Eye drops containing peceganan modified hyaluronic acid as well as preparation method and application of eye drops

Abstract

The invention provides eye drops containing peceganan modified hyaluronic acid as well as a preparation method and application of the eye drops. The eye drops are prepared from the peceganan modified hyaluronic acid, adipose tissue-derived stromal cell exosome and normal saline. The eye drops have a good wetting effect, an antibacterial effect, no toxic or side effect, drug resistance and the like, can achieve a good repairing effect on damaged eye cells, promotes the eyes to secrete tears, and enables the xerophthalmia to be rapidly recovered.

Description

Technical Field

[0001] The invention belongs to the technical field of eye drops and relates to an eye drop containing pergiganan modified hyaluronic acid, a preparation method and application thereof. Background Art

[0002] With the widespread use of electronic products and the rapid changes in people's lifestyles and entertainment, bad eye habits have become a problem faced by almost everyone. Along with this come a large number of eye discomforts and diseases, including dry eye disease, corneal damage, ulcers, etc.

[0003] Dry eye disease, also known as keratoconjunctivitis sicca, is a common and complex eye disease caused by insufficient tear secretion, excessive evaporation or abnormal tear composition. Dry eye disease is mainly caused by improper use of the eyes, and excessive use of electronic products will cause symptoms of dry eye disease in most people. Long-term dry eye disease symptoms can lead to keratitis and corneal damage, causing irreversible and permanent damage to the eyes.

[0004] At present, many existing patents disclose formulas for treating or alleviating dry eye disease, such as the Reprosa formulation disclosed in patent CN113056353 B, and the natural product extracts disclosed in patents CN 110051683 B and CN 105012404 B. However, Reprosa has obvious toxic and side effects, and long-term use can cause dizziness and even affect spleen and stomach function. Although natural product extracts have small toxic and side effects, the influence of the origin and picking date of Chinese herbal medicine on the efficacy is very obvious, and the extracted active ingredients are greatly affected by the extraction process, so it is difficult to achieve quality control. Therefore, we urgently need to develop eye drops with small toxic and side effects and stable quality. Summary of the invention

[0005] The purpose of the present invention is to provide an eye drop containing pergiganan modified hyaluronic acid, a preparation method and an application thereof, so as to solve the problems of large toxic and side effects and poor quality stability of the existing eye drops.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present application provides an eye drop containing pegapican-modified hyaluronic acid, wherein the raw materials for preparing the eye drop include, by mass fraction, 0.5-2% pegapican-modified hyaluronic acid, 0.1-0.5% adipose mesenchymal stem cell exosomes, and 97.5-99.4% normal saline.

[0007] Hyaluronic acid, also known as hyaluronic acid, is the main component of connective tissues such as the intercellular matrix, vitreous body, and synovial fluid of joints in the human body. It plays physiological functions such as water retention, maintaining extracellular space, regulating osmotic pressure, lubrication, and promoting cell repair in the body. It can be used to prepare various eye drops. Hyaluronic acid has no antibacterial properties and is easily contaminated, so additional preservatives need to be added.

[0008] Pexiganum is a natural polypeptide antibacterial agent with broad-spectrum antibacterial effects, excellent activity, and no toxic side effects. When used in the preparation of eye drops, it can avoid the problems of large toxic side effects and drug resistance of traditional antibacterial agents. When peiganum is used to modify hyaluronic acid, peiganum can combine with hyaluronic acid to form a glycopeptide conjugate, allowing sugar and peptide to work synergistically, avoiding the rapid metabolism of peiganum, while maintaining the good wetting effect of hyaluronic acid.

[0009] Adipose-derived mesenchymal stem cell exosomes are small vesicles secreted by stem cells derived from adipose tissue, which have the characteristics of natural low immunogenicity, high biocompatibility, small size, easy absorption and stable quality. When adipose-derived mesenchymal stem cell exosomes are added to eye drops, they can play a better role in repairing damaged eye cells and quickly recover from dry eye disease.

[0010] Preferably, the raw materials for preparing the eye drops include 1.2% pegastrone-modified hyaluronic acid, 0.2% adipose-derived mesenchymal stem cell exosomes and 98.6% normal saline in terms of mass fraction.

[0011] In a second aspect, the present application provides a method for preparing an eye drop containing pexiganan-modified hyaluronic acid, the method comprising: S01: Add the periganan-modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes into physiological saline respectively, stir and mix well to obtain solution a and solution b.

[0012] There is a difference in the solubility of pegasiganan-modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes in physiological saline, and they are prone to insolubility. To prevent the phenomenon of insolubility, in this application, pegasiganan-modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes are added to physiological saline, respectively, and stirred for 5-20 minutes to obtain solution a and solution b. Among them, the mass ratio of pegasiganan-modified hyaluronic acid to physiological saline is 0.5-2:40-70, and the mass ratio of adipose-derived mesenchymal stem cell exosomes to physiological saline is 0.1-0.5:5-10.

[0013] In the present application, the preparation method of pegicanam modified hyaluronic acid comprises: S011: Add hyaluronic acid to water, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide under stirring to obtain a mixed solution, wherein 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are condensing agents.

[0014] S012: After the mixed solution is stirred for 20-40 minutes, acetic acid pexigan is added and stirred for reaction for 12-48 hours. Among them, the mass ratio of hyaluronic acid, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and acetic acid pexigan is 10:500-1000:1.9-3.8:0.6-1.2:2.5-5.1. After the reaction is completed, the reaction solution is transferred to a dialysis bag with a molecular weight of 3500D and dialyzed for 24-72 hours to filter out small molecule condensation agents and unreacted acetic acid pexigan. After the dialysis is completed, the dialyzate is freeze-dried to obtain pexigan-modified hyaluronic acid.

[0015] S02: Mix solution a and solution b, stir for 5-10 minutes to mix well, then add normal saline, and continue stirring for 10-20 minutes until uniform to obtain eye drops.

[0016] On the third side, the eye drops in the present application are used to stimulate tear secretion in mice.

[0017] The present invention has the following beneficial effects: (1) The use of pegapican to modify hyaluronic acid enables pegapican to combine with hyaluronic acid to form a glycopeptide conjugate, thereby allowing the sugar and peptide to act synergistically, avoiding the rapid metabolism of pegapican, while maintaining the good wetting effect of hyaluronic acid, the antibacterial effect, non-toxic side effects and drug resistance of pegapican.

[0018] (2) After adding adipose-derived mesenchymal stem cell exosomes to eye drops, they can repair damaged eye cells and quickly recover from dry eye disease.

[0019] (3) The eye drops have good wetting, antibacterial, non-toxic and side effects, and drug resistance. They can repair damaged eye cells, promote tear secretion, and quickly recover from dry eye disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The comparison chart of tear volume of mice in each group on the 7th and 14th days; Figure 2 The ocular fluorescence detection images of mice in the model group and Example 3 group on the 7th and 14th days. DETAILED DESCRIPTION

[0021] The adipose-derived mesenchymal stem cell exosomes used in the examples of this application were purchased from Duolaimi Biotechnology (Wuhan) Co., Ltd. The technical solution of the present invention is further explained and illustrated by specific examples below.

[0022] Example 1 The embodiment of the present application provides an eye drop containing pegapicanam-modified hyaluronic acid, wherein the raw materials for preparing the eye drop include, by mass fraction, 1.5% pegapicanam-modified hyaluronic acid, 0.4% adipose-derived mesenchymal stem cell exosomes, and 98.1% normal saline.

[0023] The preparation method of the eye drops comprises: S101: 10 g of hyaluronic acid is added to 700 g of water, and 3 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 g of N-hydroxysuccinimide are added under stirring to obtain a mixed solution.

[0024] S102: After the mixture was stirred for 30 minutes, 4 g of acetic acid pegaseline was added and stirred for reaction for 36 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight of 3500D and dialyzed for 60 hours. After freeze-drying, pegaseline-modified hyaluronic acid was obtained.

[0025] S103: 1.5 g of persiganan-modified hyaluronic acid was mixed with 50 g of normal saline for 15 min to obtain solution a. 0.4 g of adipose-derived mesenchymal stem cell exosomes was mixed with 7 g of normal saline for 15 min to obtain solution b.

[0026] S104: Solution a and solution b were mixed, stirred for 8 minutes, 41.1 g of normal saline was added, and stirring was continued for 15 minutes until the mixture was uniform to obtain eye drops.

[0027] Example 2 The embodiment of the present application provides an eye drop containing pegapicanam-modified hyaluronic acid, wherein the raw materials for preparing the eye drop include, by mass fraction, 0.5% pegapicanam-modified hyaluronic acid, 0.1% adipose-derived mesenchymal stem cell exosomes, and 99.4% normal saline.

[0028] The preparation method of the eye drops comprises: S201: 10 g of hyaluronic acid is added to 500 g of water, and 1.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.6 g of N-hydroxysuccinimide are added under stirring to obtain a mixed solution.

[0029] S202: After the mixture was stirred for 20 minutes, 2.5 g of acetic acid pegaseline was added and stirred for reaction for 12 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight of 3500D and dialyzed for 24 hours. After freeze-drying, pegaseline-modified hyaluronic acid was obtained.

[0030] S203: 0.5 g of periganan-modified hyaluronic acid was mixed with 40 g of normal saline for 5 min to obtain solution a. 0.1 g of adipose-derived mesenchymal stem cell exosomes was mixed with 5 g of normal saline for 5 min to obtain solution b.

[0031] S204: Solution a and solution b were mixed, stirred for 5 minutes, and then 54.4 g of normal saline was added, and stirring was continued for 10 minutes until the mixture was uniform to obtain eye drops.

[0032] Example 3 The embodiment of the present application provides an eye drop containing pegapican-modified hyaluronic acid, wherein the raw materials for preparing the eye drop include 1.2% pegapican-modified hyaluronic acid, 0.2% adipose-derived mesenchymal stem cell exosomes, and 98.6% normal saline in terms of mass fraction.

[0033] The preparation method of the eye drops comprises: S301: 10 g of hyaluronic acid is added to 800 g of water, and 2.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.86 g of N-hydroxysuccinimide are added under stirring to obtain a mixed solution.

[0034] S302: After the mixture was stirred for 30 minutes, 3.8 g of pegiganate acetate was added and stirred for reaction for 24 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight of 3500D and dialyzed for 48 hours. After freeze-drying, pegiganate-modified hyaluronic acid was obtained.

[0035] S303: 1.2 g of persiganan modified hyaluronic acid was mixed with 60 g of normal saline for 10 min to obtain solution a. 0.2 g of adipose mesenchymal stem cell exosomes was mixed with 8 g of normal saline for 10 min to obtain solution b.

[0036] S304: Solution a and solution b were mixed, stirred for 7 minutes, and then 30.6 g of normal saline was added, and stirring was continued for 15 minutes until the mixture was uniform to obtain eye drops.

[0037] Example 4 The embodiment of the present application provides an eye drop containing pegapican-modified hyaluronic acid, wherein the raw materials for preparing the eye drop include 2% pegapican-modified hyaluronic acid, 0.5% adipose-derived mesenchymal stem cell exosomes, and 97.5% normal saline in terms of mass fraction.

[0038] The preparation method of the eye drops comprises: S401: 10 g of hyaluronic acid is added to 1000 g of water, and 3.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2 g of N-hydroxysuccinimide are added under stirring to obtain a mixed solution.

[0039] S402: After the mixture was stirred for 40 minutes, 5.1 g of pegiganate acetate was added and stirred for reaction for 48 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight of 3500D and dialyzed for 72 hours. After freeze-drying, pegiganate-modified hyaluronic acid was obtained.

[0040] S403: 2 g of persiganan modified hyaluronic acid was mixed with 70 g of normal saline for 20 min to obtain solution a. 0.5 g of adipose mesenchymal stem cell exosomes was mixed with 10 g of normal saline for 20 min to obtain solution b.

[0041] S404: Solution a and solution b were mixed, stirred for 10 minutes, and then 17.5 g of normal saline was added, and stirring was continued for 20 minutes until the mixture was uniform to obtain eye drops.

[0042] In order to test whether the eye drops prepared in the present application have the effect of stimulating mice to secrete tears, the treatment effect and recovery status of mice with dry eye disease were studied in the examples of the present application.

[0043] Specifically, 0.2% benzalkonium chloride aqueous solution (w / w) was dripped into the eyeballs of male C57BL / 6 mice weighing 20g and aged 6-7 weeks, 1 drop each time, twice a day, to construct a mouse dry eye disease model. After 14 days, the tear volume of the mice was tested using tear secretion detection filter paper, and mice whose tear volume was 1 / 2 of the tear volume when no benzalkonium chloride aqueous solution was added were selected for dry eye disease treatment experiments.

[0044] The screened dry eye mice were randomly divided into 5 groups, with no less than 6 mice in each group. The model group mice were dripped with normal saline every day, and the treatment group mice were dripped with the eye drops prepared in Examples 1-4, wherein the model group mice and the treatment group mice were dripped with 1 drop each time, twice a day, for 14 consecutive days. The tear volume of each group of mice was tested with tear secretion detection filter paper on the 7th day and the 14th day, and the attached Figure 1 Comparison chart shown.

[0045] By the attached Figure 1It can be seen that compared with the model group mice, the tear volume of the mice in Example 1-4 groups on the 7th and 14th days increased significantly, and the tear volume on the 7th day was close to 1.5mm, and the tear volume on the 14th day was more than 1.5mm, which shows that the eye drops prepared in the examples of the present application can stimulate the secretion of tears in the eyes of mice, and then be used to treat dry eyes. Among them, the tear volume of the mice in Example 3 group increased most significantly, so the eye drops prepared in Example 3 had the best effect.

[0046] On the 7th and 14th days, one mouse each from the model group and Example 3 group was taken to observe the recovery of dry eye disease in the mice. Specifically, after normal saline was dripped into the eyes of the mice in the model group for at least 6 hours, a sodium fluorescein aqueous solution with a mass concentration of 1% was dripped into the eyes. Similarly, after the eye drops prepared in Example 3 were dripped into the eyes of the Example 3 group for at least 6 hours, a sodium fluorescein aqueous solution with a mass concentration of 1% was dripped into the eyes. A microscope was used to take pictures of the eyes of the mice after the sodium fluorescein aqueous solution was added, and the attached Figure 2 .

[0047] By the attached Figure 2 It can be seen that on the 7th and 14th days, obvious green fluorescence was observed in the eyes of the mice in the model group; while a small amount of green fluorescence was observed in the eyes of the mice in the Example 3 group on the 7th day, and the green fluorescence almost disappeared on the 14th day, indicating that the dry eye disease of the mice in the Example 3 group had recovered after using the eye drops.

[0048] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An eye drop containing pexiganan-modified hyaluronic acid, characterized in that: The preparation raw materials include 0.5-2% of pegastrone-modified hyaluronic acid, 0.1-0.5% of adipose-derived mesenchymal stem cell exosomes and 97.5-99.4% of normal saline according to mass fraction.

2. The eye drops containing pergiganan-modified hyaluronic acid according to claim 1, characterized in that: The preparation raw materials include 1.2% of pegastrone-modified hyaluronic acid, 0.2% of adipose-derived mesenchymal stem cell exosomes and 98.6% of normal saline according to mass fraction.

3. A method for preparing eye drops containing pergiganan modified hyaluronic acid, characterized in that: include: Adding periganan-modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes into physiological saline, respectively, and stirring to obtain solution a and solution b; The solution a and the solution b are mixed, stirred and mixed, and then physiological saline is added, and the stirring is continued to be uniform to obtain eye drops.

4. The method for preparing the eye drops containing pergiganan modified hyaluronic acid according to claim 3, characterized in that: The preparation method of the pergiganan modified hyaluronic acid comprises: Add hyaluronic acid to water, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide under stirring to obtain a mixed solution; Pegasecan acetate is added to the mixed solution, stirred for reaction for 12-48 hours, then transferred to a dialysis bag with a molecular weight of 3500D for dialysis for 24-72 hours, and freeze-dried to obtain pegasecan modified hyaluronic acid.

5. The method for preparing the eye drops containing pergiganan modified hyaluronic acid according to claim 4, characterized in that: The mass ratio of hyaluronic acid, water, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and persiganate acetate is 10:500-1000:1.9-3.8:0.6-1.2:2.5-5.

1.

6. The method for preparing the eye drops containing pergiganan modified hyaluronic acid according to claim 3, characterized in that: The mass ratio of pergiganan modified hyaluronic acid to normal saline is 0.5-2:40-70.

7. The method for preparing the eye drops containing pergiganan modified hyaluronic acid according to claim 3, characterized in that: The mass ratio of adipose-derived mesenchymal stem cell exosomes to normal saline is 0.1-0.5:5-10.

8. The eye drops according to any one of claims 1 and 2 or the eye drops prepared by the method according to any one of claims 3 to 7 are used to stimulate tear secretion in mice.

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