An eye drop containing peceganam-modified hyaluronic acid, its preparation method and application

Abstract

This application provides an eye drop containing peceganam-modified hyaluronic acid, its preparation method, and its application. The eye drop is prepared using peceganam-modified hyaluronic acid, adipose-derived mesenchymal stem cell exosomes, and physiological saline. This eye drop exhibits good wetting and antibacterial properties, is non-toxic, has no side effects, and does not induce drug resistance. It can effectively repair damaged ocular cells, promote tear secretion, and facilitate rapid recovery from dry eye disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of eye drops, and relates to an eye drop containing pexiganan modified hyaluronic acid, a preparation method and application. BACKGROUND

[0002] With the massive popularization and application of electronic products and the rapid change of people's life and entertainment, bad eye habits have almost become a problem faced by everyone. As a result, a large number of eye discomforts and diseases, including dry eye disease, corneal damage, ulcer, etc.

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

[0004] At present, many patents disclose formulas for treating or relieving dry eye disease, such as the reproxal formulation disclosed in patent CN113056353B, and natural product extracts disclosed in patents CN110051683B and CN105012404B. However, reproxal has obvious toxic side effects, and long-term use can cause dizziness and even affect the function of the spleen and stomach. Although the natural product extract has small toxic side effects, the effect of Chinese herbal medicine origin and picking date on curative effect is very obvious, and the extracted active ingredients are greatly affected by the extraction process, so it is difficult to realize quality control. Therefore, we urgently need to develop eye drops with small toxic side effects and stable quality. SUMMARY

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

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides an eye drop containing pexiganan modified hyaluronic acid, and the preparation raw materials of the eye drop include pexiganan modified hyaluronic acid 0.5-2%, adipose mesenchymal stem cell exosomes 0.1-0.5% and physiological saline 97.5-99.4% by mass fraction.

[0008] Hyaluronic acid, also known as hyaluronic acid, is the main component of human body interstitial tissue, vitreous body, joint synovial fluid and other connective tissues, which plays physiological functions such as water retention, maintenance of extracellular space, regulation of osmotic pressure, lubrication and promotion of cell repair in vivo, and can be used to prepare various eye drops. Hyaluronic acid has no antibacterial property and is easy to be contaminated, so it needs to add additional preservatives.

[0009] Pexiganan is a natural polypeptide antibacterial agent with broad-spectrum antibacterial effect and excellent activity, and no toxic side effects. When used to prepare eye drops, it can avoid the problems of traditional antibacterial agents such as large toxic side effects and drug resistance. When pexiganan is used to modify hyaluronic acid, pexiganan can combine with hyaluronic acid to form glycopeptide conjugates, so that the sugar and peptide can synergize, avoid the rapid metabolism of pexiganan, and at the same time maintain the good wetting effect of hyaluronic acid.

[0010] Adipose-derived mesenchymal stem cell exosomes are small vesicles secreted by stem cells derived from adipose tissue, which have natural low immunogenicity, high biocompatibility, small volume, easy absorption and stable quality. After adding adipose-derived mesenchymal stem cell exosomes to eye drops, the damaged ocular cells can be repaired well, so that the dry eye disease can be quickly recovered.

[0011] More preferably, the preparation raw materials of the eye drops comprise pexiganan modified hyaluronic acid 1.2%, adipose-derived mesenchymal stem cell exosomes 0.2% and physiological saline 98.6% by mass fraction.

[0012] In a second aspect, the application provides a preparation method of the eye drops containing pexiganan modified hyaluronic acid, which comprises:

[0013] S01: Add pexiganan modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes into physiological saline respectively, stir and mix uniformly to obtain solution a and solution b.

[0014] Pexiganan modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes have different solubility in physiological saline, and are prone to appear insoluble. In order to prevent the phenomenon of insolubility, in the present application, pexiganan modified hyaluronic acid and adipose-derived mesenchymal stem cell exosomes are added into physiological saline respectively, and stirred for 5-20 minutes to obtain solution a and solution b. The mass ratio of pexiganan 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.

[0015] In the present application, the preparation method of pexiganan modified hyaluronic acid comprises:

[0016] S011: hyaluronic acid is added to water, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added under stirring conditions to obtain a mixed solution. The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are condensing agents.

[0017] S012: After the mixed solution is stirred for 20-40 min, pexiganan acetate is added, and the reaction is stirred for 12-48 h. The mass ratio of hyaluronic acid, water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, and pexiganan acetate 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 for dialysis for 24-72 h to filter out small molecule condensing agents and unreacted pexiganan acetate. After dialysis is completed, the dialysate is freeze-dried to obtain pexiganan acetate modified hyaluronic acid.

[0018] S02: Solution a and solution b are mixed, stirred for 5-10 min to mix, physiological saline is added, and stirring is continued for 10-20 min until uniform to obtain the eye drops.

[0019] In the third aspect, the eye drops in the application are applied to stimulate tear secretion in mice.

[0020] The application has the following beneficial effects:

[0021] (1) The use of pexiganan acetate modified hyaluronic acid can make pexiganan acetate and hyaluronic acid combine to form a glycopeptide conjugate, so that the sugar and the peptide can synergize, avoid rapid metabolism of pexiganan acetate, and maintain good moisturizing effect of hyaluronic acid, antibacterial effect of pexiganan acetate, no toxic side effects, and drug resistance, etc.

[0022] (2) After adding adipose mesenchymal stem cell exosomes to the eye drops, the damaged ocular cells can be repaired well, and the dry eye disease can be quickly recovered.

[0023] (3) The eye drops have good moisturizing effect, antibacterial effect, no toxic side effects, and drug resistance, etc., can repair the damaged ocular cells well, promote tear secretion of the eye, and make the dry eye disease quickly recover. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a comparison chart of tear volume of mice in each group at 7 days and 14 days;

[0025] Figure 2 is an eye fluorescence detection chart of mice in the model group and example 3 group at 7 days and 14 days. DETAILED DESCRIPTION

[0026] The adipose-derived mesenchymal stem cell exosomes used in the embodiments of this application were purchased from Dolaybio Biotechnology (Wuhan) Co., Ltd. The technical solution of the present invention will be further explained and illustrated below through specific embodiments.

[0027] Example 1

[0028] This application provides an eye drop containing peceganum-modified hyaluronic acid. The raw materials for preparing the eye drop include, by mass fraction, 1.5% peceganum-modified hyaluronic acid, 0.4% adipose-derived mesenchymal stem cell exosomes, and 98.1% physiological saline.

[0029] The preparation method of this eye drop includes:

[0030] S101: Add 10g of hyaluronic acid to 700g of water, and under stirring conditions, add 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1g of N-hydroxysuccinimide to obtain a mixture.

[0031] S102: After stirring the mixture for 30 min, add 4 g of peciganam acetate and stir for 36 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight of 3500D and dialyze for 60 h. After lyophilization, peciganam-modified hyaluronic acid is obtained.

[0032] S103: Mix 1.5g of percyganate-modified hyaluronic acid with 50g of physiological saline and stir for 15 minutes to obtain solution a. Mix 0.4g of adipose-derived mesenchymal stem cell exosomes with 7g of physiological saline and stir for 15 minutes to obtain solution b.

[0033] S104: Mix solution a and solution b, stir for 8 minutes until well mixed, then add 41.1g of physiological saline and continue stirring for 15 minutes until homogeneous to obtain eye drops.

[0034] Example 2

[0035] This application provides an eye drop containing peceganum-modified hyaluronic acid. The raw materials for preparing the eye drop include, by mass fraction, 0.5% peceganum-modified hyaluronic acid, 0.1% adipose-derived mesenchymal stem cell exosomes, and 99.4% physiological saline.

[0036] The preparation method of this eye drop includes:

[0037] S201: Add 10g of hyaluronic acid to 500g of water, and under stirring conditions, add 1.9g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.6g of N-hydroxysuccinimide to obtain a mixture.

[0038] S202: After stirring the mixture for 20 min, add 2.5 g of peciganam acetate and stir for 12 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight of 3500D and dialyze for 24 h. After lyophilization, peciganam-modified hyaluronic acid is obtained.

[0039] S203: Mix 0.5g of perceganum-modified hyaluronic acid with 40g of physiological saline and stir for 5 minutes to obtain solution a. Mix 0.1g of adipose-derived mesenchymal stem cell exosomes with 5g of physiological saline and stir for 5 minutes to obtain solution b.

[0040] S204: Mix solution a and solution b, stir for 5 minutes until well mixed, then add 54.4g of physiological saline and continue stirring for 10 minutes until homogeneous to obtain eye drops.

[0041] Example 3

[0042] This application provides an eye drop containing peceganum-modified hyaluronic acid. The raw materials for preparing the eye drop include, by mass fraction, 1.2% peceganum-modified hyaluronic acid, 0.2% adipose-derived mesenchymal stem cell exosomes, and 98.6% physiological saline.

[0043] The preparation method of this eye drop includes:

[0044] S301: Add 10g of hyaluronic acid to 800g of water, and under stirring conditions, add 2.8g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.86g of N-hydroxysuccinimide to obtain a mixture.

[0045] S302: After stirring the mixture for 30 min, add 3.8 g of peciganam acetate and stir for 24 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight of 3500D and dialyze for 48 h. After lyophilization, peciganam-modified hyaluronic acid is obtained.

[0046] S303: Mix 1.2g of perceganum-modified hyaluronic acid with 60g of physiological saline and stir for 10 minutes to obtain solution a. Mix 0.2g of adipose-derived mesenchymal stem cell exosomes with 8g of physiological saline and stir for 10 minutes to obtain solution b.

[0047] S304: Mix solution a and solution b, stir for 7 minutes until well mixed, then add 30.6g of physiological saline and continue stirring for 15 minutes until homogeneous to obtain eye drops.

[0048] Example 4

[0049] This application provides an eye drop containing peceganum-modified hyaluronic acid. The raw materials for preparing the eye drop include, by mass fraction, 2% peceganum-modified hyaluronic acid, 0.5% adipose-derived mesenchymal stem cell exosomes, and 97.5% physiological saline.

[0050] The preparation method of this eye drop includes:

[0051] S401: Add 10g of hyaluronic acid to 1000g of water, and under stirring conditions, add 3.8g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2g of N-hydroxysuccinimide to obtain a mixture.

[0052] S402: After stirring the mixture for 40 min, add 5.1 g of peciganam acetate and stir for 48 h. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight of 3500D and dialyze for 72 h. After lyophilization, peciganam-modified hyaluronic acid is obtained.

[0053] S403: Mix 2g of perceganum-modified hyaluronic acid with 70g of physiological saline and stir for 20 minutes to obtain solution a. Mix 0.5g of adipose-derived mesenchymal stem cell exosomes with 10g of physiological saline and stir for 20 minutes to obtain solution b.

[0054] S404: Mix solution a and solution b, stir for 10 minutes until well mixed, then add 17.5g of physiological saline and continue stirring for 20 minutes until homogeneous to obtain eye drops.

[0055] To test whether the eye drops prepared in this application have the effect of stimulating tear secretion in mice, the treatment effect and recovery of mice with dry eye disease were studied in the embodiments of this application.

[0056] Specifically, a 0.2% benzalkonium chloride aqueous solution (w / w) was instilled into the eyes of 6-7 week old male C57BL / 6 mice weighing 20g, one drop each time, twice daily, to establish a mouse model of dry eye disease. After 14 days, tear volume was measured using tear secretion detection filter paper. Mice with a tear volume half that of mice without benzalkonium chloride solution were selected for dry eye disease treatment experiments.

[0057] The selected dry eye mice were randomly divided into 5 groups, with no fewer than 6 mice in each group. Mice in the model group were given daily drops of physiological saline, while mice in the treatment group were given drops of the eye drops prepared in Examples 1-4, one drop each time, twice daily for 14 consecutive days. The tear volume of each group of mice was measured using tear secretion detection filter paper on days 7 and 14, respectively, to obtain the results. Figure 1 The comparison chart shown.

[0058] From the appendix Figure 1As can be seen, compared with the model group mice, the tear volume of mice in Examples 1-4 was significantly increased on days 7 and 14, with the tear volume approaching 1.5 mm on day 7 and exceeding 1.5 mm on day 14. This indicates that the eye drops prepared in the examples of this application can stimulate tear secretion in mice and thus be used to treat dry eye syndrome. Among them, the tear volume increase was most significant in the Example 3 group mice; therefore, the eye drops prepared in Example 3 have the best effect.

[0059] On days 7 and 14, one mouse from the model group and one mouse from the Example 3 group were taken to observe the recovery of dry eye disease. Specifically, after instilling physiological saline into the eyes of mice in the model group for at least 6 hours, a 1% sodium fluorescein solution was instilled into the eyes. Similarly, after instilling the eye drops prepared in Example 3 into the eyes for at least 6 hours, a 1% sodium fluorescein solution was instilled into the eyes. Microscopic photographs of the mice's eyes after the instillation of the sodium fluorescein solution were taken to obtain the attached images. Figure 2 .

[0060] From the appendix Figure 2 It can be seen that obvious green fluorescence was observed in the eyes of mice in the model group on days 7 and 14; while a small amount of green fluorescence was observed in the eyes of mice in the Example 3 group on day 7, and the green fluorescence almost disappeared on day 14. This indicates that the dry eye disease of mice in the Example 3 group has recovered after the use of eye drops.

[0061] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An eye drop solution containing pesiglan-modified hyaluronic acid, characterized by, The preparation raw materials include pesiglan modified hyaluronic acid 0.5-2%, fat mesenchymal stem cell exosome 0.1-0.5% and physiological saline 97.5-99.4% by mass fraction; wherein, the preparation method of the pesiglan modified hyaluronic acid comprises: The hyaluronic acid is added to water, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added under stirring to obtain a mixed solution; Acetic acid pesiglan is added to the mixed solution, and after stirring reaction for 12-48h, the solution is transferred to a dialysis bag with a molecular weight of 3500D for dialysis for 24-72h, and then freeze-drying is performed to obtain pesiglan modified hyaluronic acid.

2. The pesciganan-containing modified hyaluronic acid eye drop of claim 1, characterized by, The preparation raw materials include pesiglan modified hyaluronic acid 1.2%, fat mesenchymal stem cell exosome 0.2% and physiological saline 98.6% by mass fraction.

3. A method of preparing an eye drop solution containing pesiglan modified hyaluronic acid, characterized by, It comprises: The pesiglan modified hyaluronic acid and the fat mesenchymal stem cell exosome are respectively added to physiological saline, and stirring is performed to obtain solution a and solution b; The solution a and the solution b are mixed, physiological saline is added after stirring, and stirring is continued to obtain eye drops; The preparation method of the pesiglan modified hyaluronic acid comprises: The hyaluronic acid is added to water, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added under stirring to obtain a mixed solution; Acetic acid pesiglan is added to the mixed solution, and after stirring reaction for 12-48h, the solution is transferred to a dialysis bag with a molecular weight of 3500D for dialysis for 24-72h, and then freeze-drying is performed to obtain pesiglan modified hyaluronic acid.

4. The method of preparing a pesciganan-containing hyaluronic acid modified eyedrop solution according to claim 3, characterized by, The mass ratio of hyaluronic acid, water, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and acetic acid pesiglan is 10:500-1000:1.9-3.8:0.6-1.2:2.5-5.

1.

5. The method of claim 3, wherein the preparation of the pexiganan-modified hyaluronic acid-containing eye drop is characterized by, The mass ratio of pesiglan modified hyaluronic acid and physiological saline is 0.5-2:40-70.

6. The method of claim 3, wherein the preparation of the pesciganan-containing hyaluronic acid modified eye drop is characterized by, The mass ratio of fat mesenchymal stem cell exosome and physiological saline is 0.1-0.5:5-10.

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