Nanometer vesicle and cytoskeleton for treating Parkinson's disease

Abstract

The invention discloses a nano vesicle and a cytoskeleton for treating Parkinson's disease, and belongs to the field of Parkinson's disease transplantation therapy, and the preparation method comprises the following steps: 1, mixing a drug solution and a micelle solution at 0-4 DEG C to form a mixed solution, putting the mixed solution into a rotary evaporator, and carrying out rotary evaporation to remove dimethyl sulfoxide so as to obtain a drug-loaded micelle; 2, adopting a cell nano perforation technology, and selecting bv2 cells to obtain exosomes; and step 3, uniformly mixing and stirring the drug-loaded micelle and the exosome at the stirring speed of 200-300rpm, performing post-ultrasonic treatment, adding the mixture into a liposome extruder after the post-ultrasonic treatment, and extruding the mixed solution for 10-20 times to obtain the nano-vesicle for treating the Parkinson's disease. The nano-vesicle disclosed by the invention treats the Parkinson's disease by utilizing the synergistic effect of various effective components, has an active oxygen responsive intelligent drug release property, is high in targeting property, and has remarkable advantages and wide application prospects in the aspect of treatment of the Parkinson's disease.

Description

Technical Field

[0001] The present invention relates to the field of transplantation treatment for Parkinson's disease, and particularly to a nanovesicle and cell scaffold for treating Parkinson's disease. Background Art

[0002] Parkinson's disease is a neurodegenerative disease that seriously affects the quality of life of patients. Its pathological characteristics are the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which in turn leads to a lack of dopamine in the striatum, causing motor symptoms such as tremors, rigidity, bradykinesia, and postural balance disorders, as well as non-motor symptoms such as hyposmia, sleep disorders, autonomic nervous system dysfunction, and cognitive impairment.

[0003] Currently, the commonly used clinical treatment methods are mainly drug treatment and surgical treatment, but both have obvious limitations. Although drug treatment can relieve symptoms in the initial stage, long-term use will result in reduced efficacy and serious adverse reactions, such as end-of-dose phenomena, on-off phenomena, dyskinesia, etc., and it is not effective in improving non-motor symptoms. Although surgical treatment can improve motor symptoms to a certain extent, it has high surgical risks, high costs, and cannot prevent the progression of the disease. After surgery, drugs still need to be maintained, and the long-term effect will also gradually decrease.

[0004] Cell transplantation treatment has received attention as a new strategy. CN102091056B discloses a cell microcapsule and composite cell scaffold for treating Parkinson's disease, which uses a microcapsule and composite scaffold containing retinal epithelial cells and neurotrophic factors to solve the problems of immune rejection and cell survival in the treatment of Parkinson's disease. However, the treatment components are single and the targeting is poor. Summary of the Invention

[0005] The present invention discloses a nanovesicle and cell scaffold for treating Parkinson's disease to solve the technical problems of single treatment components and poor targeting in the treatment of Parkinson's disease in the prior art.

[0006] The present invention discloses a method for preparing a nanovesicle for treating Parkinson's disease, comprising the following steps: Step 1: Preparation of drug-loaded micelles: Mix a drug solution and a micelle solution at 0-4°C to form a mixed solution, and at the same time use a magnetic stirrer to stir at a speed of 300-500 rpm for 20-30 minutes. Then put the mixed solution into a rotary evaporator to rotary evaporate and remove dimethyl sulfoxide to obtain drug-loaded micelles.

[0007] Step 2: Preparation of exosomes: Adopt the cell nanoelectroporation technology and select bv2 cells to obtain exosomes.

[0008] Step 3: Preparation of nanovesicles: Mix the drug-loaded micelles and exosomes evenly by stirring at a stirring speed of 200 - 300 rpm, then perform ultrasonic treatment. After ultrasonic treatment, add the mixture into a liposome extruder, select a polycarbonate membrane with a pore size of 200 - 400 nm, and extrude the mixture 10 - 20 times at 25 - 40 °C to obtain the nanovesicles for treating Parkinson's disease.

[0009] Preferably, the preparation method of the micelle solution is as follows: Mix 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide in a mass ratio of 0.1 - 0.3:0.2 - 0.4:0.1 - 0.3:5, and perform ultrasonic treatment at an ultrasonic power of 40 W for 20 - 30 minutes to obtain the micelle solution.

[0010] Among them, 4-hydroxymethyl-phenylboronic acid pinacol ester has reactive oxygen responsiveness. In the high reactive oxygen environment at the lesion site of Parkinson's disease, it can trigger the change of the micelle structure and realize the intelligent release of the drug. Poly(lactic-co-glycolic acid) is a biodegradable polymer material, and its degradation products are harmless to the human body and can be degraded into carbon dioxide and water in the body. N-trimethyl chitosan has good biocompatibility and water solubility, which helps to improve the stability and biocompatibility of the micelles; 4-hydroxymethyl-phenylboronic acid pinacol ester is combined with poly(lactic-co-glycolic acid) to ensure the stable encapsulation of the drug in the normal physiological environment while realizing the precise release of the drug at the lesion site. The addition of chitosan enhances the biocompatibility of the material, and the three materials work synergistically to construct a micelle shell with good biocompatibility and good drug release performance.

[0011] Preferably, the preparation method of the drug solution is as follows: Mix gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide in a mass ratio of 0.05 - 0.15:0.05 - 0.1:0.1 - 0.3:2, and stir evenly to obtain the drug solution.

[0012] Among them, gastrodin has the properties of scavenging free radicals and nerve sedation. Its antioxidant stress characteristics help to neutralize excessive reactive oxygen free radicals and protect neurons from oxidative damage. As an iron chelator, deferoxamine can specifically bind to excess iron ions in cells, reduce the production of reactive oxygen species, lower the level of oxidative stress, and help maintain the normal function of neurons. Levodopa is a precursor of dopamine, which can cross the blood-brain barrier and enter the brain. It is converted into dopamine through a series of enzymatic reactions in neurons, thereby directly supplementing the lacking dopamine in the brains of Parkinson's disease patients, effectively improving the motor symptoms of patients, and enhancing the patients' motor ability and quality of life. In the regulation of oxidative stress, gastrodin reduces the production and damage of reactive oxygen species by scavenging free radicals, while deferoxamine reduces the generation of reactive oxygen species mediated by iron ions from the source. The two work synergistically to more comprehensively reduce the intracellular oxidative stress level and protect the normal physiological function of neurons. In terms of therapeutic effects, levodopa directly improves the motor symptoms of Parkinson's disease. Gastrodin and deferoxamine help maintain the therapeutic effect of levodopa, extend its action time, and reduce the efficacy decline and motor complications that may occur due to long-term use of levodopa by protecting neurons and improving the intracellular environment. The three work synergistically to more comprehensively improve the motor and non-motor symptoms of patients.

[0013] Preferably, the specific operation of step two is as follows: S1. Inoculate bv2 cells in a cell culture dish and culture them in an incubator at 25 - 40 °C and 5% carbon dioxide until the cell density reaches 80% - 90%. Then digest the cells with 0.25% trypsin, wash and resuspend the cells with phosphate buffer, and adjust the cell concentration to 5×10 6 cells / mL to obtain a cell suspension.

[0014] S2. Mix the cell suspension with a PBS buffer solution containing plasmid RVG-Lamp2b-HA at a concentration of 1 g / L at a mass ratio of 7 - 10:1, put it into an electroporation container, and perform electroporation at a voltage of 200 V, using 5 pulses, each pulse for 10 ms, and an interval of 0.1 s.

[0015] S3. Continue to culture the electroporated cells under the conditions in S1 for 24 - 48 hours, collect the cell culture supernatant, transfer the supernatant to a centrifuge tube, first centrifuge at 1000×g for 10 minutes, then transfer the supernatant to a new centrifuge tube and centrifuge at 100000×g for 2 - 3 hours. Collect the precipitate, which is the exosome. Resuspend the exosome with PBS buffer solution and store it in a -80 °C refrigerator for later use.

[0016] Preferably, in step one, the mass ratio of the drug solution to the micelle solution is 1:1.

[0017] Preferably, in the first step, the temperature in the rotary evaporator is 50 °C and the pressure is 0.08 - 0.1 MPa.

[0018] Preferably, in the third step, the mass ratio of the drug-loaded micelles to exosomes is 1:1.

[0019] Preferably, in the third step, the ultrasonic power is 30 - 50 W and the ultrasonic time is 5 - 10 minutes.

[0020] Preferably, in S1, the cell culture dish contains a culture medium, and the culture medium is high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0021] The present invention also discloses the nanovesicles prepared by the above method for preparing nanovesicles for treating Parkinson's disease.

[0022] The present invention also discloses a cell scaffold for treating Parkinson's disease, and the preparation method of the cell scaffold includes the following steps: A1. Mix poly(lactic-co-glycolic acid) and an organic solvent at a mass ratio of 1:7 - 10, stir evenly to obtain a scaffold solution, pour the solution into a circular mold with a diameter of 15 mm and a thickness of 3 mm, and place the mold in a fume hood for 24 - 48 hours to form a porous poly(lactic-co-glycolic acid) scaffold.

[0023] A2. Wash the porous poly(lactic-co-glycolic acid) scaffold 2 - 3 times with PBS solution, disperse the above nanovesicles in PBS solution to prepare a nanovesicle suspension, soak the porous poly(lactic-co-glycolic acid) scaffold in the nanovesicle suspension, let it stand at 4 °C for 18 hours, and rinse the surface 3 times with PBS solution to obtain a cell scaffold loaded with nanovesicles.

[0024] Preferably, the concentration of the nanovesicle suspension is 5×10 8 cells / mL.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) In the present invention, a micelle solution is prepared by mixing 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide; a drug solution is prepared by mixing gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide; a drug-loaded micelle is prepared by mixing the drug solution and the micelle solution; the cell nano-poration technology is used to obtain exosomes by selecting bv2 cells; and a nanovesicle for treating Parkinson's disease is prepared by mixing the drug-loaded micelles and exosomes.

[0026] (2) In the present invention, 4-hydroxymethylphenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), and N-trimethyl chitosan are used as the micelle materials of the nanocapsule. Among them, 4-hydroxymethylphenylboronic acid pinacol ester has reactive oxygen responsiveness. In the high reactive oxygen environment at the lesion site of Parkinson's disease, it can trigger the change of the micelle structure to achieve the intelligent release of drugs. Poly(lactic-co-glycolic acid) is a biodegradable polymer material, and its degradation products are harmless to the human body and can be degraded into carbon dioxide and water in the body. N-trimethyl chitosan has good biocompatibility and water solubility, which helps to improve the stability and biocompatibility of the micelle. The combination of 4-hydroxymethylphenylboronic acid pinacol ester and poly(lactic-co-glycolic acid) ensures the stable encapsulation of drugs in the normal physiological environment while achieving the precise release of drugs at the lesion site. The addition of chitosan enhances the biocompatibility of the material. The three materials work synergistically to construct a micelle shell with good biocompatibility and drug release performance.

[0027] (3) In the present invention, gastrodin, deferoxamine, and levodopa are used as the drug materials of the nanocapsule. Among them, gastrodin has the properties of scavenging free radicals and nerve sedation. Its antioxidant stress characteristics help to neutralize excessive reactive oxygen free radicals and protect neurons from oxidative damage. Deferoxamine, as an iron chelator, can specifically bind to the excess iron ions in cells, reduce the production of reactive oxygen species, and lower the oxidative stress level, which helps to maintain the normal function of neurons. Levodopa is a precursor of dopamine, which can cross the blood-brain barrier and enter the brain. It is converted into dopamine through a series of enzymatic reactions in neurons, thus directly supplementing the lacking dopamine in the brains of Parkinson's disease patients, effectively improving the motor symptoms of patients, and enhancing their motor ability and quality of life. In the regulation of oxidative stress, gastrodin reduces the production and damage of reactive oxygen species by scavenging free radicals, while deferoxamine reduces the generation of reactive oxygen species mediated by iron ions from the source. The two work synergistically to more comprehensively reduce the intracellular oxidative stress level and protect the normal physiological function of neurons. In terms of the therapeutic effect, levodopa directly improves the motor symptoms of Parkinson's disease. Gastrodin and deferoxamine help to maintain the therapeutic effect of levodopa, extend its action time, and reduce the reduction of efficacy and motor complications caused by long-term use of levodopa by protecting neurons and improving the intracellular environment. The three work synergistically to more comprehensively improve the motor and non-motor symptoms of patients.

[0028] (4) The nanovesicles of the present invention effectively improve the motor and non-motor symptoms of Parkinson's disease patients through the synergistic action of multiple drug components, achieve the precise release of drugs at the lesion site, improve the drug utilization efficiency, and reduce the potential side effects on normal tissues, showing broad application prospects in the field of Parkinson's disease treatment. Description of the Drawings

[0029] Figure 1Flow chart of the preparation method of nanovesicles for treating Parkinson's disease in a specific embodiment of the present invention. Detailed implementation mode

[0030] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.

[0031] The preparation method of nanovesicles for treating Parkinson's disease in the present application will be described below through a specific embodiment. As Figure 1 shown, the preparation method of the nanovesicles for treating Parkinson's disease includes the following steps: S1. Prepare a micelle solution: Mix 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide in a mass ratio of 0.1-0.3:0.2-0.4:0.1-0.3:5, and ultrasonicate for 20-30 minutes at an ultrasonic power of 40W to obtain a micelle solution.

[0032] It can be understood that in the process of preparing the micelle solution in the embodiment of the present application, 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), and N-trimethyl chitosan are used as the raw materials of the micelle solution. Among them, 4-hydroxymethyl-phenylboronic acid pinacol ester has active oxygen responsiveness and can trigger the change of the micelle structure in the high active oxygen environment at the lesion site of Parkinson's disease to achieve the intelligent release of drugs. Poly(lactic-co-glycolic acid) is a biodegradable polymer material, and its degradation products are harmless to the human body and can be degraded into carbon dioxide and water in the body. N-trimethyl chitosan has good biocompatibility and water solubility, which helps to improve the stability and biocompatibility of the micelles; 4-hydroxymethyl-phenylboronic acid pinacol ester is combined with poly(lactic-co-glycolic acid) to ensure the stable encapsulation of drugs in the normal physiological environment while achieving the precise release of drugs at the lesion site. The addition of chitosan enhances the biocompatibility of the material, and the three materials work together to construct a micelle shell with good biocompatibility and drug release performance.

[0033] S2. Prepare a drug solution: Mix gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide in a mass ratio of 0.05-0.15:0.05-0.1:0.1-0.3:2, and stir evenly to obtain a drug solution.

[0034] It can be understood that in the process of preparing the drug solution in the embodiments of the present application, gastrodin, deferoxamine, and levodopa are used as raw materials. Among them, gastrodin has the properties of scavenging free radicals and nerve sedation. Its antioxidant stress characteristics help to neutralize excessive reactive oxygen free radicals and protect neurons from oxidative damage. Deferoxamine, as an iron chelator, can specifically bind to excess iron ions in cells, reduce the production of reactive oxygen species, lower the level of oxidative stress, and help maintain the normal function of neurons. Levodopa is a precursor of dopamine, which can cross the blood-brain barrier and enter the brain. It is converted into dopamine through a series of enzymatic reactions in neurons, thereby directly supplementing the lacking dopamine in the brains of Parkinson's disease patients, effectively improving the motor symptoms of patients, and enhancing the patients' motor ability and quality of life. In the regulation of oxidative stress, gastrodin reduces the production and damage of reactive oxygen species by scavenging free radicals, while deferoxamine reduces the generation of reactive oxygen species mediated by iron ions from the source. The two work synergistically to more comprehensively reduce the intracellular oxidative stress level and protect the normal physiological function of neurons. In terms of treatment effect, levodopa directly improves the motor symptoms of Parkinson's disease. Gastrodin and deferoxamine help maintain the therapeutic effect of levodopa, prolong its action time, and reduce the efficacy decline and motor complications that may occur due to long-term use of levodopa by protecting neurons and improving the intracellular environment. The three work synergistically to more comprehensively improve the motor and non-motor symptoms of patients.

[0035] S3. Prepare drug-loaded micelles: Mix the drug solution and the micelle solution at a mass ratio of 1:1 at 0 - 4°C to form a mixed solution, and at the same time use a magnetic stirrer to stir at a speed of 300 - 500 rpm for 20 - 30 minutes. Then put the mixed solution into a rotary evaporator, set the temperature in the rotary evaporator to 50°C and the pressure to 0.08 - 0.1 MPa, and rotary evaporate to remove dimethyl sulfoxide to obtain drug-loaded micelles.

[0036] S4. Inoculate bv2 cells in a cell culture dish. The culture medium in the culture dish is high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Culture the cells in an incubator at 25 - 40°C and 5% carbon dioxide until the cell density reaches 80% - 90%. Then digest the cells with 0.25% trypsin, wash and resuspend the cells with phosphate buffer, and adjust the cell concentration to 5×10 6cells / mL to obtain a cell suspension. The cell suspension was mixed with a PBS buffer solution containing plasmid RVG-Lamp2b-HA at a concentration of 1 g / L at a mass ratio of 7-10:1, placed in an electroporation container, and electroporated under the conditions of a voltage of 200 V, using 5 pulses, each pulse being 10 ms, with an interval of 0.1 s. The electroporated cells were further cultured under the conditions in S1 for 24-48 hours. The cell culture supernatant was collected, transferred to a centrifuge tube, first centrifuged at 1000×g for 10 minutes, then the supernatant was transferred to a new centrifuge tube and centrifuged at 100000×g for 2-3 hours. The precipitate was collected, which was exosomes. The exosomes were resuspended with PBS buffer solution and stored in a -80°C refrigerator for later use.

[0037] S5. Preparation of nanovesicles: The drug-loaded micelles and exosomes were mixed at a mass ratio of 1:1 and stirred evenly at a stirring speed of 200-300 rpm, followed by ultrasonic treatment with an ultrasonic power of 30-50 W for 5-10 minutes. After ultrasonic treatment, the mixture was added to a liposome extruder, and a polycarbonate membrane with a pore size of 200-400 nm was selected. At 25-40°C, the mixture was extruded 10-20 times to obtain nanovesicles for the treatment of Parkinson's disease.

[0038] In summary, through the synergistic effect of various drug components, the present application effectively improves the motor and non-motor symptoms of Parkinson's disease patients, realizes the precise release of drugs at the lesion site, improves the drug utilization efficiency, and reduces the potential side effects on normal tissues.

[0039] Next, with reference to specific embodiments, the present application will be described. It should be noted that these embodiments are merely descriptive and do not limit the present application in any way.

[0040] Example 1 This example provides a method for preparing nanovesicles for the treatment of Parkinson's disease S1. Preparation of micelle solution: 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide were mixed in a mass ratio of 0.2:0.3:0.2:5 and ultrasonicated at an ultrasonic power of 40 W for 30 minutes to obtain a micelle solution.

[0041] S2. Preparation of drug solution: Gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide were mixed at a mass ratio of 0.1:0.05:0.2:2 and stirred evenly to obtain a drug solution.

[0042] S3. Preparation of drug-loaded micelles: At 4°C, mix the drug solution and the micelle solution at a mass ratio of 1:1 to form a mixed solution. Meanwhile, use a magnetic stirrer to stir at a speed of 400 rpm for 30 minutes. Then, place the mixed solution in a rotary evaporator, set the temperature in the rotary evaporator to 50°C and the pressure to 0.08 MPa, and rotary evaporate to remove dimethyl sulfoxide to obtain drug-loaded micelles.

[0043] S4. Inoculate bv2 cells in a cell culture dish. The culture medium in the culture dish is high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Culture the cells in an incubator at 37°C and 5% carbon dioxide until the cell density reaches 80%. Then, digest the cells with 0.25% trypsin, wash and resuspend the cells with phosphate buffer, and adjust the cell concentration to 5×10 6 cells / mL to obtain a cell suspension. Mix the cell suspension and the PBS buffer solution containing plasmid RVG-Lamp2b-HA with a concentration of 1 g / L at a mass ratio of 10:1, place it in an electroporation container, and perform electroporation at a voltage of 200 V, using 5 pulses, each pulse being 10 ms and the interval being 0.1 s. Continue to culture the electroporated cells under the conditions in S1 for 48 hours, collect the cell culture supernatant, transfer the supernatant to a centrifuge tube, first centrifuge at 1000×g for 10 minutes, then transfer the supernatant to a new centrifuge tube and centrifuge at 100000×g for 3 hours. Collect the precipitate, which is exosomes. Resuspend the exosomes with PBS buffer and store them in a -80°C refrigerator for later use.

[0044] S5. Preparation of nanovesicles: Mix the drug-loaded micelles and exosomes at a mass ratio of 1:1 and stir evenly at a stirring speed of 300 rpm. Then, perform ultrasonic treatment with an ultrasonic power of 50 W for 10 minutes. After ultrasonic treatment, add the mixture to a liposome extruder, select a polycarbonate membrane with a pore size of 400 nm, and extrude the mixture 20 times at 37°C to obtain nanovesicles for the treatment of Parkinson's disease.

[0045] Example 2 This example provides a method for preparing nanovesicles for the treatment of Parkinson's disease S1. Preparation of micelle solution: Mix 4-hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide in a mass ratio of 0.1:0.2:0.1:5, and perform ultrasonic treatment at an ultrasonic power of 40 W for 20 minutes to obtain a micelle solution.

[0046] S2. Preparation of drug solution: Mix gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide in a mass ratio of 0.05:0.05:0.1:2, and stir evenly to obtain a drug solution.

[0047] S3. Preparation of drug-loaded micelles: At 0 °C, the drug solution and the micelle solution were mixed at a mass ratio of 1:1 to form a mixed solution, and at the same time, a magnetic stirrer was used to stir at a speed of 300 rpm for 20 minutes. Then, the mixed solution was placed in a rotary evaporator, and the temperature in the rotary evaporator was set at 50 °C and the pressure was 0.08 MPa. Dimethyl sulfoxide was removed by rotary evaporation to obtain drug-loaded micelles.

[0048] S4. Inoculate bv2 cells into a cell culture dish. The culture medium in the culture dish was high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were cultured in an incubator at 25 °C and 5% carbon dioxide until the cell density reached 80%. Then, the cells were digested with 0.25% trypsin, washed with phosphate buffer and resuspended. The cell concentration was adjusted to 5×10 6 cells / mL to obtain a cell suspension. The cell suspension was mixed with a PBS buffer solution containing plasmid RVG-Lamp2b-HA at a concentration of 1 g / L at a mass ratio of 7:1, placed in an electroporation container, and electroporation was performed under the conditions of a voltage of 200 V, using 5 pulses, each pulse being 10 ms and the interval being 0.1 s. The electroporated cells were continuously cultured under the conditions in S1 for 24 hours. The cell culture supernatant was collected, transferred to a centrifuge tube, centrifuged at 1000×g for 10 minutes first, and then the supernatant was transferred to a new centrifuge tube and centrifuged at 100000×g for 2 hours. The precipitate was collected, which was exosomes. The exosomes were resuspended with PBS buffer and stored in a -80 °C refrigerator for later use.

[0049] S5. Preparation of nanovesicles: The drug-loaded micelles and exosomes were mixed and stirred evenly at a mass ratio of 1:1, and the stirring speed was 200 rpm. Then, ultrasonic treatment was carried out with an ultrasonic power of 30 W for 5 minutes. After ultrasonic treatment, the mixture was added to a liposome extruder, and a polycarbonate membrane with a pore size of 200 nm was selected. At 25 °C, the mixture was extruded 10 times to obtain nanovesicles for the treatment of Parkinson's disease.

[0050] Example 3 This example provides a method for preparing nanovesicles for the treatment of Parkinson's disease S1. Preparation of micelle solution: 4-Hydroxymethyl-phenylboronic acid pinacol ester, poly(lactic-co-glycolic acid), N-trimethyl chitosan, and dimethyl sulfoxide were mixed in a mass ratio of 0.3:0.4:0.3:5, and ultrasonic treatment was carried out at an ultrasonic power of 40 W for 30 minutes to obtain a micelle solution.

[0051] S2. Preparation of drug solution: Gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide were mixed at a mass ratio of 0.15:0.1:0.3:2 and stirred evenly to obtain a drug solution.

[0052] S3. Prepare drug-loaded micelles: At 4°C, mix the drug solution and the micelle solution at a mass ratio of 1:1 to form a mixed solution. At the same time, use a magnetic stirrer to stir at a speed of 500 rpm for 30 minutes. Then, put the mixed solution into a rotary evaporator, set the temperature in the rotary evaporator to 50°C and the pressure to 0.1 MPa, and rotary evaporate to remove dimethyl sulfoxide to obtain the drug-loaded micelles.

[0053] S4. Inoculate bv2 cells into a cell culture dish. The culture medium in the culture dish is high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Culture the cells in an incubator at 40°C and 5% carbon dioxide until the cell density reaches 90%. Then, digest the cells with 0.25% trypsin, wash and resuspend the cells with phosphate buffer, and adjust the cell concentration to 5×10 6 cells / mL to obtain a cell suspension. Mix the cell suspension with a PBS buffer solution containing plasmid RVG-Lamp2b-HA at a concentration of 1 g / L at a mass ratio of 10:1, put it into an electroporation container, and perform electroporation under the conditions of a voltage of 200 V, using 5 pulses, each pulse being 10 ms, and an interval of 0.1 s. Continue to culture the electroporated cells under the conditions in S1 for 48 hours, collect the cell culture supernatant, transfer the supernatant to a centrifuge tube, first centrifuge at 1000×g for 10 minutes, then transfer the supernatant to a new centrifuge tube and centrifuge at 100000×g for 3 hours. Collect the precipitate, which is the exosome. Resuspend the exosome with PBS buffer solution and store it in a -80°C refrigerator for later use.

[0054] S5. Prepare nanovesicles: Mix the drug-loaded micelles and exosomes at a mass ratio of 1:1 and stir evenly at a stirring speed of 300 rpm. Then, perform ultrasonic treatment with an ultrasonic power of 50 W for 10 minutes. After ultrasonic treatment, add it to a liposome extruder, select a polycarbonate membrane with a pore size of 400 nm, and extrude the mixed solution 20 times at 40°C to obtain the nanovesicles for treating Parkinson's disease.

[0055] Example 4 This example provides a preparation method of a cell scaffold for treating Parkinson's disease A1. Mix 200 ul of polylactic acid-glycolic acid and 2 ml of an organic solvent, stir evenly to obtain a scaffold solution, pour the solution into a circular mold with a diameter of 15 mm and a thickness of 3 mm, and place the mold in a fume hood for 48 hours to form a porous polylactic acid-glycolic acid scaffold; A2. Wash the porous polylactic acid-glycolic acid scaffold 3 times with PBS solution, disperse the nanovesicles prepared in Example 1 in PBS solution, and prepare a concentration of 5×10 8A nanovesicle suspension of [[ID=]] cells / mL was prepared. A porous poly(lactic-co-glycolic acid) scaffold was immersed in the nanovesicle suspension and left standing at 4 °C for 18 hours. The surface was rinsed 3 times with PBS solution to obtain a cell scaffold loaded with nanovesicles.

[0056] Performance detection Particle size detection of nanovesicles The particle sizes of the nanovesicles prepared in Examples 1-3 for the treatment of Parkinson's disease were detected using dynamic light scattering technology.

[0057] In vivo targeting detection Parkinson's disease mice induced by MPTP were selected. Fluorescently labeled nanovesicles prepared in Example 1 for the treatment of Parkinson's disease were intravenously injected at a dose of 100 μL. At 1, 3, 6, and 24 hours after the injection of the nanovesicles, whole-body fluorescence imaging of the mice was performed using a small animal in vivo imaging system to observe the distribution of the nanovesicles in vivo.

[0058] Detection results Table 1 shows the particle size detection results of the nanovesicles

[0059] As can be seen from Table 1, the average particle size of the nanovesicles is between 200-400 nm, and the particle size distribution is relatively uniform, which is helpful for the circulation of the nanovesicles in vivo and their effective delivery to the lesion site, avoiding rapid clearance by the reticuloendothelial system.

[0060] Table 2 is a record table of whole-body fluorescence imaging of mice in the in vivo targeting detection

[0061] As can be seen from Table 2, 24 hours after the injection, the fluorescence intensity in the brain tissue of the mice is significantly higher than that in other organs, indicating that the nanovesicles can specifically target the brain lesion area in vivo and have good in vivo targeting.

[0062] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made within the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. A method for preparing nanovesicles for treating Parkinson's disease, characterized in that: The following steps are involved: Step 1, preparation of drug-loaded micelles: mixing the drug solution and the micelle solution at 0-4°C to form a mixed solution, and stirring the solution at 300-500 rpm using a magnetic stirrer for 20-30 minutes, then placing the mixed solution in a rotary evaporator to remove dimethyl sulfoxide by rotary evaporation to obtain drug-loaded micelles; Step 2: Preparation of exosomes: Using cell nanoporation technology, BV2 cells were selected to obtain exosomes; Step 3, preparation of nanovesicles: the drug-loaded micelles and exosomes are mixed and stirred evenly at a stirring speed of 200-300 rpm, and then ultrasonicated. After ultrasonication, they are added to a liposome extruder, a polycarbonate membrane with a pore size of 200-400 nm is selected, and the mixed solution is extruded 10-20 times at 25-40° C. to obtain nanovesicles for the treatment of Parkinson's disease; The preparation method of the micelle solution is as follows: 4-hydroxymethyl-phenylboronic acid pinacol ester, polylactic acid-glycolic acid, N-trimethyl chitosan, and dimethyl sulfoxide are mixed in a mass ratio of 0.1-0.3:0.2-0.4:0.1-0.3:5, and ultrasonically treated at an ultrasonic power of 40 W for 20-30 minutes to obtain a micelle solution; The preparation method of the drug solution is as follows: gastrodin, deferoxamine, levodopa, and dimethyl sulfoxide are mixed in a mass ratio of 0.05-0.15:0.05-0.1:0.1-0.3:2, and stirred evenly to obtain the drug solution.

2. The method for preparing nanovesicles for treating Parkinson's disease according to claim 1, characterized in that: The specific operation of step 2 is as follows: S1. Inoculate BV2 cells in a cell culture dish and culture them in an incubator at 25-40°C and 5% carbon dioxide until the cell density reaches 80%-90%. Then digest the cells with 0.25% trypsin, wash and resuspend the cells with phosphate buffer, and adjust the cell concentration to 5×10 6 / mL, and obtain cell suspension; S2, the cell suspension was mixed with PBS buffer containing plasmid RVG-Lamp2b-HA at a concentration of 1 g / L at a mass ratio of 7-10:1, placed in an electroporation container, and electroporated at a voltage of 200 V, using 5 pulses, each pulse of 10 ms, and an interval of 0.1 s; S3. Continue to culture the electroporated cells under the conditions in S1 for 24-48 hours, collect the cell culture supernatant, transfer the supernatant to a centrifuge tube, centrifuge at 1000×g for 10 minutes, transfer the supernatant to a new centrifuge tube, centrifuge at 100000×g for 2-3 hours, collect the precipitate, which is the exosomes, resuspend the exosomes with PBS buffer, and store in a -80°C refrigerator for later use.

3. The method for preparing nanovesicles for treating Parkinson's disease according to claim 1, characterized in that: In the step 1, the mass ratio of the drug solution to the micelle solution is 1:

1.

4. The method for preparing nanovesicles for treating Parkinson's disease according to claim 1, characterized in that: In the step 1, the temperature in the rotary evaporator is 50° C. and the pressure is 0.08-0.1 MPa.

5. The method for preparing nanovesicles for treating Parkinson's disease according to claim 1, characterized in that: In the step three, the mass ratio of the drug-loaded micelles to the exosomes is 1:

1.

6. The method for preparing nanovesicles for treating Parkinson's disease according to claim 1, characterized in that: In the step 3, the ultrasonic power is 30-50W, and the ultrasonic time is 5-10 minutes.

7. The method for preparing nanovesicles for treating Parkinson's disease according to claim 2, characterized in that: The cell culture dish contains a culture medium, which is a high-glucose Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin.

8. A nanovesicle prepared according to the method for preparing nanovesicles for treating Parkinson's disease according to any one of claims 1 to 7.

9. A cell scaffold for treating Parkinson's disease, characterized in that: The preparation method of the cell scaffold comprises the following steps: A1, polylactic acid-glycolic acid and an organic solvent are mixed in a mass ratio of 1:7-10, stirred evenly to obtain a scaffold solution, the solution is poured into a circular mold with a diameter of 15 mm and a thickness of 3 mm, and the mold is placed in a fume hood for 24-48 hours to form a porous polylactic acid-glycolic acid scaffold; A2. Wash the porous polylactic acid-glycolic acid scaffold 2-3 times with PBS solution, disperse the nanovesicles according to claim 8 in PBS solution to prepare a nanovesicle suspension, immerse the porous polylactic acid-glycolic acid scaffold in the nanovesicle suspension, let it stand at 4°C for 18 hours, rinse the surface with PBS solution 3 times, and obtain a cell scaffold loaded with nanovesicles.

10. The cell scaffold for treating Parkinson's disease according to claim 9, characterized in that: The concentration of the nanovesicle suspension is 5×10 8 Pieces / mL.

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