Exosome composition, application thereof and exosome eye drops

Abstract

The invention relates to an exosome composition, application thereof and exosome eye drops, and relates to the technical field of pharmaceutical preparations, and the exosome composition is composed of a mesenchymal stem cell exosome and a rabdosia lingua extract in a weight ratio of (5-10): (13-21). The exosome composition is composed of the mesenchymal stem cell exosome and the rabdosia lophanthoides extract in a proper proportion, and the mesenchymal stem cell exosome and the rabdosia lophanthoides extract are matched for use, so that the exosome composition has an excellent treatment effect on cataract and can be applied to preparation of products such as eye drops and the like, and a new treatment means is provided for cataract patients. In addition, the exosome eye drops provided by the invention do not contain side-effect components such as preservatives and hormones, have no obvious irritation and can be used for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to an exosome composition and application thereof, and exosome eye drops. Background Art

[0002] Cataract, as one of the leading causes of vision loss and blindness worldwide, has a complex pathogenesis, involving multiple links such as decreased lens transparency, protein aggregation and denaturation. With age, the lens gradually loses its original transparency, resulting in the inability of light to pass normally, thus affecting vision. At present, traditional methods of treating cataracts mostly rely on surgery, such as phacoemulsification combined with intraocular lens implantation. Although this method is effective, factors such as surgical risks, postoperative recovery and patient acceptance limit its widespread application. Therefore, the development of a safe, effective and non-invasive cataract treatment drug has become an important issue that needs to be urgently addressed in the medical community.

[0003] In recent years, with the rapid development of regenerative medicine and biotechnology, mesenchymal stem cells (MSCs) and their derivatives, exosomes, have become a new hot spot in various fields of research due to their unique biological characteristics and potential therapeutic value. As an important medium for intercellular communication, exosomes carry a wealth of bioactive molecules, such as proteins and nucleic acids, and can participate in regulating various physiological processes such as cell proliferation, differentiation, and apoptosis, showing great potential in the treatment of eye diseases. At the same time, traditional Chinese medicine also shows unique advantages in the treatment of cataracts. As a plant with a long history of medicinal and edible properties, the extract of Echeveria serrata is rich in a variety of bioactive ingredients, such as flavonoids and triterpenes, which have anti-inflammatory, antioxidant, and cell protection from damage effects. However, there are no reports in the prior art on the combined application of exosomes and Echeveria serrata in the treatment of cataracts. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an exosome composition and its application, and exosome eye drops.

[0005] In a first aspect, the present invention provides an exosome composition, which is composed of mesenchymal stem cell exosomes and Ipomoea australis extract in a weight ratio of 5~10:13~21.

[0006] Furthermore, the weight ratio of the mesenchymal stem cell exosomes to the Ipomoea australis extract is 7:18.

[0007] Furthermore, the extract of the Herba Lycopersicon esculentum L. is a water extract of the Herba Lycopersicon esculentum L.

[0008] Furthermore, the preparation method of the water extract of the narrow-lined striated schizonepeta includes the following steps: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; The working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 40-68°C, the reduced pressure reflux extraction pressure is 5-22 kPa, and the reduced pressure reflux extraction time is 60-90 minutes.

[0009] Furthermore, the reduced pressure reflux extraction temperature is 51° C., and the reduced pressure reflux extraction pressure is 9 kPa.

[0010] In a second aspect, the present invention provides a use of the exosome composition described in any one of the first aspects in the preparation of a drug for treating cataracts.

[0011] In a third aspect, the present invention provides an exosome eye drops, comprising the exosome composition described in any one of the first aspects.

[0012] Furthermore, the exosome eye drops include the following components in parts by weight: 2.5-8.5 parts of the exosome composition according to any one of the first aspects, 0.3-0.8 parts of vitamin E, 0.1-0.4 parts of carbomer, 1-5 parts of mannitol and 86-93 parts of normal saline.

[0013] Furthermore, the exosome eye drops include the following components in parts by weight: 5.5 parts of the exosome composition according to any one of the first aspects, 0.6 parts of vitamin E, 0.2 parts of carbomer, 2 parts of mannitol and 90 parts of normal saline.

[0014] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art: The embodiment of the present invention provides an exosome composition and its application, and exosome eye drops. The present invention uses an appropriate ratio of mesenchymal stem cell exosomes and an extract of Ipomoea australis to form an exosome composition. The combination of the two has an excellent therapeutic effect on cataracts and can be used to prepare products such as eye drops, providing a new treatment method for cataract patients. In addition, the exosome eye drops provided by the present invention do not contain preservatives, hormones and other side effect ingredients, are non-irritating and can be used for a long time. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0017] In a first aspect, the present invention provides an exosome composition, which is composed of mesenchymal stem cell exosomes and Ipomoea australis extract in a weight ratio of 5~10:13~21.

[0018] The embodiment of the present invention provides an exosome composition. The present invention uses an appropriate ratio of mesenchymal stem cell exosomes and an extract of Ipomoea australis to form an exosome composition. The combination of the two has an excellent therapeutic effect on cataracts and can be used to prepare products such as eye drops, providing a new treatment method for cataract patients. In addition, the exosome eye drops provided by the present invention do not contain preservatives, hormones and other side effect ingredients, have no obvious irritation and can be used for a long time.

[0019] In some specific embodiments, the mesenchymal stem cell exosomes described in the present invention can be directly obtained from commercially available products, or can be obtained by conventional methods, and the specific steps are as follows: 1. Collection and processing of cell culture supernatant: The third generation mesenchymal stem cells with good activity were selected and 40 mL of their culture supernatant was collected.

[0020] Centrifuge at 4000×g for 20 minutes at 4°C to remove cells and cell debris.

[0021] After centrifugation, transfer the supernatant to a new centrifuge tube and place it on ice to keep it cold to avoid sample degradation.

[0022] 2. Preliminary precipitation of exosomes: Take 10.0 mL of the treated cell culture supernatant and place it in a 15 mL centrifuge tube.

[0023] Add 1.25 mL of exosome precipitation reagent to the centrifuge tube and gently invert to mix to ensure that the reagent is in full contact with the supernatant.

[0024] The mixed sample was placed at 4°C for at least 30 min to facilitate the precipitation of exosomes.

[0025] 3. Collection and cleaning of exosome precipitates: After the standing period, the mixture was centrifuged at 12,000 × g for 30 minutes at 4°C.

[0026] After centrifugation, white precipitate, i.e. exosomes, can be observed at the bottom of the tube. Carefully aspirate the supernatant to avoid destroying the exosome precipitate.

[0027] 4. Resuspension and purification of exosomes: Add 100 μl of 1× PBS solution to the centrifuge tube containing the exosome pellet.

[0028] Use a pipette to repeatedly pipette or vortex to mix to ensure that the precipitate is completely dissolved to form a heavy suspension of exosomes.

[0029] The resuspended exosomes were transferred to the purification column and placed in a 1.5 mL collection tube.

[0030] Exosomes were purified by centrifugation at 3000 × g for 10 min at 4 °C.

[0031] After centrifugation, the purification column was discarded and the liquid in the collection tube was the extracted exosomes.

[0032] 5. Save: The extracted exosomes can be stored at 2-8°C for one week to maintain their activity.

[0033] If long-term storage is required, the exosomes can be placed in a -80°C refrigerator and can be stored for about three months.

[0034] In some specific embodiments, the weight ratio of the mesenchymal stem cell exosomes to the Isodonis stylosa extract is 7:18.

[0035] In some specific embodiments, the extract of Ipomoea acuta is an aqueous extract of Ipomoea acuta.

[0036] In some specific embodiments, the method for preparing the water extract of Ipomoea australis comprises the following process: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; The working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 40-68°C, the reduced pressure reflux extraction pressure is 5-22 kPa, and the reduced pressure reflux extraction time is 60-90 minutes.

[0037] In some specific embodiments, the reduced pressure reflux extraction temperature is 51° C., and the reduced pressure reflux extraction pressure is 9 kPa.

[0038] In some specific embodiments, the added weight of water is 10 to 15 times the weight of the whole plant of the narrow-lined striated camellia.

[0039] In a second aspect, based on the same inventive concept, the present invention provides a use of the exosome composition described in any one of the first aspects in the preparation of a drug for treating cataracts.

[0040] The exosome composition of the present invention provides a new and unconventional drug strategy for the treatment of cataracts. By utilizing the natural delivery characteristics and biocompatibility of exosomes, the composition can more effectively deliver therapeutic ingredients to the damaged area of ​​the eye, thereby significantly improving the therapeutic effect and reducing the stimulation and damage of drugs to eye tissues, reducing the occurrence of side effects, and improving the patient's treatment comfort and safety.

[0041] In a third aspect, based on the same inventive concept, the present invention provides an exosome eye drops, wherein the exosome eye drops include the exosome composition described in any one of the first aspects.

[0042] In some specific embodiments, the exosome eye drops include the following components in parts by weight: 2.5-8.5 parts of the exosome composition described in any one of 5 of the first aspect, 0.3-0.8 parts of vitamin E, 0.1-0.4 parts of carbomer, 1-5 parts of mannitol and 86-93 parts of normal saline.

[0043] In some specific embodiments, the exosome eye drops include the following components in parts by weight: 5.5 parts of the exosome composition according to any one of the first aspects, 0.6 parts of vitamin E, 0.2 parts of carbomer, 2 parts of mannitol and 90 parts of normal saline.

[0044] In some specific embodiments, the components in the exosome eye drops are stirred and mixed to prepare the exosome eye drops.

[0045] It should be noted that the components and raw materials involved in the exosome composition and its application, and the exosome eye drops provided in the embodiments of the present invention, unless otherwise specified or specified, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specified, can be carried out according to existing processing techniques or directly using existing equipment, and the present invention document will not repeat them one by one.

[0046] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0047] Example 1 This example provides an exosome composition, which is composed of mesenchymal stem cell exosomes and Isodonis serrata extract in a weight ratio of 7:18; The preparation method of the extract of narrow-lined striated radix serrata comprises the following steps: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; Among them, the working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 51°C, the reduced pressure reflux extraction pressure is 9kPa, the reduced pressure reflux extraction time is 75 minutes, and the added weight of water is 12 times the weight of the whole plant of the narrow-lined camellia.

[0048] Example 2 This example provides an exosome composition, and this example provides an exosome composition, wherein the exosome composition is composed of mesenchymal stem cell exosomes and Isodon serrata extract in a weight ratio of 5:21; The preparation method of the extract of narrow-lined striated radix serrata comprises the following steps: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; Among them, the working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 40°C, the reduced pressure reflux extraction pressure is 5kPa, the reduced pressure reflux extraction time is 90 minutes, and the added weight of the water is 15 times that of the whole plant of the narrow-lined camellia.

[0049] Example 3 This example provides an exosome composition, and this example provides an exosome composition, wherein the exosome composition is composed of mesenchymal stem cell exosomes and Ipomoea australis extract in a weight ratio of 10:13; The preparation method of the extract of narrow-lined striated radix serrata comprises the following steps: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; Among them, the working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 68°C, the reduced pressure reflux extraction pressure is 22kPa, the reduced pressure reflux extraction time is 60 minutes, and the added weight of the water is 10 times that of the whole plant of the narrow-lined camellia.

[0050] Example 4 This example provides an exosome composition, which is composed of mesenchymal stem cell exosomes and Isodonis serrata extract in a weight ratio of 7:18; The preparation method of the extract of narrow-lined striated radix serrata comprises the following steps: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; Among them, the working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 65°C, the reduced pressure reflux extraction pressure is 20kPa, the reduced pressure reflux extraction time is 75 minutes, and the added weight of the water is 12 times the weight of the whole plant of the narrow-lined camellia.

[0051] Example 5 This example provides an exosome eye drop, which includes the following components in parts by weight: 5.5 parts of the exosome composition described in Example 1, 0.6 parts of vitamin E, 0.2 parts of carbomer, 2 parts of mannitol and 90 parts of normal saline.

[0052] Example 6 This example provides an exosome eye drop, which includes the following components in parts by weight: 2.5 parts of the exosome composition described in Example 2, 0.3 parts of vitamin E, 0.4 parts of carbomer, 1 part of mannitol and 93 parts of normal saline.

[0053] Example 7 This example provides an exosome eye drop, which includes the following components in parts by weight: 8.5 parts of the exosome composition described in Example 3, 0.8 parts of vitamin E, 0.1 parts of carbomer, 5 parts of mannitol and 86 parts of normal saline.

[0054] Example 8 This example provides an exosome eye drop, which includes the following components in parts by weight: 5.5 parts of the exosome composition described in Example 4, 0.6 parts of vitamin E, 0.2 parts of carbomer, 2 parts of mannitol and 90 parts of normal saline.

[0055] Comparative Example 1 This example provides an exosome eye drop, which is different from Example 5 only in that: the exosome composition is composed of mesenchymal stem cell exosomes and Ipomoea australis extract in a weight ratio of 7:30; the remaining steps and parameters are the same.

[0056] Test Case In this example, the exosome eye drops obtained in Examples 5 to 8 and Comparative Example 1 were used to test the therapeutic effects on cataracts.

[0057] The test method is as follows: Experimental animals: Healthy pure strain Wistar rats were selected, half male and half female, with consistent weight and age to reduce the impact of individual differences on the experimental results.

[0058] Modeling method: Cataract modeling was performed by subcutaneous injection of a small dose (4 mg / kg) of sodium selenite once every other day for 3 consecutive times. The health status of the rats should be closely observed during the modeling period to ensure that the modeling was successful and no other complications occurred.

[0059] Grouping and administration: 60 rats with successful modeling were randomly divided into 6 groups, with 10 rats in each group (the total number of eyeballs was 20). Model control group: physiological saline was administered by eye drops. Experimental group: the exosome eye drops obtained in Examples 5 to 8 and Comparative Example 1 were administered respectively, 3 times a day, 2 drops / eye / time, 4 hours between drops, and continued for 7 days.

[0060] Observation and evaluation: After 7 consecutive days of dripping, the lens opacity of rats in each group was observed using a slit lamp. The standard grading of lens opacity is as follows: No opacity: The lens is completely transparent without any opacity. Nuclear opacity: The lens nucleus is opaque, but the cortex remains clear. Total opacity: Both the lens nucleus and the cortex are opaque and completely lose transparency.

[0061] Data statistics and analysis: The lens opacity of rats in each group was recorded in detail, and the number of lenses with different degrees of opacity in each group was counted. The test results are shown in Table 1.

[0062] Table 1 As can be seen from Table 1, compared with the model control group, the exosome eye drops provided in the embodiment of the present invention have excellent therapeutic effects on cataracts, and are significantly better than comparative example 1. In addition, the eye drops obtained in Example 5 of the present invention were respectively dripped into the conjunctival sac of healthy New Zealand rabbits with no damage in the eye examination, compressing the nasolacrimal duct and passively closing the eyes, 4 times a day, 1-2 drops each time, for 14 consecutive days. After the experiment, the cornea, iris, and conjunctiva were observed to show no swelling, congestion, turbidity, etc., indicating that the exosome eye drops provided in the embodiment of the present invention have no obvious irritation to the eye tissue.

[0063] In summary, the embodiments of the present invention provide an exosome composition and its application, and an exosome eye drop. The exosome composition is composed of an appropriate proportion of mesenchymal stem cell exosomes and an extract of the narrow-lined striated ...

[0064] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0065] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An exosome composition, characterized in that: The exosome composition is composed of mesenchymal stem cell exosomes and Ipomoea australis extract in a weight ratio of 5-10:13-21.

2. The exosome composition according to claim 1, characterized in that The weight ratio of the mesenchymal stem cell exosomes to the Isodon serrata extract is 7:

18.

3. The exosome composition according to claim 1 or 2, characterized in that The extract of the Herba Lycopersicon esculentum L. is a water extract of the Herba Lycopersicon esculentum L.

4. The exosome composition according to claim 3, characterized in that The preparation method of the water extract of narrow-lined schizonepeta tenuifolia The process includes: The whole herb of the narrow-lined schisandrae is crushed, and then added into water for vacuum reflux extraction to obtain an extract; The extract is concentrated and dried to obtain the water extract of Ipomoea acuta; The working condition parameters of the reduced pressure reflux extraction include: the reduced pressure reflux extraction temperature is 40-68°C, the reduced pressure reflux extraction pressure is 5-22 kPa, and the reduced pressure reflux extraction time is 60-90 minutes.

5. The exosome composition according to claim 4, characterized in that The reduced pressure reflux extraction temperature is 51° C., and the reduced pressure reflux extraction pressure is 9 kPa.

6. Use of the exosome composition according to any one of claims 1 to 5 in the preparation of a drug for treating cataracts.

7. An exosome eye drop, characterized in that: The exosome eye drops include the exosome composition according to any one of claims 1 to 5.

8. The exosome eye drops according to claim 7, characterized in that The exosome eye drops include the following components in parts by weight: 2.5-8.5 parts of the exosome composition according to any one of claims 1 to 5, 0.3-0.8 parts of vitamin E, 0.1-0.4 parts of carbomer, 1-5 parts of mannitol and 86-93 parts of normal saline.

9. The exosome eye drops according to claim 7, characterized in that The exosome eye drops include the following components in parts by weight: 5.5 parts of the exosome composition according to any one of claims 1 to 5, 0.6 parts of vitamin E, 0.2 parts of carbomer, 2 parts of mannitol and 90 parts of normal saline.

Images