5 cm fixed at the top. The wooden pole was wrapped with gauze to prevent slipping and placed in the cage where the mice lived at an inclination of 45°. The tested mouse was placed head-down on the top of the softwood ball and allowed to climb down the pole naturally. Timing started when the mouse touched the ball and ended when the mouse's front paws touched the ground. Observe the behavior of the mouse during the downward process and record the time it took for the mouse to climb from the top of the wooden pole to the bottom.

[0118] Each mouse had to complete 3 pole tests, with an interval of 10 min between each test. The average value of the three pole-climbing times was taken as the experimental score of the mouse. The results are as Figure 7 shown.

[0119] As Figure 7 can be seen, compared with the control group, the total pole-climbing time of the mice in the MPTP model group increased (P<0.001). The total pole-climbing time of the mice in each treatment group was close to that of the normal control group, but only the ELNs-Mix combined treatment group reached a significant difference with statistical significance.

[0120] 4. Cross Beam Test: A sturdy parallel bar (1 cm in diameter and 115 cm in length) was used, with its end connected to a small black light-tight box (15 cm × 15 cm × 15 cm) placed on the platform. The height of the balance bar from the ground was 30 cm. The time taken by the tested mice to pass through the parallel bar was recorded, as well as the situations such as one hind limb falling off, falling, or swaying. The time for the mice to pass through the balance beam was recorded to evaluate the coordination ability of the hind limbs of the experimental animals. The results are as Figure 8 shown.

[0121] As Figure 8 can be seen, compared with the control group, the total time for the mice in the MPTP model group to pass through the balance beam was significantly increased, while the total time for the mice in the SHED-Exo, several ELNs, and the combined treatment group of SHED-Exo and ELNs-Mix to pass through the balance beam was significantly shortened.

[0122] 5. Hang Test: Before the experiment, a stainless-steel bar was prepared for the hanging test of the mice. It could be wrapped with gauze to prevent slipping, and both ends were placed on the shelf for easy fixation. The height of the stainless-steel bar from the ground was 30 cm. After the tested mice were inverted and hung at the midpoint of the stainless-steel bar, the mice were released, and the condition of the hind limbs of the mice grasping the rope was observed for evaluation. Scoring criteria: Both hind limbs grasping the stainless-steel bar were scored 3 points, one hind limb grasping the bar was scored 2 points, and neither hind limb grasping the bar was scored 1 point. The experiment was repeated 3 times and the average value was taken, with an interval of 10 min between each experiment. The results are as Figure 9 shown.

[0123] As Figure 9 can be seen, compared with the control group, the score of the mice in the MPTP model group decreased (P < 0.001); while compared with the MPTP model group, the scores of the mice in the SHED-Exo and the combined treatment group of SHED-Exo and ELNs-Mix increased significantly.

[0124] Example 7 Detection of Nissl staining, immunohistochemistry, and Western blot of mouse brain tissue

[0125] The mouse model in Example 6 was subjected to Nissl staining, immunohistochemistry, and Western blot of brain tissue. The results are as Figure 10 shown in A. The number of neurons in the midbrain region of the mice in the MPTP model group decreased, while the number of neurons in the midbrain region of the mice in the treatment groups of SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and the exosome composition (ELNs-Mix) recovered; the expression level of pathological α-synuclein (P-αsyn) in the midbrain region of the mice in the MPTP model group increased, and the expression level of P-αsyn in each treatment group decreased ( Figure 10 B).

[0126] The results of Western blot analysis showed that the expression level of tyrosine hydroxylase (TH) decreased significantly in the MPTP model group, indicating a reduction in dopaminergic neurons, while the TH expression level increased significantly in each treatment group ( Figure 10 C,D); the expression level of P-αsyn detected by Western blot was consistent with the Figure 10 results of immunohistochemical detection. The expression level of P-αsyn in the midbrain region of mice in the MPTP model group increased significantly, while the expression level of P-αsyn in each treatment group decreased significantly ( Figure 10 C,E).

[0127] Example 8 Sequencing of Intestinal Microbiota in Mice

[0128] The intestinal microbiota of the mouse model in Example 6 was sequenced for 16S rRNA, and the results are as Figures 11 - 14 shown.

[0129] As Figures 11 - 14 can be seen, in the mouse intestinal microbiota of the Y-ELN, GQ-ELN, B-ELN, and exosome composition (ELNs-Mix) treatment groups, the abundance of Akkermansia increased at each taxonomic level; while the Streptococcus in the feces of the model group mice increased significantly. Streptococcus is a genus of Gram-positive cocci, belonging to the Streptococcaceae family and the Lactobacillales order in the phylum Firmicutes. In the Y-ELN, GQ-ELN, B-ELN, and exosome composition (ELNs-Mix) treatment groups, the abundances of the genus Streptococcus and Streptococcus_equinus were reversed. The s_Lactobacillus_reuteri in the feces of the model group mice increased, and after treatment with Y-ELN, GQ-ELN, B-ELN, SHED-Exo, and exosome composition (ELNs-Mix), it recovered to the level of the relative normal control group.

[0130] The functional prediction analysis of the intestinal microbiota is as Figure 15 shown. As Figure 15 can be seen, the functional enrichment at the Level1 level showed that the microbial functions in the ELNs-Mix treatment group were mainly enriched in the metabolic pathway ( Figure 15A), in which the functional enrichment at the Level 2 level shows that compared with the model group, the microbial functions in the ELNs-Mix treatment group are significantly enriched in lipid metabolism, transport and catabolism, metabolism of cofactors and vitamins, and glycan biosynthesis and metabolism ( Figure 15 B).

[0131] Example 9 Histopathological detection, immunohistochemical detection, and Western blot detection of mouse colon tissue

[0132] The mouse model of Example 6 was subjected to histopathological detection, immunohistochemical detection, and Western blot detection of colon tissue. The results are as follows Figure 16 shown in A. The colon morphology of the mice in the MPTP model group changed, with obvious inflammatory infiltration and increased expression of P-αsyn. The colon morphology of the mice in the SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and exosome composition (ELNs-Mix) treatment groups was good, and the expression of P-αsyn decreased; Detection of the peripheral blood lipopolysaccharide level showed that ( Figure 16 B), the level of LPS in the peripheral blood of the mice in the MPTP model group was significantly increased, and the level of LPS in the peripheral blood of the mice in the SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and exosome composition (ELNs-Mix) treatment groups was significantly decreased; suggesting the restoration of intestinal barrier function.

[0133] The results of Western blot detection showed that the expression level of TLR4 in the colon tissue of the mice in the MPTP model group was significantly increased, and the expression level of TLR4 in the colon of the mice in the SHED-Exo, Y-ELN, GQ-ELN, B-ELN, and ELNs-Mix treatment groups was significantly inhibited ( Figure 16 C, D); the expression levels of ZO-1 and Claudin were reversed in each exosome treatment group, and the expression levels of ZO-1 and Claudin in the ELNs-Mix group were significantly upregulated ( Figure 16 C-F).

[0134] In summary, various exosomes and exosome compositions prepared in this application can maintain neuronal activity, inhibit the effect of the neurotoxin MPP+ on the activity of neurons SH-SY5Y, and prevent neuronal aging. It can be used to prepare health products or drugs with the effect of promoting nerve regeneration, and can also be used to prepare health products or drugs for preventing and / or treating neurodegenerative diseases such as age-related physical aging, Alzheimer's disease, and Parkinson's syndrome.

[0135] Various exosomes and exosome compositions can improve the motor dysfunction in a mouse model of MPTP-induced Parkinson's disease. They can be used to prepare health products or drugs for improving neurodegenerative diseases such as age-related physical aging and Parkinson's syndrome caused by environmental factors.

[0136] The miRNAs in PELNVs can regulate the gene expression of Lactobacillus in the mouse intestine, induce the production of more interleukin (IL)-22, thereby maintaining intestinal homeostasis and improving the inflammation level (Zhao B, Lin H, Jiang X, et al. Exosome-like nanoparticles derived from fruits, vegetables, and herbs: innovative strategies of therapeutic and drug delivery. Theranostics. 2024 Aug 1;14(12):4598-4621.). The examples of this application confirm that Y-ELN, GQ-ELN, and B-ELN can significantly increase the abundance of anti-inflammatory Akkermansia and inhibit the pro-inflammatory Streptococcus. Therefore, the effect of the ELNs-Mix treatment group is the result of the "dual-pronged approach" of the brain targeting of SHED-Exo and the intestinal targeting of PELNVs.

[0137] The exosome composition prepared in this application can form a multi-dimensional treatment network of "nerve regeneration (SHED-Exo, ginsenoside Rg1) + antioxidant (GQ-ELN) + metabolic regulation (B-ELN) + anti-inflammatory (Y-ELN) + anti-pathological protein aggregation (curcumin)", significantly enhancing the comprehensive intervention effect on neurodegenerative diseases. Overcome the inherent defects of ginsenoside Rg1 and curcumin through the exosome delivery system, and use excipient modification and sequential release strategies to achieve precise targeting and long-term treatment of different dosage forms.

[0138] The above is only the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An exosome composition for anti - neurodegeneration, characterized in that, It includes human exfoliated deciduous tooth stem cell exosomes, plant-derived exosome-like nanovesicles, ginsenoside Rg1, curcumin and excipients.

2. The exosome composition for anti - neurodegeneration according to claim 1, characterized in that, It includes the following components in parts by number: Among them, the unit part is 10^8 human exfoliated deciduous tooth stem cell exosomes or plant-derived exosome-like nanovesicles, and the unit of the part by mass is g.

3. The exosome composition for anti - neurodegeneration according to claim 1, characterized in that, The plant-derived exosome-like nanovesicles include the following components in parts by number: 10 - 50 unit parts of yam exosome-like nanovesicles, 5 - 15 unit parts of blueberry exosome-like nanovesicles, and 10 - 20 unit parts of wolfberry exosome-like nanovesicles.

4. The exosome composition for anti - neurodegeneration according to claim 1, characterized in that, It includes the following components in parts by number: 7 unit parts of the human exfoliated deciduous tooth stem cell exosomes, 30 unit parts of the plant-derived exosome-like nanovesicles, 5 parts by mass of ginsenoside Rg1, 7 parts by mass of curcumin, and 10 parts by mass of excipients.

5. The exosome composition for resisting nerve degeneration according to claim 1, wherein, The excipients include a solubilizer and a lyoprotectant, and the mass ratio of the solubilizer to the lyoprotectant is (1 - 5):(5 - 9).

6. The preparation method of the exosome composition for anti - neurodegeneration according to any one of claims 1 to 5, characterized in that, It includes the following steps: Mix the human exfoliated deciduous tooth stem cell exosomes, plant-derived exosome-like nanovesicles, ginsenoside Rg1, curcumin and excipients evenly to prepare the exosome composition for anti-neural degeneration.

7. The preparation method of the exosome composition for anti - neurodegeneration according to claim 6, characterized in that, The preparation method of the human exfoliated deciduous tooth stem cell exosomes is: culture, digest and passage human exfoliated deciduous tooth stem cells, collect the supernatant of the human exfoliated deciduous tooth stem cell culture solution, and centrifuge to obtain the human exfoliated deciduous tooth stem cell exosomes.

8. The preparation method of the exosome composition for anti - neurodegeneration according to claim 7, characterized in that, Collect human exfoliated deciduous tooth stem cells with passage numbers of 3 - 6 generations.

9. Use of the exosome composition against neurodegeneration according to any one of claims 1 to 5, characterized in that, It is applied to the preparation of health products or drugs with the function of promoting nerve regeneration, and health products or drugs for preventing and / or treating age-related physical aging, Alzheimer's disease, Parkinson's syndrome and other neurodegenerative diseases.

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