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

Abstract

The invention relates to the technical field of biological medicines, in particular to an exosome-containing eye drop as well as a preparation method and application thereof. The eye drops comprise an agent A and an agent B, the agent A comprises exosome freeze-dried powder; the exosome freeze-dried powder comprises exosome, mannitol and trehalose; the agent B comprises trehalose, hydrolyzed sodium hyaluronate, vitamin B12, vitamin B6, dendrobium officinale polysaccharide, taurine, dipotassium glycyrrhizinate and water. By optimizing the components of the eye drops and the freeze-drying process, the structure stability of the exosome is maintained, the exosome is prevented from being damaged by ice crystals in the freeze-drying process, and the damage to the structure of the exosome is avoided. Nitrogen protection is introduced into the preparation method, so that the influence of oxidative stress on the exosome is reduced. In addition, the prepared eye drops are beneficial to eye tissue repair, eye fatigue can be relieved, and the eye drops have high comfort after being used.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an eye drop containing exosomes, and a preparation method and application thereof. Background Art

[0002] Dry eye refers to a type of disease caused by abnormal tear quantity or quality, which leads to tear film instability and damage to the ocular surface, resulting in ocular discomfort. It is the most common ocular surface disease.

[0003] As an important medium for intercellular communication, exosomes carry abundant bioactive molecules such as proteins and nucleic acids, and show great potential in the treatment of eye diseases, tissue repair and vision protection. However, exosomes are easily degraded in liquid state, and the long-term storage and transportation costs are high. Direct application to the eye may cause certain irritation.

[0004] Therefore, it is necessary to provide an eye drop containing exosomes and a preparation method and application thereof, wherein the prepared eye drops can effectively protect the activity of exosomes, reduce storage and transportation costs and are safe to use. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes an eye drop containing exosomes and a preparation method and application thereof. The prepared eye drops can effectively protect the activity of exosomes, reduce storage and transportation costs and are safe to use.

[0006] A first aspect of the present invention provides an eye drop containing exosomes.

[0007] Specifically, the eye drops include agent A and agent B;

[0008] The agent A includes lyophilized exosome powder;

[0009] The exosome lyophilized powder comprises exosomes, mannitol and trehalose;

[0010] The B agent includes trehalose, hydrolyzed sodium hyaluronate, vitamin B12, vitamin B6, Dendrobium officinale polysaccharide, taurine, dipotassium glycyrrhizinate and water.

[0011] Preferably, the exosome lyophilized powder comprises 20 to 60 parts of exosomes, 5 to 15 parts of mannitol, and 1 to 5 parts of trehalose in parts by weight;

[0012] The agent B comprises 0.01-15 parts of trehalose, 0.01-5 parts of hydrolyzed sodium hyaluronate, 0.0001-0.1 parts of vitamin B12, 0.001-1 parts of vitamin B6, 0.001-1 parts of dendrobium officinale polysaccharide, 0.01-1 parts of taurine, 0.01-1 parts of dipotassium glycyrrhizinate, and 75-99 parts of water.

[0013] Further preferably, the exosome lyophilized powder comprises 20-50 parts of exosomes, 8-10 parts of mannitol, and 1-5 parts of trehalose in parts by weight;

[0014] The agent B comprises 0.01-15 parts of trehalose, 0.01-5 parts of hydrolyzed sodium hyaluronate, 0.0001-0.1 parts of vitamin B12, 0.001-1 parts of vitamin B6, 0.001-1 parts of dendrobium officinale polysaccharide, 0.01-1 parts of taurine, 0.01-1 parts of dipotassium glycyrrhizinate, and 75-99 parts of water.

[0015] A second aspect of the present invention provides a method for preparing eye drops containing exosomes.

[0016] Specifically, the following steps are included:

[0017] (1) Pre-freeze agent A into a solution at a constant temperature;

[0018] (2) After pre-freezing, drying is performed;

[0019] (3) After the primary drying is completed, secondary drying is performed, and after the secondary drying, protective gas is filled to obtain agent A;

[0020] (4) After dissolving agent B, add it to agent A and mix them to prepare eye drops containing exosomes.

[0021] Preferably, in step (1), the constant temperature pre-freezing temperature is -40 to -60°C, and the time is 120 to 240 minutes.

[0022] Further preferably, in step (1), the constant temperature pre-freezing temperature is -45 to -55°C, and the time is 150 to 240 minutes.

[0023] More preferably, in step (1), the constant temperature pre-freezing temperature is -50°C and the time is 180 to 240 minutes.

[0024] Preferably, in step (2), the primary drying includes a primary drying first stage and a primary drying second stage.

[0025] Preferably, the temperature of the first stage of primary drying is -35 to -45°C, the heating time is 10 to 20 minutes, the constant temperature time is 1000 to 1500 minutes, and the target vacuum degree is 0.1 to 0.5 mbar;

[0026] The temperature of the second stage of the primary drying is -20 to -30°C, the heating time is 10 to 20 minutes, the constant temperature time is 120 to 180 minutes, and the target vacuum degree is 0.1 to 0.5 mbar.

[0027] Further preferably, the temperature of the first stage of the primary drying is -35 to -40°C, the heating time is 10 to 18 minutes, the constant temperature time is 1200 to 1500 minutes, and the target vacuum degree is 0.1 to 0.3 mbar;

[0028] The temperature of the second stage of the primary drying is -20 to -28°C, the heating time is 10 to 15 minutes, the constant temperature time is 120 to 160 minutes, and the target vacuum degree is 0.1 to 0.3 mbar.

[0029] More preferably, the temperature of the first stage of the primary drying is -38 to -40°C, the heating time is 10 to 15 minutes, the constant temperature time is 1200 to 1300 minutes, and the target vacuum degree is 0.1 to 0.3 mbar;

[0030] The temperature of the second stage of the primary drying is -20 to -25°C, the heating time is 10 to 12 minutes, the constant temperature time is 120 to 125 minutes, and the target vacuum degree is 0.1 to 0.3 mbar.

[0031] Preferably, in step (3), the secondary drying includes a first secondary drying stage and a second secondary drying stage.

[0032] Preferably, the temperature of the first stage of the secondary drying is -10 to -20°C, the heating time is 10 to 30 minutes, the constant temperature time is 120 to 180 minutes, and the target vacuum degree is 0.1 to 0.5 mbar;

[0033] The temperature of the second stage of the secondary drying is 20-30° C., the heating time is 1-10 min, the constant temperature time is 120-180 min, and the target vacuum degree is 0.1-0.5 mbar.

[0034] Further preferably, the temperature of the first stage of the secondary drying is -20 to 10°C, the heating time is 20 to 30 minutes, the constant temperature time is 120 to 160 minutes, and the target vacuum degree is 0.1 to 0.3 mbar;

[0035] The temperature of the second stage of the secondary drying is 20-28° C., the heating time is 1-8 min, the constant temperature time is 120-150 min, and the target vacuum degree is 0.1-0.3 mbar.

[0036] More preferably, the temperature of the first stage of the secondary drying is 5-10°C, the heating time is 25-30 min, the constant temperature time is 150-160 min, and the target vacuum degree is 0.1-0.3 mbar;

[0037] The temperature of the second stage of the secondary drying is 20-25° C., the heating time is 1-5 min, the constant temperature time is 140-150 min, and the target vacuum degree is 0.1-0.3 mbar.

[0038] A third aspect of the present invention provides use of an eye drop containing exosomes in the preparation of a medicament for treating eye diseases.

[0039] Preferably, the eye diseases include but are not limited to dry eye, conjunctivitis, and keratitis.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] The present invention optimizes the components of the eye drops and the freeze-drying process to maintain the stability of the exosome structure, prevent the exosomes from being damaged by ice crystals during the freeze-drying process, and avoid the destruction of the exosome structure. Nitrogen protection is introduced into the preparation method to reduce the impact of oxidative stress on exosomes. In addition, the eye drops prepared by the present invention help repair eye tissues, reduce eye fatigue, and have a high degree of comfort after use. DETAILED DESCRIPTION

[0042] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0043] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0044] Example 1

[0045] An eye drop containing exosomes and a preparation method thereof.

[0046] The exosome-containing eye drops consist of two parts, A and B. A is a freeze-dried powder containing exosomes, which includes 50 parts of exosomes, 8 parts of mannitol, and 1 part of trehalose by weight. B is the solvent of A, which includes 1 part of trehalose, 0.1 part of hydrolyzed sodium hyaluronate, 0.01 part of vitamin B12, 0.01 part of vitamin B6, 0.01 part of Dendrobium officinale polysaccharide, 0.1 part of taurine, 0.1 part of dipotassium glycyrrhizinate, and 98.67 parts of water by weight.

[0047] The steps of the preparation method are as follows:

[0048] (1) After the components of Agent A are prepared into a 1.5 mL solution according to the proportions, the solution is added to a sterile vial in a sterile environment, placed in a freeze dryer, and pre-frozen at -50°C for 180 min.

[0049] (2) After prefreezing, perform primary drying; set the freeze dryer temperature for the first stage of primary drying to -40°C, the heating time to 15 min, the constant temperature time to 1300 min, and the target vacuum degree to 0.3 mbar; set the freeze dryer temperature for the second stage to -20°C, the heating time to 10 min, the constant temperature time to 120 min, and the target vacuum degree to 0.3 mbar.

[0050] (3) After the primary drying is completed, secondary drying is performed. The temperature of the freeze dryer in the first stage of secondary drying is set to 10°C, the heating time is 30 min, the constant temperature time is 160 min, and the target vacuum degree is 0.3 mbar; the temperature of the freeze dryer in the second stage is set to 25°C, the heating time is 5 min, the constant temperature time is 150 min, and the target vacuum degree is 0.3 mbar; after the secondary drying, nitrogen is filled and the cap is pressed to obtain Agent A.

[0051] (4) After dissolving the components of agent B in sterile purified water, stirring at room temperature, the mixture is placed in a sterile vial, and then added to agent A before use, mixed and dissolved to prepare exosome-containing eye drops.

[0052] Example 2

[0053] An eye drop containing exosomes and a preparation method thereof.

[0054] The exosome-containing eye drops consist of two parts, A and B. A is a freeze-dried powder containing exosomes, which includes 20 parts of exosomes, 10 parts of mannitol, and 1 part of trehalose by weight. B is the solvent of A, which includes 0.5 parts of trehalose, 0.1 parts of hydrolyzed sodium hyaluronate, 0.1 parts of vitamin B12, 0.1 parts of vitamin B6, 0.01 parts of Dendrobium officinale polysaccharide, 0.1 parts of taurine, 0.1 parts of dipotassium glycyrrhizinate, and 98.99 parts of water by weight.

[0055] The steps of the preparation method are as follows:

[0056] (1) After the components of Agent A are prepared into a 1.5 mL solution according to the proportions, the solution is added to a sterile vial in a sterile environment, placed in a freeze dryer, and pre-frozen at -50°C for 240 min.

[0057] (2) After prefreezing, perform primary drying; set the freeze dryer temperature of the first stage of primary drying to -40°C, the heating time to 15 min, the constant temperature time to 1200 min, and the target vacuum degree to 0.3 mbar; set the freeze dryer temperature of the second stage to -20°C, the heating time to 10 min, the constant temperature time to 120 min, and the target vacuum degree to 0.3 mbar.

[0058] (3) After the primary drying is completed, secondary drying is performed. The temperature of the freeze dryer in the first stage of secondary drying is set to 10°C, the heating time is 30 min, the constant temperature time is 160 min, and the target vacuum degree is 0.3 mbar; the temperature of the freeze dryer in the second stage is set to 25°C, the heating time is 5 min, the constant temperature time is 150 min, and the target vacuum degree is 0.3 mbar; after the secondary drying, nitrogen is filled and the cap is pressed to obtain Agent A.

[0059] (4) After dissolving the components of agent B in sterile purified water, stirring at room temperature, the mixture is placed in a sterile vial, and then added to agent A before use, mixed and dissolved to prepare exosome-containing eye drops.

[0060] Comparative Example 1

[0061] An eye drop containing exosomes and a preparation method thereof.

[0062] The difference from Example 1 is that the exosome-containing eye drops A do not contain mannitol and trehalose; the constant temperature time of the first stage of primary drying in the preparation method is 600 minutes, and nitrogen is not charged after the secondary drying. The remaining components and preparation method are the same as those in Example 1.

[0063] Comparative Example 2

[0064] An eye drop containing exosomes and a preparation method thereof.

[0065] The difference from Example 1 is that the exosome-containing eye drops B do not contain vitamin B12, vitamin B6, or Dendrobium officinale polysaccharide. The remaining components and preparation method are the same as those in Example 1.

[0066] Product effect test:

[0067] 1. The morphology and color of agent A in exosome-containing eye drops.

[0068] The product form and color of Agent A (exosome lyophilized powder) in the exosome-containing eye drops is one of the core performances of the product. It will not only seriously affect the product's appearance and customer acceptance, but also indicate the stability of the product.

[0069] Table 1 Morphology and color of lyophilized exosome powder

[0070]

[0071]

[0072] As can be seen from Table 1, the morphology of the eye drop exosome freeze-dried powder prepared by the present invention is significantly better than that of comparative example 1, indicating that the exosome freeze-dried powder of the present invention has good molding ability. After being placed at room temperature for 1 month, the color of the eye drop exosome freeze-dried powder prepared by the present invention still maintains the initial white color, while the exosome freeze-dried powder of comparative example 1 gradually turns into brown-yellow, indicating that a chemical reaction occurs in the product, resulting in a change in product performance.

[0073] In addition, as the core active substance of this product, it is necessary to evaluate the change in the exosome content after freeze-drying. By comparing the exosomes before and after freeze-drying for one month, it can be found that the content of exosomes in the exosome freeze-dried powder is also affected, and the exosome content of Examples 1 to 2 of the present invention does not change much. However, the exosome content of Comparative Example 1 without the addition of trehalose decreased by 24%, indicating that the exosome-containing eye drop component of the present invention and the preparation method (freeze-drying curve) have a protective effect on the active ingredient exosomes, greatly improving its stability.

[0074] 2. OSDI dry eye score.

[0075] The OSDI dry eye rating scale is a tool used to diagnose dry eye and assess its severity. The scale quantifies the severity of dry eye symptoms by asking patients about their eye symptoms and the impact on their visual function in the past week. The effect on the dry eye population was evaluated by measuring the change in dry eye scores before and after the use of the product. Three groups of subjects with the same average initial score were tested, with 5 people in each group for a period of 4 weeks. The results were averaged and are shown in Table 2.

[0076] Table 2 OSDI dry eye scores

[0077] Example 1 Example 2 Comparative Example 2 0 Weeks 20.0 20.0 20.0 4 weeks 10.3 11.2 15.8

[0078] As can be seen from Table 2, after the subjects used the products of Examples 1 to 2 of the present invention for four weeks, the rate of decrease in dry eye scores was significantly higher than that of Comparative Example 2, indicating that the eye repair effect of the present invention is significant and can effectively promote eye repair after use.

[0079] 3. Height of the River of Tears

[0080] Tear meniscus height is one of the important indicators for evaluating dry eye. Under normal circumstances, the tear meniscus height is between 0.4 and 1 mm. If it is lower than 0.35 mm, it indicates the possibility of dry eye. By using a slit lamp microscope to directly observe the changes in tear meniscus height of dry eye patients before and after using the product, its effect on dry eye patients was evaluated. The left and right eyes of four groups of subjects were tested separately, with 4 people in each group for a period of 4 weeks. The results were averaged and the results are shown in Table 3.

[0081] Table 3 Changes in the height of the River of Tears

[0082]

[0083] As can be seen from Table 3, after four weeks of using the products of Examples 1 to 2 of the present invention, the tear meniscus height of the subjects recovered to the normal range, while the tear meniscus height of Comparative Examples 1 and 2 recovered to some extent, but had not yet recovered to the normal level, indicating that the freeze-drying process curve and formula of the present invention have a good protective effect on the active ingredients, and the selected formula ingredients have significant eye lacrimal gland repair effect, and can effectively promote eye tear secretion after use.

[0084] 4. Tear film breakup time

[0085] Tear film breakup time is one of the key indicators for the diagnosis of dry eye. The normal tear film breakup time is 10 to 45 seconds. If the tear film breakup time is less than 10 seconds, it indicates that the tear film is unstable and the patient is likely to have dry eye. The test subject was touched lightly with a sodium fluorescein test paper to make a small amount of sodium fluorescein adhere to the ocular surface. The test subject blinked several times and then opened his eyes naturally, and then observed under the slit lamp. The time was counted from the last blink of the test subject until the first black spot appeared on the corneal surface, that is, the tear film broke. The recorded time was the tear film breakup time. The tear film breakup time was graded and scored: normal, tear film breakup time is 10 to 45 seconds; mild abnormality: when the tear film breakup time is 6 to 9 seconds; moderate abnormality: tear film breakup time is 3 to 5 seconds; severe abnormality: if the tear film breakup time is less than 3 seconds. The left and right eyes of the four groups of subjects were tested separately, with 4 people in each group for 4 weeks. The results were averaged, and the results are shown in Table 4.

[0086] Table 4 Tear film breakup time

[0087]

[0088] As can be seen from Table 4, after four weeks of using the products of Examples 1 to 2 of the present invention, the tear film breakup time of the subjects recovered to the normal range of 10s, while although the tear film stability of Comparative Examples 1 and 2 was restored, it had not yet recovered to the normal level, indicating that the freeze-drying process curve and formula of the present invention have a good protective effect on the active ingredients, and the selected formula ingredients may have a repairing effect on the meibomian glands, which increases the secretion of lipid components by repairing the meibomian glands, thereby improving the stability of the tear film.

[0089] 5. Conjunctival hyperemia score

[0090] Conjunctival congestion scoring is commonly used in the diagnosis, condition observation and treatment effect evaluation of various eye diseases such as dry eye and conjunctivitis. The present invention adopts a comprehensive evaluation method based on area and color to estimate the proportion of conjunctival area occupied by congestion; observe the color depth of the conjunctiva, the closer to normal white, the lower the score; the closer to dark purple, the higher the score. The scoring result of grade 0 means that the conjunctiva is normal and there is no congestion; the more obvious the congestion is, the more severe the symptoms are, and the higher the score result is, with the highest being grade 3. The left and right eyes of four groups of subjects were tested separately, with 4 people in each group for a period of 4 weeks, and the results were averaged. The results are shown in Table 5.

[0091] Table 5 Conjunctival hyperemia scores

[0092]

[0093] As can be seen from Table 5, after four weeks of using the products of Examples 1 to 2 of the present invention, the conjunctival congestion of the subjects was significantly relieved, while although the conjunctival congestion of Comparative Examples 1 and 2 was alleviated, it was not as obvious as that of the embodiments of the present invention, indicating that the freeze-drying process curve and formula of the present invention have a good protective effect on the active ingredients, and the selected formula ingredients can effectively relieve conjunctival congestion.

[0094] 6. Corneal repair score

[0095] Dry eye generally causes a certain degree of corneal damage, so corneal staining score can also be used as an important criterion for dry eye repair. Scoring criteria: Divide the cornea into 4 quadrants, use sodium fluorescein to stain, and observe the staining of each quadrant under the slit lamp. 0 points means that there is no fluorescein staining on the cornea, which means that the corneal epithelium is intact and undamaged; 1 point means that there are scattered punctate staining in a single quadrant, indicating that there is very slight corneal epithelial damage in this area; 2 points means that there are punctate staining in 2 to 3 quadrants, indicating that the scope of damage has expanded; 3 points means that all 4 quadrants have punctate or fused staining, indicating that the corneal epithelium is severely damaged. The left and right eyes of the four groups of subjects were tested separately, with 4 people in each group for 4 weeks, and the results were averaged. The results are shown in Table 6.

[0096] Table 6 Corneal staining scores

[0097]

[0098]

[0099] As shown in Table 6, after four weeks of using the products of Examples 1 and 2 of the present invention, the cornea of ​​the subjects basically returned to normal. Although the corneal damage of Comparative Examples 1 and 2 was restored, the repair effect was not as obvious as that of the examples of the present invention, indicating that the freeze-drying process curve and formula of the present invention have a good protective effect on the active ingredients, and the selected formula ingredients have a better repair effect on the cornea.

[0100] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution obtained by any modification, equivalent replacement, improvement, etc. made by a person skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection determined by the claims.

Claims

1. An eye drop containing exosomes, characterized in that: The eye drops include agent A and agent B; The agent A includes lyophilized exosome powder; The exosome lyophilized powder comprises exosomes, mannitol and trehalose; The B agent includes trehalose, hydrolyzed sodium hyaluronate, vitamin B12, vitamin B6, Dendrobium officinale polysaccharide, taurine, dipotassium glycyrrhizinate and water.

2. The eye drops according to claim 1, characterized in that In terms of weight, the exosome lyophilized powder includes 20 to 60 parts of exosomes, 5 to 15 parts of mannitol, and 1 to 5 parts of trehalose; The agent B comprises 0.01-15 parts of trehalose, 0.01-5 parts of hydrolyzed sodium hyaluronate, 0.0001-0.1 parts of vitamin B12, 0.001-1 parts of vitamin B6, 0.001-1 parts of dendrobium officinale polysaccharide, 0.01-1 parts of taurine, 0.01-1 parts of dipotassium glycyrrhizinate, and 75-99 parts of water.

3. The method for preparing the exosome-containing eye drops according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Pre-freeze agent A into a solution at a constant temperature; (2) After pre-freezing, drying is performed; (3) After the primary drying is completed, secondary drying is performed, and after the secondary drying, protective gas is filled to obtain agent A; (4) After dissolving agent B, add it to agent A and mix them to prepare eye drops containing exosomes.

4. The preparation method according to claim 3, characterized in that: In step (1), the constant temperature pre-freezing temperature is -40 to -60°C, and the time is 120 to 240 minutes.

5. The preparation method according to claim 3, characterized in that: In step (2), the primary drying includes a primary drying first stage and a primary drying second stage.

6. The preparation method according to claim 5, characterized in that: The temperature of the first stage of primary drying is -35 to -45°C, the heating time is 10 to 20 minutes, the constant temperature time is 1000 to 1500 minutes, and the target vacuum degree is 0.1 to 0.5 mbar; The temperature of the second stage of the primary drying is -20 to -30°C, the heating time is 10 to 20 minutes, the constant temperature time is 120 to 180 minutes, and the target vacuum degree is 0.1 to 0.5 mbar.

7. The preparation method according to claim 3, characterized in that: In step (3), the secondary drying includes a secondary drying first stage and a secondary drying second stage.

8. The preparation method according to claim 7, characterized in that: The temperature of the first stage of the secondary drying is -20 to 10°C, the heating time is 10 to 30 minutes, the constant temperature time is 120 to 180 minutes, and the target vacuum degree is 0.1 to 0.5 mbar; The temperature of the second stage of the secondary drying is 20-30° C., the heating time is 1-10 min, the constant temperature time is 120-180 min, and the target vacuum degree is 0.1-0.5 mbar.

9. Use of the exosome-containing eye drops according to any one of claims 1 to 2 in the preparation of drugs for treating eye diseases.

10. The use according to claim 9, characterized in that: The eye diseases include but are not limited to dry eyes, conjunctivitis, and keratitis.

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