阅读说明：本技术 一种含外泌体滴眼液及其制备方法和应用 (Eye drops containing exosome and preparation method and application thereof ) 是由 李阿峰 明磊国 王清霞 于 2021-08-03 设计创作，主要内容包括：本发明涉及滴眼液制备方法领域,具体公开了一种含外泌体滴眼液及其制备方法和应用,所述滴眼液由间充质干细胞外泌体,β-葡聚糖,渗透压调节剂,pH调节剂和营养补充剂组成。本发明制备的滴眼液对眼部刺激小,稳定性高,对外泌体活性具有保护作用。(The invention relates to the field of eye drops preparation methods, and particularly discloses an eye drop containing an exosome, and a preparation method and an application thereof. The eye drops prepared by the invention have small irritation to eyes and high stability, and have a protective effect on the activity of exosome.)

1. An eye drop containing exosome is characterized by being prepared from the following components in percentage by mass:

osmotic pressure regulator 0.19-0.21%, ph regulator 0.019-0.021%, nutritional supplement 0.01-5%, and balance of mesenchymal stem cell exosome/beta-glucan aqueous solution;

the mass fraction of the beta-glucan aqueous solution is 0.1-3%.

2. The exosome-containing ophthalmic solution according to claim 1, wherein the mesenchymal stem cell exosome is umbilical cord mesenchymal stem cell or adipose mesenchymal stem cell.

3. The exosome-containing ophthalmic solution according to claim 2, wherein said mesenchymal stem cell exosomes are exosomes induced by cultured mesenchymal stem cells in human limbal stromal cell conditioned medium.

4. An eye drop containing exosome according to claim 3, wherein said β -glucan is β -1, 3 glucan.

5. The exosome-containing ophthalmic solution according to claim 4, wherein said β -1, 3 glucan has a molecular weight of 1-2600 kDa.

6. An eye drop containing exosome according to claim 5, wherein the osmotic pressure regulator is any one or a mixture of dextran, mannitol, glycerol and physiological saline.

7. A method for producing an eye drop containing exosome according to any one of claims 1 to 6, comprising the steps of:

s1, preparation of mesenchymal stem cell exosomes:

first, human limbal stromal cells were collected according to 0.2-1 x 10⁵/cm²Inoculating the cells in a complete culture medium for culture, collecting supernatant when the cells grow to reach the cell density of 80-85%, obtaining conditioned culture solution of the human corneal limbus stromal cells, adding the conditioned culture solution into mesenchymal stem cells for induced culture, discarding the supernatant when the cells are cultured to reach the cell density of 85-90%, cleaning the cells, continuously culturing for 24-48h, collecting the supernatant, and centrifuging to obtain mesenchymal stem cell exosomes for later use;

s2, adding the beta-glucan into water for injection, heating to about 80 ℃, stirring to dissolve, and cooling to room temperature to obtain a beta-glucan aqueous solution for later use;

s3, uniformly mixing the mesenchymal stem cell exosomes prepared in the S1 and the beta-glucan aqueous solution prepared in the S2, adding an osmotic pressure regulator, a ph regulator and a nutritional supplement, stirring and dissolving completely, and filtering and sterilizing to obtain the exosome-containing eye drops.

8. The method for producing an ophthalmic solution containing exosome according to claim 7, wherein in S1, the culture condition of human limbal stromal cells is 37 ℃ and 5% CO₂。

9. The method for preparing an eye drop containing exosome according to claim 7, wherein in S1, the specific process of centrifugation is: centrifuging at 1000rpm for 10 min, removing dead cells and cell debris, taking the supernatant, centrifuging the obtained supernatant for 10,000 Xg for 30min, taking the supernatant, 100,000 Xg, continuously centrifuging at 4 ℃ for 60-120 min, removing the supernatant, and re-suspending the left precipitate PBS to obtain the mesenchymal stem cell exosome.

10. Use of beta-glucan according to any one of claims 1 to 6 in the preparation of an exosome-containing eye drop, wherein the beta-glucan is used for protecting the stability of exosomes and enhancing the efficacy of exosomes.

Technical Field

The invention relates to the field of eye drops preparation methods, and in particular relates to eye drops containing exosomes and a preparation method and application thereof.

Background

The eye is often considered an immune privileged organ due to the presence of a blood-eye barrier, lack of a lymphatic drainage system, and the like. Nevertheless, the eye still has serious immune-mediated ophthalmopathy, such as thyroid-associated ophthalmopathy, autoimmune uveitis and Sjogren's syndrome, which may affect the daily life of the patient, and cause serious visual impairment or even blindness. At the present stage, clinical treatment methods mainly using glucocorticoids and immunosuppressants have poor curative effects and are easy to relapse, so that the diagnosis and treatment ideas and strategies for the diseases need to be developed urgently. Exosomes are lipid bilayer vesicles secreted by cells, containing bioactive molecules such as proteins, lipids, and nucleic acids, which play important mediators of intercellular communication. The previous research shows that the exosome has important inflammation and immunoregulation functions.

The limbal stem cells are a group of stem cells located in the Vogt fence at the limbal basal part to maintain the corneal epithelium stable and renewed, and the conditioned medium of the limbal stem cells contains a large amount of cytokines, growth factors and exosomes secreted by the limbal stem cells. The mesenchymal stem cells are cultured by using the corneal limbal stem cell conditioned medium, the activity of the mesenchymal stem cells can be promoted, the mesenchymal stem cells can be induced to obtain the characteristics of the corneal limbal stem cells, and the secreted exosomes have the characteristics of the corneal limbal stem cell exosomes, so that the stability and the renewal of corneal epithelium are promoted, and meanwhile, the important inflammation and immunoregulation functions can be regulated. The beta-glucan can activate macrophages, neutrophils and the like, so that the contents of leukocytin, cytokinin and special antibodies can be improved, the immune system of an organism can be comprehensively stimulated, the ability of lymphocytes of the injured organism to generate cell factors (IL-1) can be quickly recovered to be normal, and the immune function of the organism can be effectively regulated. Beta-glucan can help improve the immune response potential of inflammatory and inflammatory disease patients.

At present, exosome can only be stably stored for 3 days at the temperature of 2-8 ℃ and for 1 month at the temperature of-20 ℃. The long-term storage needs low temperature of-80 ℃, and the storage conditions are high, and the defects influence the intensive research of exosomes.

Disclosure of Invention

In order to solve the technical problems, the invention provides an exosome-containing eye drop and a preparation method and application thereof.

The invention provides an exosome-containing eye drop which is prepared from the following components in parts by mass:

beta-glucan 0.1-3%, osmotic pressure regulator 0.19-0.21%, ph regulator 0.019-0.021%, nutritional supplement 0.01-5%, and the balance of mesenchymal stem cell exosome/beta-glucan aqueous solution;

the mass fraction of the beta-glucan aqueous solution is 0.1-3%.

Further, the mesenchymal stem cell exosome is an umbilical cord mesenchymal stem cell or an adipose mesenchymal stem cell.

Further, the mesenchymal stem cell exosome is an exosome for inducing and culturing mesenchymal stem cell secretion in a human corneal limbal stromal cell conditioned medium.

Further, the beta-glucan is beta-1, 3 glucan.

Further, the molecular weight of the beta-1, 3 glucan is 1-2600 kDa.

Further, the osmotic pressure regulator is any one or a mixture of more of glucan, mannitol, glycerol and physiological saline.

The invention also provides a preparation method of the eye drops containing the exosome, which comprises the following steps:

s1, preparation of mesenchymal stem cell exosomes:

first, human limbal stromal cells were collected according to 0.2-1 x 10⁵/cm²Inoculating the cells in a complete culture medium for culture, collecting supernatant when the cells grow to reach the cell density of 80-85%, obtaining conditioned culture solution of the human corneal limbus stromal cells, adding the conditioned culture solution into mesenchymal stem cells for induced culture, discarding the supernatant when the cells are cultured to reach the cell density of 85-90%, cleaning the cells, continuously culturing for 24-48h, collecting the supernatant, and centrifuging to obtain mesenchymal stem cell exosomes for later use;

s2, adding the beta-glucan into water for injection, heating to about 80 ℃, stirring to dissolve, and cooling to room temperature to obtain a beta-glucan aqueous solution for later use;

s3, uniformly mixing the mesenchymal stem cell exosomes prepared in the S1 and the beta-glucan aqueous solution prepared in the S2, adding an osmotic pressure regulator, a ph regulator and a nutritional supplement, stirring and dissolving completely, and filtering and sterilizing to obtain the exosome-containing eye drops.

Further, in S1, the culture conditions of the human limbal stromal cells were 37 ℃ and 5% CO₂。

Further, in S1, the specific process of centrifugation is: centrifuging at 1000rpm for 10 min, removing dead cells and cell debris, taking the supernatant, centrifuging the obtained supernatant for 10,000 Xg for 30min, taking the supernatant, 100,000 Xg, continuously centrifuging at 4 ℃ for 60-120 min, removing the supernatant, and re-suspending the left precipitate PBS to obtain the mesenchymal stem cell exosome.

The invention also provides application of the beta-glucan in preparation of the exosome-containing eye drops, and the beta-glucan is used for protecting the stability of exosomes and improving the efficacy of exosomes.

Compared with the prior art, the invention has the beneficial effects that:

1. the exosome eye drops prepared by the invention has small irritation to eyes, high stability and stable exosome activity;

2. in the prepared exosome eye drops, glucan and exosome can effectively activate or inhibit the immune response of an organism, and the exosome eye drops have obvious effects on uveitis, Sjogren's syndrome, corneal wound healing and corneal rejection alleviation;

3. the invention combines exosomes and beta-glucan, protects the stability of exosomes and synergistically promotes the efficacy of exosomes by utilizing the activity and the characteristics of glucan, and has good prevention effect on eye diseases.

Detailed Description

The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.

Example 1:

this example 1 provides an eye drop containing exosome, which is specifically prepared by the following steps:

s1, preparation of mesenchymal stem cell exosomes:

(1) human limbal stromal cells were counted as 0.5 x 10⁵/cm²The density of (2) was inoculated into a sterile cell culture flask, a complete medium (90% DMEM (H) + 10% FBS) was added thereto, and the mixture was left at 37 ℃ with 5% CO₂Culturing in an incubator, and aseptically collecting cell culture supernatant when the cells grow to reach the cell density of 82.5%, thus obtaining the human corneal limbal stromal cell conditioned medium;

(2) after the mesenchymal stem cells prepared in the laboratory are subjected to recovery culture, the recovery culture is carried out according to the weight ratio of 1.5 x 10⁴/cm²The cells were inoculated into a sterile cell culture flask, and a mesenchymal stem cell medium (90% α -MEM + 10% FBS) was added thereto, and the mixture was incubated at 37 ℃ and 5% CO₂Culturing in an incubator for 12.5h, removing the mesenchymal stem cell culture medium, adding the human limbal stromal cell conditioned medium obtained in the step (1) to induce and culture, removing the supernatant when the cell density is 87.5%, washing the cells twice by using a phosphate buffer solution preheated to 37 ℃ in advance, removing the phosphate buffer solution, adding a serum-free mesenchymal stem cell culture medium (100% alpha-MEM) to continue culturing for 36h, and collecting the supernatant.

(3) Extraction of exosomes: and (3) carrying out differential centrifugation on the supernatant obtained in the step (2) to obtain the mesenchymal stem cell exosome. The specific centrifugal operation is as follows: the mixture was centrifuged at 1000rpm for 10 minutes to remove dead cells and cell debris, and the supernatant was collected. The obtained supernatant was centrifuged at 10,000 Xg for 30 minutes, and the supernatant was collected. 100,000 Xg, continuously centrifuging at 4 ℃ for 90 minutes, removing supernatant, and re-suspending the remaining precipitated PBS, wherein the PBS is PBS with pH value of 7.2-7.4, and the concentration of the PBS is 0.01M, so as to obtain the mesenchymal stem cell exosome. The BCA assay kit determines exosome concentration.

S2, aqueous β -glucan solution preparation:

adding 1.55g of beta-1, 3-glucan into 98.45g of water for injection, heating to about 80 ℃, stirring for dissolving, and cooling to room temperature to obtain a beta-glucan water solution with the mass fraction of 1.55% for later use;

the molecular weight of the beta-1, 3 glucan is 40 kDa;

s3, obtaining the eye drops containing the exosome:

adding 50g of the mesenchymal stem cell exosome prepared in the S1 into a beta-glucan aqueous solution prepared by 50g S2, uniformly mixing to obtain the mesenchymal stem cell exosome with the mass fraction of 50%, then adding 0.2g of osmotic pressure regulator (sodium chloride), 0.02g of pH regulator (sodium citrate) and 0.25g of nutritional supplement (taurine), stirring and completely dissolving, regulating the osmotic pressure to be within the range of 200 mOsm/L by using the osmotic pressure regulator, controlling the pH to be between 6 and 7, then filtering and sterilizing by using a 0.22 microporous filter membrane to obtain the exosome-containing eye drops, and canning.

Example 2

This example provides an eye drop containing exosome, the specific preparation steps are substantially the same as those in example 1, except that:

in S2, the beta-glucan is beta-1, 3 glucan and has a molecular weight of 2600 kDa.

Example 3

This example provides an eye drop containing exosome, the specific preparation steps are substantially the same as those in example 1, except that:

in S2, the molecular weight of the beta-1, 3 glucan is 1 kDa.

Example 4

This example provides an eye drop containing exosome, the specific preparation steps are substantially the same as those in example 1, except that:

in S2, the molecular weight of the beta-1, 3 glucan is 1300 kDa.

Example 5

This example provides an eye drop containing exosome, the specific preparation steps are substantially the same as those in example 1, except that:

s1 human limbus in (1)Density inoculation of stromal cells was 0.2 x 10⁵/cm²Collecting the cells when the cells grow to reach the cell density of 80%;

(2) after the mesenchymal stem cells are subjected to recovery culture, the inoculation density is 1 x 10⁴/cm²The culture time is 10h, the cells are cultured until the cell density is 85%, and the continuous culture time is 24h after the serum-free mesenchymal stem cell culture medium is added;

(3) centrifuging for 60 min;

in S2, the prepared beta-glucan aqueous solution accounts for 0.1 percent of the mass fraction;

in S3, the mass fraction of the osmotic pressure regulator is 0.19%, the mass fraction of the ph regulator is 0.019%, the mass fraction of the nutritional supplement (sodium hyaluronate) is 0.01%, and the balance is the prepared mesenchymal stem cell exosome/beta-glucan aqueous solution.

Example 6

This example provides an eye drop containing exosome, the specific preparation steps are substantially the same as those in example 1, except that:

s1, the density of the stromal cells on human limbus in (1) was seeded at 1 x 10⁵/cm²Collecting the cells when the cells grow to 85% of cell density;

(2) after the mesenchymal stem cells are subjected to recovery culture, the inoculation density is 2 x 10⁴/cm²The culture time is 15h, the cells are cultured until the cell density is 90%, and the serum-free mesenchymal stem cell culture medium is added for continuous culture for 48 h;

(3) centrifuging for 120 min;

in S2, the prepared beta-glucan aqueous solution accounts for 0.3 percent of the mass fraction;

in S3, the weight percentage of osmotic pressure regulator is 0.21%, the weight percentage of ph regulator is 0.021%, the weight percentage of nutritional supplement (vitamin C) is 5%, and the balance is the mesenchymal stem cell exosome/beta-glucan aqueous solution.

Comparative example 1

Comparative example 1 provides an eye drop, which is prepared substantially in the same manner as in example 1 except that:

contains no beta glucan.

It should be noted that the nutritional supplement in the present invention may be any one of taurine, vitamin B12, sodium hyaluronate, lutein, vitamin C, and vitamin E.

Since the eye drops prepared in examples 1 to 6 have similar properties, the present invention is represented by example 1 and the following test experiments were carried out.

Firstly, the eye drops containing exosome prepared by the invention can detect the degree of eye irritation

Taking 27 healthy New Zealand rabbits with no damage on eye examination as a representative, the eye drops containing exosomes prepared in the above example 1 were randomly divided into nine groups, three groups/group according to body weight, and the eye drops prepared in the example 1 were given to the left eye and the right eye; respectively dripping normal saline and eye drops into conjunctival sac of rabbit eye, pressing nasolacrimal duct, passively closing eye, dripping one to two drops each time for 14 days four times a day, observing and recording stimulation of cornea, iris, conjunctiva, edema and secretion, and scoring the result as shown in Table 1.

TABLE 1 cornea, iris, conjunctiva, edema, secretion stimulation score table

Ocular irritation score criteria: 0-3 min, no stimulation: 4-8 points, mild stimulation: 9-12 points, moderate stimulation.

TABLE 2 eye drops containing exosome prepared in example 1 have irritation to eye

Secondly, the stability detection of the eye drops containing exosome prepared in the embodiment 1 of the invention

1. Exosome CD63 protein immune activity assay

Diluting a mouse anti-human CD63 monoclonal antibody (purchased from Beijing Donggong Boye Biotechnology Co., Ltd.) to 0.22mg/mL by using 10mmol/L PBS buffer solution, printing the diluted mouse anti-human CD63 monoclonal antibody on a protein chip substrate by a micro-protein printer GeSim Nano-Plotter TM 2.1, repeating the printing for 4 times on each point, finally obtaining a circular spot with the diameter of about 400um, and incubating for 12h at room temperature to obtain the CD63 antibody chip.

The eye drops are taken out at months 0, 1, 3, 6, 12, 18 and 24 respectively, and the prepared CD63 antibody chip is used for detecting the immunological activity of exosome CD63 protein, which comprises the following specific steps:

the preserved eye drops containing the exosome are taken out and restored to the room temperature, 100uL of eye drops are added into each hole, the shaking is carried out for 40 minutes, the chip is washed by PBS for three times, then 100uL of fluorescein labeled mouse anti-human CD63 monoclonal antibody (4nmol/L) is added, the shaking is carried out for 40 minutes, and then the chip is washed by PBS for three times, washed by pure water for one time and dried in the air.

Scanning the chip by using a biochip reading scanner, obtaining a 16-bit gray level image after scanning, analyzing the image by using MidaScan Software, wherein the fluorescence intensity of each point is obtained by dividing the total fluorescence intensity of a selected area by the area, and the average fluorescence intensity of 4 parallel points on the image is defined as the fluorescence intensity of the test. The activity of CD63 protein was positively correlated with the fluorescence intensity on the images obtained. The results of the detection of the exosome CD63 protein are shown in table 3.

TABLE 3 detection of fluorescence intensity of eye drops CD63 protein with different storage time

As can be seen from Table 3, no matter under which conditions, the exosome activity in the eye drops in the first six months is hardly affected, and at the storage temperature of 25-40 ℃, the exosome activity in the eye drops is slightly reduced in the 12 th month and later, but the reduction degree is small, which indicates that the eye drops containing exosome prepared in the embodiment 1 of the present application have low requirement on the storage temperature, and the exosome activity can be basically maintained for two years.

2. Stability test of exosome-containing eye drops

(1) Accelerated test

The exosome eye drops prepared in the embodiment 1 of the invention and the eye drops prepared in the comparative example 1 are respectively placed in a constant temperature and humidity box under the condition of commercial package, placed under the conditions of the temperature of 40 ℃ plus or minus 2 ℃ and the relative humidity of 25 percent plus or minus 5 percent, sampled at 1 st month, 2 nd month, 3 rd month and 6 th month respectively, and measured according to the related requirements of Chinese pharmacopoeia 2015 edition, and the results are shown in table 4.

TABLE 4 stability of eye drops prepared according to the invention

As can be seen from Table 4, the stability of exosome was protected by adding β -glucan in example 1, and the stability of eye drops was greatly improved.

(2) Long term test

Taking example 1 of the invention as a representative, comparing with comparative example 1, placing the product in a constant temperature and humidity box under the condition of commercial package, placing the product under the conditions of 25 ℃ plus or minus 2 ℃ and 40% plus or minus 5% of relative humidity, respectively sampling at 3 rd, 6 th, 9 th and 12 th months, and measuring according to the relevant requirements of Chinese pharmacopoeia 2015 edition. The measurement results were compared with the measurement results of 0 month, and the results are shown in Table 5.

TABLE 5 Long-term stability of eye drops prepared in example 1 of the present invention

As can be seen from tables 1 to 5, the properties of the eye drops containing exosome provided by the invention are not changed, and visible foreign matters meet the regulations; in the related substance examination, the content of the related substance has no obvious increasing trend, no other impurities appear, the content measurement and pH value examination results have no obvious change, and the eye drops are examined aseptically and grow aseptically, which shows that the eye drops containing exosome provided by the invention have good stability, while the eye drops lacking beta glucan and having p-bengli 1 have faster impurity content increase and poorer stability.

Secondly, the eye drops containing exosome prepared in the embodiment of the invention has immunosuppressive effect on early reaction phase of mouse pollen allergic conjunctivitis

1. Test materials:

40 Balb/c female mice 6-8 weeks old were selected and randomly divided into 4 groups of 10 mice each. The mouse pollen allergic conjunctivitis test was performed on the normal control group, the model control group, the exosome eye drops prepared in example 1, and the commercially available eye drops.

2. The test method comprises the following steps:

(1) establishing a ragweed pollen allergic conjunctivitis model:

mixing PBS containing 50 μ g ragweed pollen with Freund's complete adjuvant at volume ratio of 1: 1 completely, injecting other components except normal control group subcutaneously at the root of mouse tail and left hind foot pad to perform primary sensitization, each part is 150 μ L, mixing identical ragweed pollen PBS with Freund's incomplete adjuvant at volume ratio of 1: 1 at 7d after injection, injecting into abdominal cavity to perform enhanced sensitization, and performing enhanced sensitization again at 14d in the same way. In the treatment process, the exosome eye drops (marked as the eye drop group) prepared by the method in the embodiment 1 of the invention are adopted 7 days after the last enhancement sensitization and 21-27 days, and rats in a normal control group and a model control group are dropped with purified water 3 times a day and 2 drops at a time. After the treatment, each mouse was administered 300. mu.g of the pollen protein crude extract to each test group for eye drop stimulation. And (3) anesthetizing with 10% chloral hydrate within 1h after excitation, killing the mouse by using eyeball bleeding, taking down the eyeball for manufacturing a pathological section for mast cell detection, and collecting serum for ELISA detection of ragweed pollen specific IgE.

(2) Evaluation of clinical symptoms:

after treatment with the corresponding drugs, mice in all experimental groups were challenged with ragweed pollen allergen and observed under a microscope for signs within 30min for clinical evaluation. The evaluation index includes: the conjunctival edema, conjunctival congestion, eyelid swelling and lacrimation are scored according to 0-3 points, and the scoring standard is established: none: score 0, mild: score 1, medium: 2 points, severe; and 3 minutes. The sum of the respective scores of these four categories is the total score: the eyes had severe conjunctival congestion, edema, eyelid swelling, lacrimation with a maximum score of 12, moderate with 8 and mild with 4.

After the treatment of each group of mice is finished, eye drop excitation is carried out by using ragweed pollen protein crude extract, and the clinical symptoms of each group of mice are scored within 30min after excitation.

The results are shown in Table 6: the mice in the model control group (model eye physiological saline) have obvious allergic symptoms, including conjunctival congestion, edema, eyelid swelling and lacrimation; the eye drop group prepared in the embodiment 1 of the invention can observe the clinical symptoms of mild allergic conjunctivitis, and compared with a model control group, the difference has significant statistical significance (P is less than 0.01); the normal saline control group was treated with normal saline for eyes of normal mice.

Table 6 comparison of allergen-challenged posterior ocular signs scores for each group of mice (n ═ 10)

Group of	Eyelid swelling	Lacrimation	Conjunctival congestion	Edema of conjunctiva	Total score
						Saline control	0.5	0.6	0.4	0.7	2.2
Model control group	2.3	2.1	1.9	2.5	8.8
						The eye drop group of the present invention	1.0	0.9	1.2	0.8	3.9

Influence on preparation of xerophthalmia rabbit model by injecting atropine into lacrimal gland

1. Test materials:

40 common-grade New Zealand rabbits (80 eyes) are respectively female and male, 1% atropine sulfate eye drops are dropped into both eyes, 1% atropine sulfate eye drops are dropped into eyes at 8, 12, 16 and 20 days every day, and 1-2 drops are dropped each time to induce rabbit xerophthalmia model eyes.

2. The test method comprises the following steps:

randomly selecting 10 rabbits as group A, treating with the eye drop solution of example 13 times a day with 2 drops each time, and continuously using for 14 days as a test group;

randomly selecting 10 rabbits as group B, treating with eye drop prepared in comparative example 13 times a day with 2 drops each time for 14 days as control group 1;

10 rabbits were randomly selected as group C, and commercially available 0.1% sodium hyaluronate eye drops (Ali, Japan ginseng day) were used 2 times a day, 2 drops each time, as a positive control, and was designated as control group 2. The animals in the test group and the control group are given 2.5 g.L-1 chloramphenicol eye drops for anti-infection treatment for two eyes every day, 1 time every day, 2 drops each time, and the eye drops are continuously used for 14 days.

The eye drops prepared in example 1 were used as test articles; the treatment and improvement effects of the exosome-containing eye drops described in example 1 on the rabbit dry eye model eyes were observed using comparative example 1 (control 1) and commercially available 0.1% sodium hyaluronate eye drops as positive controls (control 2).

Fourth, experimental results

1. Eye observation

The symptoms of congestion and edema of eyes in the test group are reduced compared with the model eyes in the control group 1 and the control group 2, and the animals with conjunctival secretion appearing in the rabbit eyes in the test group are fewer in number and reduced in degree.

2. Corneal fluorescein staining score

Compared with the control group before atropine is dripped for molding, the fluorescein staining score of the membrane before dripping the medicines is obviously increased (P is less than 0.01) at each observation time point after the control group 1 and the control group 2 drip the medicines; the time points after dropping the drug in the test group are not statistically different (P is more than 0.05) compared with the time points before dropping the drug. The test groups were compared with control group 1 and control group 2, except for 14d before and after instillation, the test groups were significantly lower than control group 1 and control group 2(P < 0.01) at the other observation time points, as shown in Table 7.

TABLE 7 inhibitory Effect of eye drops prepared in example 1 on Dry eye of rabbits

	Control group 1 (comparative example 1)	Control group 2	Test set (example 1)
				Before molding	1.04	1.23	1.20
1d	1.26	1.47**	1.38△△
				3d	1.54**	1.63**	1.49△△
5d	1.74**	1.82**	1.73△△
				7d	2.08**	2.01**	1.94△△
11d	2.52**	2.31**	2.25△△
				14d	2.32**	1.97**	1.84△△

Note: p < 0.05, P < 0.01, compared to the composition before molding; compared with the control group 1, the Δ P was less than 0.01.

The test results are shown in table 6, the eye drops prepared in example 1 have certain curative effect on prevention and treatment of the rabbit dry eye with atropine dripped locally, can inhibit the appearance of the rabbit dry eye induced by the atropine eye drops, promote the secretion of the rabbit tears, remarkably increase the secretion amount, and are expected to become a novel ophthalmic preparation applied to clinic.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.