Exosome-containing eye drops as well as preparation method and application thereof

Abstract

The invention relates to eye drops containing exosomes and a preparation method and application thereof, and belongs to the technical field of eye drop preparation, the eye drops comprise exosome freeze-dried powder, the exosome freeze-dried powder is prepared from the exosomes obtained after mesenchymal stem cells are treated with TNF-alpha and IFN-alpha, and a herba houttuyniae extract, the eye drops provided by the invention can inhibit the phenotype of macrophage M1 and promote the M1 to be converted into the phenotype of M2; human corneal cell proliferation, migration and damage repair are promoted; mouse cornea injury repair is accelerated; the eye drops can be used for treating xerophthalmia, conjunctivitis and keratitis, and can also be used for preventing eye diseases and relieving asthenopia; the eye drops disclosed by the invention contain the exosome obtained after the mesenchymal stem cells are treated by the TNF-alpha and the IFN-alpha, so that the anti-inflammatory ability is stronger; the mesenchymal stem cell exosome and the herba houttuyniae extract are stored in a freeze-dried form, so that the activity is stable, and the storage time is long; in addition, the cordate houttuynia is cooperated with the exosome, so that the effect playing time can be prolonged, and the use frequency is reduced.

Description

Technical Field

[0001] The present invention relates to an eye drop, and particularly to an exosome-containing eye drop applied to the technical field of eye drop preparation, and its preparation method and application. Background Art

[0002] As a novel treatment method, in the past decade, significant progress has been made in the understanding of exosome biology and its application in the field of ophthalmic treatment. Exosomes can be used to treat various eye diseases, including traumatic diseases, autoimmune diseases, neovascular diseases, glaucoma and other ophthalmic diseases. Exosomes have the advantages of low immunogenicity, reducing the clearance rate of the body; high stability, multiple drug release; the ability to penetrate biological barriers, targeting tissues to play a better role; safety and easy transportation.

[0003] The invention patent with the publication number of CN118453656B discloses an eye drop composition containing stem cell exosomes and its preparation method. The invention provides an eye drop composition containing stem cell exosomes, including exosomes derived from mesenchymal stem cells and a pharmaceutically acceptable carrier.

[0004] The invention patent with the publication number of CN115337325A discloses a stem cell exosome ophthalmic derivative and a preparation method of stem cell exosomes, which can integrate health care functions and the functions of preventing and treating eye diseases.

[0005] Some eye drops in the prior art, although containing exosomes, still have certain limitations in the treatment effect and fail to fully exert the advantages of exosomes in the treatment of ophthalmic diseases. Therefore, we propose an exosome-containing eye drop and its preparation method and application. Summary of the Invention

[0006] Aiming at the above-mentioned prior art, the technical problem to be solved by the present invention is: how to further improve the treatment effect of eye drops on ophthalmic diseases.

[0007] To solve the above problem, the present invention provides an exosome-containing eye drop, which contains exosome freeze-dried powder. The exosome freeze-dried powder is prepared from exosomes obtained by treating mesenchymal stem cells with TNF-α and IFN-α and houttuynia cordata extract.

[0008] As a further improvement of the present application, the preparation method of the exosome freeze-dried powder includes the following steps:

[0009] S11. Culture mesenchymal stem cells at passages P3 - P5 in MEMα medium containing 10% FBS, TNF-α, and IFN-α. After culturing until the cell confluence reaches 80 - 90%, wash the cells three times with PBS, and then change to serum-free MEMα medium without TNF-α and IFN-α and culture for 40 - 50 h.

[0010] S12. Collect the supernatant of the culture medium and isolate exosomes from the supernatant to obtain an exosome resuspension.

[0011] S13. Prepare 1 L of exosome lyophilized powder excipient: Weigh 1 - 10 g of mannitol, stir and dissolve it, then add 2 - 8 g of trehalose, dissolve it with ultrapure water to a total volume of 1 L, and sterilize it at high temperature to obtain an excipient solution.

[0012] S14. Add Houttuynia cordata extract to the exosome resuspension to make the protein content of the exosomes 1 mg / L and the content of methyl nonyl ketone 2 - 5 mg / L.

[0013] S15. Mix the exosome resuspension after adding Houttuynia cordata extract with the excipient solution, and then perform low-temperature freeze-drying on it by vacuum freeze-drying technology to obtain exosome lyophilized powder.

[0014] As a further improvement of this application, the mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, or umbilical cord mesenchymal stem cells.

[0015] As a further improvement of this application, the use concentration of TNF-α in the culture medium is 10 - 100 ng / ml, and the use concentration of IFN-α in the culture medium is 10 - 100 ng / ml.

[0016] As a further improvement of this application, the eye drops contain 1 mg / L of exosomes, and the Houttuynia cordata extract contains 2 - 5 mg / L of methyl nonyl ketone.

[0017] As a further improvement of this application, the concentration of exosomes is measured by the protein concentration determined by the BCA method.

[0018] As a further improvement of this application, the components of the solvent solution of the exosome lyophilized powder include carbomer, normal saline, pH regulator, and preservative.

[0019] As a further improvement of this application, when using the eye drops, add the solvent solution to the exosome lyophilized powder and mix well.

[0020] A preparation method of eye drops containing exosomes, comprising the following steps:

[0021] S21. Culture passage 3 - 5 mesenchymal stem cells in MEMα medium containing 10% FBS with TNF-α and IFN-α. After culturing until the cell confluence reaches 80 - 90%, wash the cells three times with PBS, and then change to serum-free MEMα medium without TNF-α and IFN-α and culture for 40 - 50 h;

[0022] S22. Collect the supernatant of the culture medium. Centrifuge the supernatant at a relative centrifugal force of 300 g for 15 min to remove cells and cell debris in the supernatant. Then centrifuge the supernatant at a relative centrifugal force of 1500 g for 30 min, and then transfer the supernatant to a 100 kDa ultrafiltration membrane for filtration to obtain a filtrate;

[0023] S23. Centrifuge the filtrate at a temperature of 4°C and a relative centrifugal force of 150000 g for 2 h. Discard the supernatant, add an appropriate amount of PBS or sterile injection water, and pipette the precipitate to obtain an exosome resuspension;

[0024] S24. Use a BCA protein assay kit to detect the exosome concentration. The detection result shows that the protein content in the exosomes is 11.05 g / L;

[0025] S25. Prepare 1 L of exosome lyophilized powder excipient: Weigh 1 - 10 g of mannitol, stir and dissolve it, then add 2 - 8 g of trehalose, dissolve it with ultrapure water to a total volume of 1 L, and sterilize at high temperature to obtain an excipient solution;

[0026] S26. Add Houttuynia cordata extract to the exosome resuspension to make the protein content in the exosomes 1 mg / L and the methyl nonyl ketone content 2 - 5 mg / L, and then add the excipient solution to make the total volume of the liquid 1 L.

[0027] The application of an eye drop containing exosomes. The eye drop can be used for the treatment of dry eye, conjunctivitis, keratitis, or for the prevention of eye diseases and the relief of visual fatigue.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The eye drop prepared by the present invention contains exosomes obtained after treating mesenchymal stem cells with TNF-α and IFN-α, and has stronger anti-inflammatory ability;

[0030] 2. The eye drop prepared by the present invention preserves the mesenchymal stem cell exosomes and Houttuynia cordata extract in a lyophilized form, with stable activity and a long preservation time;

[0031] 3. In the eye drop prepared by the present invention, Houttuynia cordata synergizes with exosomes to prolong the effective time and reduce the frequency of use. Brief Description of the Drawings

[0032] Figure 1 It is the process flow chart of the preparation method of exosome freeze-dried powder in this application;

[0033] Figure 2 It is the process flow chart of the preparation method of the eye drops containing exosomes in this application;

[0034] Figure 3 It is the detection chart of IDO-1 gene expression after TNF-α and / or IFN-α stimulate mesenchymal stem cells for 48h in this application;

[0035] Figure 4 It is the detection chart of RNA expression of M1 phenotype (IL-6, IL-1β) of eye drops inhibiting RAW264.7 in this application;

[0036] Figure 5 It is the detection chart of RNA expression of M2 phenotype (IL-10, ARG1) of eye drops promoting RAW264.7 in this application;

[0037] Figure 6 It is the graph of eye drops promoting the proliferation of human corneal epithelial cells HCE-T in this application;

[0038] Figure 7 It is the graph of eye drops promoting the scratch repair of human corneal epithelial cells HCE-T in this application;

[0039] Figure 8 It is the graph of eye drops promoting the corneal repair of injured mice in this application. Detailed implementation manners

[0040] The following will make a detailed description of two implementation manners of this application with reference to the accompanying drawings.

[0041] The first implementation manner:

[0042] An eye drop containing exosomes, comprising exosome freeze-dried powder, and the exosome freeze-dried powder is prepared from exosomes obtained by treating mesenchymal stem cells with TNF-α and IFN-α and houttuynia cordata extract.

[0043] Please refer to Figure 1 , the preparation method of the exosome freeze-dried powder includes the following steps:

[0044] S11. Cultivate passage 3-5 mesenchymal stem cells with MEMα culture medium containing 10% FBS with TNF-α and IFN-α. After culturing until the cell confluence reaches 80-90%, wash with PBS for three times, and then change to serum-free MEMα culture medium without TNF-α and IFN-α and culture for 40-50h;

[0045] S12. Collect the supernatant of the culture medium and isolate exosomes from the supernatant to obtain an exosome resuspension;

[0046] S13. Prepare 1 L of exosome lyophilized powder excipient: Weigh 1 - 10 g of mannitol, stir and dissolve it, then add 2 - 8 g of trehalose, dissolve it with ultrapure water to a total volume of 1 L, and sterilize it at high temperature to obtain an excipient solution;

[0047] S14. Add houttuynia cordata extract to the exosome resuspension to make the protein content of the exosomes 1 mg / L and the content of methyl nonyl ketone 2 - 5 mg / L;

[0048] S15. Mix the exosome resuspension after adding houttuynia cordata extract with the excipient solution, and then perform low-temperature freeze-drying on it by vacuum freeze-drying technology to obtain exosome lyophilized powder.

[0049] The mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells.

[0050] The usage concentration of TNF-α in the culture medium is 10 - 100 ng / ml, and the usage concentration of IFN-α in the culture medium is 10 - 100 ng / ml.

[0051] The eye drops contain 1 mg / L of exosomes, and the houttuynia cordata extract contains 2 - 5 mg / L of methyl nonyl ketone.

[0052] The concentration of exosomes is measured by the protein concentration determined by the BCA method.

[0053] The components of the solvent solution of the exosome lyophilized powder include carbomer, normal saline, pH regulator, and preservative.

[0054] When using the eye drops, add the solvent solution to the exosome lyophilized powder and mix well.

[0055] Please refer to Figure 2 , A preparation method of an eye drop containing exosomes, comprising the following steps:

[0056] S21. Culture P3 - P5 generation mesenchymal stem cells with MEMα culture medium containing 10% FBS and TNF-α and IFN-α. After culturing until the cell confluence reaches 80 - 90%, wash it three times with PBS, and then change to serum-free MEMα culture medium without TNF-α and IFN-α and culture for 40 - 50 h;

[0057] S22. Collect the supernatant of the culture medium, centrifuge the supernatant at a relative centrifugal force of 300 g for 15 min to remove cells and cell debris in the supernatant, then centrifuge the supernatant at a relative centrifugal force of 1500 g for 30 min, and then transfer the supernatant to a ultrafiltration membrane with a molecular weight of 100 kDa for filtration to obtain a filtrate;

[0058] S23. Centrifuge the filtrate at a temperature of 4 °C and a relative centrifugal force of 150000 g for 2 h, discard the supernatant, add an appropriate amount of PBS or sterile injection water, and pipette the precipitate to obtain an exosome resuspension;

[0059] S24. Use a BCA protein assay kit to detect the exosome concentration, and the detection result shows that the protein content in the exosome is 11.05 g / L;

[0060] S25. Prepare 1 L of exosome lyophilized powder excipient: Weigh 1 - 10 g of mannitol, stir and dissolve it, then add 2 - 8 g of trehalose, dissolve it with ultrapure water to a total volume of 1 L, and sterilize it at high temperature to obtain an excipient solution;

[0061] S26. Add Houttuynia cordata extract to the exosome resuspension to make the protein content in the exosome 1 mg / L and the methyl nonyl ketone content 2 - 5 mg / L, and then add the excipient solution to make the total volume of the liquid 1 L.

[0062] The application of an eye drop containing exosomes, and the eye drop can be used for the treatment of dry eye, conjunctivitis, keratitis, or for the prevention of eye diseases and the relief of visual fatigue.

[0063] The eye drop prepared by the present invention contains exosomes obtained after treating mesenchymal stem cells with TNF-α and IFN-α, and has stronger anti-inflammatory ability; moreover, the mesenchymal stem cell exosomes and Houttuynia cordata extract are stored in a lyophilized form, with stable activity and a long storage time; in addition, Houttuynia cordata synergizes with exosomes to prolong the effective time and reduce the frequency of use.

[0064] The second embodiment:

[0065] Please refer to Figure 3 , after stimulating mesenchymal stem cells with TNF-α or IFN-a for 48 h, detect the IDO-1 gene expression:

[0066] 1. Inoculate canine mesenchymal stem cells in a 6-well plate at a density of 1x10^4 / cm 2 , after culturing for 24 h, divide them into 4 groups, with three replicates in each group. The unstimulated group is the control group; the culture medium group containing 100 ng / ml TNF-α; the culture medium group containing 100 ng / ml IFN-α; the culture medium group containing both 100 ng / ml TNF-α and 100 ng / ml IFN-α, and the stimulation time is 48 h;

[0067] 2. According to the product instruction manual of the RNA extraction kit, extract the RNA of 4 groups of canine mesenchymal stem cells, and use a nucleic acid content detector (OneDrop TM ) to detect the RNA content;

[0068] 3. Reverse transcribe the extracted RNA to obtain cDNA. Using the HPRT1 gene as an internal reference (F-5’-ACGCTGAGGATTTGGAAAAAGT-3’; R-5’-ACACAGAGGGCTACGATGTG-3’), and canine IDO1 specific primers (F-5’-TGCACCGAGCCCATAAAGAG-3’; R-5’-GCCCAGCAGAATGTCAAAGC-3’) amplify the IDO1 gene under the following reaction conditions: 95°C - 2 minutes, 95°C - 15 seconds (40 cycles), 55°C - 30 seconds (40 cycles), 72°C - 30s (40 cycles), with 3 replicates for each sample;

[0069] 4. Count the relative expression level of the IDO1 gene by the method;

[0070] 5. Detection found that 100 ng / ml TNF-α or 100 ng / ml IFN-α promoted the expression of the IDO1 gene, and when the two stimulated the cells simultaneously, the expression level of the IDO1 gene in canine mesenchymal stem cells was higher than that in the group stimulated separately.

[0071] Please refer to Figure 4 , Detection of the RNA expression of the M1 phenotype (IL-6, IL-1β) of the eye drops inhibiting mouse mononuclear macrophages RAW264.7:

[0072] 1. Inoculate mouse mononuclear macrophages RAW264.7 in a 6-well plate at a density of 2x10^5 / cm 2 . After culturing for 24 h, add 1 μg / ml LPS to the culture medium and divide it into four groups. Add 10 μl of the solvent solution as the control group; the eye drop group prepared from exosomes after stimulation with 10 μl of 100 ng / ml TNF-α; the eye drop group containing exosomes after stimulation with 10 μl of 100 ng / ml IFN-α; the eye drop group stimulated with 10 μl of 100 ng / ml TNF-α and 100 ng / ml IFN-α simultaneously, and continue to culture for 48 h.

[0073] 2. According to the product instruction manual of the RNA extraction kit, extract the RNA of 4 groups of canine mesenchymal stem cells, and use a nucleic acid content detector (OneDrop TM ) to detect the RNA content.

[0074] 3. Reverse transcribe the extracted RNA to obtain cDNA. Using the HPRT1 gene as an internal reference (F - 5’-ACGCTGAGGATTTGGAAAAAGT-3’; R - 5’-ACACAGAGGGCTACGATGTG-3’), canine IL-6 specific primers (F - 5’-gtgaccactcctgacccaac-3’; R - 5’-atccgaacagccctcagact-3’), and IL-1β specific primers, amplify the IL-6 and IL-1β genes under the following reaction conditions: 95°C for 2 minutes, 95°C for 15 seconds (40 cycles), 55°C for 30 seconds (40 cycles), 72°C for 30 seconds (40 cycles), with 3 replicates for each sample;

[0075] 4. Use the method to count the relative expression levels of the IL-6 and IL-1β genes;

[0076] 5. Detection found that 100 ng / ml TNF-α or 100 ng / ml IFN-α inhibited the expression of the IL-6 and IL-1β genes in the M1 phenotype of macrophages, and when both stimulated the cells simultaneously, the expression levels of the IL-6 and IL-1β genes in canine mesenchymal stem cells were lower than those in the respective stimulation groups.

[0077] Please refer to Figure 5 for the detection of the RNA expression of the M2 phenotype (IL-10, ARG1) of mouse mononuclear macrophages RAW264.7 promoted by eye drops:

[0078] 1. Seed mouse mononuclear macrophages RAW264.7 in a 6-well plate at a density of 2x10^5 / cm 2 . After culturing for 24 hours, add 1 μg / ml LPS to the culture medium and divide it into four groups. Add 10 μl of the solvent solution as the control group; 10 μl of the eye drops prepared from exosomes after stimulation with 100 ng / ml TNF-α; 10 μl of the eye drops prepared from exosomes after stimulation with 100 ng / ml IFN-α; 10 μl of the eye drops with simultaneous stimulation with 100 ng / ml TNF-α and 100 ng / ml IFN-α, and continue to culture for 48 hours;

[0079] 2. According to the product manual of the RNA extraction kit, extract the RNA of the 4 groups of canine mesenchymal stem cells, and use a nucleic acid content detector (OneDrop TM ) to detect the RNA content;

[0080] 3. Reverse transcribe the extracted RNA to obtain cDNA. Using the HPRT1 gene as an internal reference (F - 5’-ACGCTGAGGATTTGGAAAAAGT-3’; R - 5’-ACACAGAGGGCTACGATGTG-3’), canine IL-10 specific primers (F - 5’-ctgagaaccacgacccagac-3’; R - 5’-ctccaccgccttgctcttat-3’), and ARG1 (F - 5’-TCTCTGTGTCATTTGGGTGG-3’; R - 5’-CAAGAGAATCCTGGTACATCGG-3’), amplify the IL-10 and ARG1 genes under the following reaction conditions: 95°C for 2 minutes, 95°C for 15 seconds (40 cycles), 55°C for 30 seconds (40 cycles), 72°C for 30 seconds (40 cycles), with 3 replicates for each sample.

[0081] 4. Count the relative expression levels of the IL-6 and IL-1β genes by the method.

[0082] 5. Detection found that 100 ng / ml TNF-α or 100 ng / ml IFN-α promoted the expression of the IL-10 and ARG1 genes in the M2 phenotype of macrophages, and the expression levels of the IL-10 and ARG1 genes in canine mesenchymal stem cells were higher when stimulated by both of them simultaneously than in the groups stimulated separately.

[0083] Please refer to Figure 6 for the detection of the effect of the eye drops on the proliferation of human corneal epithelial cells HCE-T by the CCK-8 method:

[0084] 1. Seed 1x10^4 / cm 2 human corneal epithelial cells HCE-T (100 μl / well) in a 96-well plate. Divide them into 3 groups and use 10 μl of DMEM / F12 complete culture medium as a control (vehicle solution), DMEM / F12 complete culture medium + 10 μl of eye drops for culture for 72 h for cell proliferation detection, with 6 replicates for each group.

[0085] 2. When the culture plate is incubated in the incubator for 72 h, add 10 μl of CCK-8 solution to each well and incubate the culture plate in the incubator for 2 h.

[0086] 3. Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

[0087] 4. Calculate the cell proliferation rate of each treatment group relative to the control group and analyze the effect of the eye drops on the proliferation of HCE-T cells.

[0088] 5. When the eye drops were incubated with HCE-T cells for 72 h, the proliferation rate of the exosome group was significantly higher than that of the control group, indicating that the eye drops of the present invention promoted the in vitro proliferation of human corneal cells.

[0089] Please refer to Figure 7 , in vitro repair damage detection of eye drops (scratch test to detect the in vitro repair of human corneal epithelial cells by the eye drops of the present invention):

[0090] 1. Culture HCE-T cells in a 12-well plate. When the density reaches 80-90%, use a sterile 200 μl pipette tip to scratch the cells, and wash them twice with PBS to remove floating cells;

[0091] 2. Add 10 μl of control (solvent) to each well in the control group, and add 10 μl of eye drops to each well in the eye drop treatment group, and continue to culture for 24 h;

[0092] 3. Take pictures of the scratched area under the microscope at 0 h and 24 h, and measure the wound area through ImageJ software;

[0093] 4. Calculate the wound area at 24 h / the wound area at 0 h in the two groups to evaluate the wound repair ability of the eye drops;

[0094] 5. It was found that the speed of the eye drops in repairing the wound was significantly faster than that of the control group.

[0095] Please refer to Figure 8 , corneal wound treatment with eye drops:

[0096] 1. Anesthetize 6-month-old C57BL / 6J mice by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (5 mg / kg). After topical anesthesia of the eyes with 0.5% prilocaine, remove a 2 mm area of the central corneal epithelium of the mice, immediately drop in the eye drops or control (solvent), and then keep dropping 3-5 times a day;

[0097] 2. Monitor wound closure at 0 and 24 hours, measure the wound area through ImageJ software, compare with the baseline, and measure the percentage of wound healing;

[0098] 3. It was found that the speed of the eye drops in repairing the corneal wound of mice was significantly faster than that of the control group.

[0099] Combined with the current actual needs, the above-mentioned implementation manners adopted in this application, the scope of protection is not limited thereto. Within the knowledge scope of those skilled in the art, various changes made without departing from the concept of this application still fall within the protection scope of the present invention.

Claims

1. An eye drop containing exosomes, characterized in that: The invention comprises lyophilized exosome powder, wherein the lyophilized exosome powder is prepared from exosomes obtained after mesenchymal stem cells are treated with TNF-α and IFN-α and houttuynia cordata extract.

2. An eye drop containing exosomes according to claim 1, characterized in that: The method for preparing the exosome freeze-dried powder comprises the following steps: S11, culturing P3-P5 generation mesenchymal stem cells with MEMα culture medium containing 10% FBS containing TNF-α and IFN-α until the cell confluence reaches 80-90%, washing with PBS for three times, and then changing to serum-free MEMα culture medium without TNF-α and IFN-α for 40-50h; S12, collecting the supernatant of the culture solution, and separating exosomes from the supernatant to obtain an exosome heavy suspension; S13. Prepare 1 L of exosome lyophilized powder excipient: weigh 1-10 g of mannitol, stir to dissolve, add 2-8 g of trehalose, dissolve with ultrapure water to a total volume of 1 L, and sterilize at high temperature to obtain an excipient solution; S14, adding Houttuynia cordata extract to the exosome heavy suspension, so that the protein content of the exosomes is 1 mg / L and the methyl nonanone content is 2-5 mg / L; S15. Mix the exosome heavy suspension after adding Houttuynia cordata extract with the auxiliary material solution, and then freeze-dry it at low temperature by vacuum freeze-drying technology to obtain exosome freeze-dried powder.

3. An eye drop containing exosomes according to claim 2, characterized in that: The mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells or umbilical cord mesenchymal stem cells.

4. The exosome-containing eye drops according to claim 2, characterized in that: The concentration of TNF-α in the culture medium is 10-100 ng / ml, and the concentration of IFN-α in the culture medium is 10-100 ng / ml.

5. The exosome-containing eye drops according to claim 2, characterized in that: The eye drops contain 1 mg / L of the exosomes, and the Houttuynia cordata extract contains 2-5 mg / L of methyl nonanone.

6. The exosome-containing eye drops according to claim 2, characterized in that: The concentration of the exosomes was measured by the BCA method.

7. The exosome-containing eye drops according to claim 2, characterized in that: The components of the solvent liquid of the exosome freeze-dried powder include carbomer, physiological saline, a pH regulator, and a preservative.

8. The exosome-containing eye drops according to claim 7, characterized in that: When using the eye drops, the solvent solution is added to the exosome freeze-dried powder and mixed evenly.

9. A method for preparing the exosome-containing eye drops according to any one of claims 1 to 8, characterized in that: The following steps are involved: S21, culturing P3-P5 generation mesenchymal stem cells with MEMα culture medium containing 10% FBS containing TNF-α and IFN-α until the cell confluence reaches 80-90%, washing with PBS for three times, and then changing to serum-free MEMα culture medium without TNF-α and IFN-α for 40-50h; S22, collecting the supernatant of the culture solution, centrifuging the supernatant for 15 min at a relative centrifugal force of 300 g to remove cells and cell debris in the supernatant, then centrifuging the supernatant for 30 min at a relative centrifugal force of 1500 g, and then transferring the supernatant to an ultrafiltration membrane with a molecular weight of 100 kDa for filtration to obtain a filtrate; S23, centrifuging the filtrate for 2 h at 4°C and a relative centrifugal force of 150,000 g, discarding the supernatant, adding an appropriate amount of PBS or sterile water for injection, and blowing the precipitate to obtain a heavy suspension of exosomes; S24, using the BCA protein assay kit to detect the exosome concentration, the test result showed that the protein content in the exosomes was 11.05 g / L; S25. Prepare 1 L of exosome lyophilized powder excipient: weigh 1-10 g of mannitol, stir to dissolve, add 2-8 g of trehalose, dissolve with ultrapure water to a total volume of 1 L, and sterilize at high temperature to obtain an excipient solution; S26. Add Houttuynia cordata extract to the exosome heavy suspension to make the protein content in the exosomes 1 mg / L and the methyl nonanone content 2-5 mg / L, and then add the auxiliary material solution to make the total volume of the liquid 1L.

10. Use of the exosome-containing eye drops according to any one of claims 1 to 8, characterized in that: The eye drops can be used for the treatment of dry eyes, conjunctivitis, keratitis, or for the prevention of eye diseases and the relief of visual fatigue.

Images