Carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops as well as preparation method and application thereof

Abstract

The invention discloses carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops as well as a preparation method and application thereof, and relates to the technical field of eye drops and medical biogel. The carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops are prepared from tannic acid modified carboxymethyl chitosan and adipose tissue-derived stromal cell exosome freeze-dried powder, the carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops provided by the invention can effectively treat xerophthalmia, and are stable in components, safe and reliable. The three components including the tannic acid modified carboxymethyl chitosan, the hyaluronic acid and the adipose tissue-derived stromal cell exosome coexist, so that the functions of all the components can be fully played, a synergistic effect is achieved, and a better treatment effect on xerophthalmia is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of eye drops and medical biogels, in particular to a carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drop, and a preparation method and application thereof. Background Art

[0002] Dry eye disease is an eye condition caused by abnormal tear production. With the accelerated pace of modern life and the extensive use of electronic devices, the incidence of dry eye disease is increasing year by year. Prolonged dry eye symptoms can lead to corneal infection and thinning, and in severe cases, corneal ulcers, perforations, and even blindness. Traditional hyaluronic acid eye drops have a good moisturizing effect and can effectively prevent the occurrence of dry eye disease, but their therapeutic effect is relatively poor.

[0003] Chinese patent CN 113056353 B discloses a formulation containing Reproza ​​for treating dry eye disease, but Reproza ​​has significant side effects on the human body. Chinese patent CN 110051683 B discloses eye drops containing an extract of Anemarrhena asphodeloides polysaccharides, and Chinese patent CN 105012404 B discloses eye drops containing a bear bile powder extract. Both can be used to treat dry eye disease, but the key extracts, Anemarrhena asphodeloides polysaccharides and bear bile powder, are complex, making quality control of the eye drops a challenge.

[0004] Therefore, there is an urgent need to develop an eye drop product that is effective in treating dry eye, which can ensure that it has no toxic side effects and can effectively control product quality. Summary of the Invention

[0005] To solve the above problems, the purpose of the present invention is to provide a carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops and a preparation method and application thereof.

[0006] To achieve the above-mentioned purpose, the present invention is implemented through the following technical solutions:

[0007] A carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drop containing tannic acid-modified carboxymethyl chitosan and adipose-derived mesenchymal stem cell exosome freeze-dried powder.

[0008] Preferably, the tannic acid modified carboxymethyl chitosan is prepared according to the following steps:

[0009] Add tannic acid and n-dodecanoic acid to an organic solvent, add a condensing agent and N-hydroxysuccinimide thereto, stir at 5-30° C. for 20-40 minutes to obtain a mixed solution, add the obtained mixed solution dropwise to a carboxymethyl chitosan aqueous solution at a dropping rate of 1-5 ml / min, stir at 20-40° C. for 8-24 hours after the dropwise addition, dialyze with a dialysis bag with a molecular weight of 3500D, and freeze-dry to obtain tannic acid-modified carboxymethyl chitosan;

[0010] The mass concentration of the carboxymethyl chitosan aqueous solution is 5-10%;

[0011] The mass ratio of tannic acid, n-dodecanoic acid, organic solvent, condensing agent, N-hydroxysuccinimide and carboxymethyl chitosan aqueous solution is 2-4:3-5:30-50:8-16:4-6:500-1000.

[0012] The present invention also includes a method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, comprising the following steps:

[0013] ① Preparation of tannic acid-modified carboxymethyl chitosan: tannic acid and n-dodecanoic acid are added to an organic solvent, a condensing agent and N-hydroxysuccinimide are added thereto, and the mixture is stirred at 5-30° C. for 20-40 minutes to obtain a mixed solution, which is then added dropwise to a carboxymethyl chitosan aqueous solution at a dropping rate of 1-5 ml / min. After the dropwise addition is completed, the mixture is stirred at 20-40° C. for 8-24 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0014] The mass concentration of the carboxymethyl chitosan aqueous solution is 5-10%;

[0015] wherein the mass ratio of tannic acid, n-dodecanoic acid, organic solvent, condensing agent, N-hydroxysuccinimide and carboxymethyl chitosan aqueous solution is 2-4:3-5:30-50:8-16:4-6:500-1000;

[0016] ② Add hyaluronic acid and the tannic acid-modified carboxymethyl chitosan obtained in step ① to normal saline, stir and mix for 0.5 to 2 hours, add the adipose-derived mesenchymal stem cell exosome freeze-dried powder, and stir for 10 to 20 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops;

[0017] The mass ratio of hyaluronic acid, tannic acid-modified carboxymethyl chitosan obtained in step ①, normal saline, and adipose-derived mesenchymal stem cell exosome freeze-dried powder is 1-3:1-3:8900-9100:0.2-0.5.

[0018] Preferably, the organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

[0019] Preferably, the molecular weight of the hyaluronic acid is 40-100KD.

[0020] Preferably, the condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide.

[0021] Preferably, the carboxymethyl chitosan aqueous solution is obtained by dissolving carboxymethyl chitosan with a substitution degree of ≥90% in water.

[0022] Preferably, the mass concentration of the carboxymethyl chitosan aqueous solution is 6-8%.

[0023] The present invention also includes the use of carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops in treating dry eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Calcein AM / PI live / dead cell test results after HCE-T cells were cultured with a medium solution at a concentration of 200 μg / mL for 48 hours;

[0025] Figure 2 The cell survival rate of HCE-T cells after culturing with culture medium solutions with total sample mass concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL for 24 hours;

[0026] Figure 3 is the cell viability after 48 h of culture;

[0027] Figure 4 Images of eye drops (HSE) tested for eye irritation in the chorioallantoic membrane test of fertilized chicken eggs;

[0028] Figure 5 The tear volume of mice after adding 0.2% benzalkonium chloride;

[0029] Figure 6 The tear volume of mice with dry eye disease after being treated with HSE eye drops, 10-fold diluted HSE eye drops, and 100-fold diluted HSE eye drops for 14 days.

[0030] Figure 7 The results of sodium fluorescein staining of the eyes of mice with dry eye disease after 7 and 14 days of treatment with eye drops;

[0031] Figure 8 HE staining of mouse cornea;

[0032] Figure 9 This is a PAS staining image of mouse conjunctiva;

[0033] Figure 10is the number of goblet cells in the conjunctiva of mice in the blank group and the model group on day 0;

[0034] Figure 11 The number of goblet cells in the conjunctiva of mice in the model group and the experimental group on the 7th day;

[0035] Figure 12 These are MMP-3 immunofluorescence images (green) of mouse corneal epithelial cells, where A is the image on day 0 and B is the image on day 7.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] The carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops of the present invention are effective in treating dry eye disease, and feature stable ingredients, safety, and reliability. The coexistence of tannic acid-modified carboxymethyl chitosan, hyaluronic acid, and adipose-derived mesenchymal stem cell exosomes maximizes the functions of each component, creating a synergistic effect and achieving a better therapeutic effect for dry eye disease.

[0038] First, the structure of carboxymethyl chitosan is modified with tannic acid. Tannic acid is a natural antimicrobial substance with no toxic side effects that effectively inhibits and kills a variety of bacteria harmful to the human body. Modifying carboxymethyl chitosan significantly enhances its antimicrobial activity, effectively preventing the proliferation of microorganisms in eye drops and replacing the traditional addition of antibacterial agents. Second, adipose-derived mesenchymal stem cell exosomes are extracellular vesicles secreted by adipose-derived mesenchymal stem cells. They have a saucer-like structure, are stable, and easily stored. They regulate local immune function, reduce inflammatory responses, and promote the repair of damaged tissue. The presence of adipose-derived mesenchymal stem cell exosomes can significantly promote the repair of damaged corneal cells and have a good therapeutic effect on dry eye disease. Finally, tannic acid-modified carboxymethyl chitosan contains a large number of amino groups that can ionically bond with the carboxyl groups of hyaluronic acid. The resulting cross-linked network effectively encapsulates adipose-derived mesenchymal stem cell exosomes, further improving their stability. DETAILED DESCRIPTION

[0039] The purpose of the present invention is to provide a carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops and its preparation method and application. The present invention is further described below with reference to specific examples.

[0040] The adipose-derived mesenchymal stem cell exosome freeze-dried powder used in the embodiment of the present invention was purchased from Duolei Mi Biotechnology (Wuhan) Co., Ltd., item number DL22074.

[0041] Example 1 A method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, comprising the following steps:

[0042] ① Preparation of tannic acid-modified carboxymethyl chitosan: 2 g of tannic acid and 3 g of n-dodecanoic acid were added to 30 g of an organic solvent, 8 g of a condensing agent and 4 g of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 5°C for 40 minutes to obtain a mixed solution. The obtained mixed solution was added dropwise to 500 g of a carboxymethyl chitosan aqueous solution at a dropping rate of 1 ml / min. After the dropwise addition, the mixture was stirred at 20°C for 24 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0043] The mass concentration of the carboxymethyl chitosan aqueous solution is 5%;

[0044] ② Add 1 g of hyaluronic acid and 3 g of tannic acid-modified carboxymethyl chitosan obtained in step ① to 8900 g of normal saline, stir and mix for 0.5 hours, add 0.2 g of adipose-derived mesenchymal stem cell exosome freeze-dried powder, stir and mix for 10 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops.

[0045] Example 2 A method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, comprising the following steps:

[0046] ① Preparation of tannic acid-modified carboxymethyl chitosan: 4 g tannic acid and 5 g n-dodecanoic acid were added to 50 g organic solvent, 16 g condensing agent and 6 g N-hydroxysuccinimide were added thereto, and the mixture was stirred at 30°C for 20 minutes to obtain a mixed solution, which was then added dropwise to 1000 g carboxymethyl chitosan aqueous solution at a dropping rate of 5 ml / min. After the addition was complete, the mixture was stirred at 40°C for 8 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0047] The mass concentration of the carboxymethyl chitosan aqueous solution is 10%;

[0048] ② Add 3 g of hyaluronic acid and 1 g of tannic acid-modified carboxymethyl chitosan obtained in step ① to 9100 g of normal saline, stir and mix for 2 hours, add 0.5 g of adipose-derived mesenchymal stem cell exosome freeze-dried powder, stir and mix for 20 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops.

[0049] Example 3 A method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, comprising the following steps:

[0050] ① Preparation of tannic acid-modified carboxymethyl chitosan: 2.5 g of tannic acid and 3.5 g of n-dodecanoic acid were added to 35 g of an organic solvent, 9 g of a condensing agent and 4.5 g of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 10°C for 25 minutes to obtain a mixed solution, which was then added dropwise to 600 g of a carboxymethyl chitosan aqueous solution at a dropping rate of 3 ml / min. After the addition was complete, the mixture was stirred at 30°C for 10 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0051] The mass concentration of the carboxymethyl chitosan aqueous solution is 6%;

[0052] ② Add 1.5 g of hyaluronic acid and 2.5 g of tannic acid-modified carboxymethyl chitosan obtained in step ① to 8950 g of normal saline, stir and mix for 1 hour, add 0.3 g of adipose-derived mesenchymal stem cell exosome freeze-dried powder, and stir for 12 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops.

[0053] Example 4 A method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops comprises the following steps:

[0054] ① Preparation of tannic acid-modified carboxymethyl chitosan: 3.5 g of tannic acid and 4 g of n-dodecanoic acid were added to 45 g of an organic solvent, 12 g of a condensing agent and 5.5 g of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 25° C. for 35 minutes to obtain a mixed solution, which was then added dropwise to 800 g of a carboxymethyl chitosan aqueous solution at a dropping rate of 4 ml / min. After the addition was complete, the mixture was stirred at 35° C. for 12 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0055] The mass concentration of the carboxymethyl chitosan aqueous solution is 9%;

[0056] ② Add 2.5 g of hyaluronic acid and 1.5 g of tannic acid-modified carboxymethyl chitosan obtained in step ① to 9080 g of normal saline, stir and mix for 1.5 hours, add 0.4 g of adipose-derived mesenchymal stem cell exosome freeze-dried powder, and stir for 15 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops.

[0057] Example 5 A method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, comprising the following steps:

[0058] ① Preparation of tannic acid-modified carboxymethyl chitosan: 3 g of tannic acid and 4 g of n-dodecanoic acid were added to 40 g of an organic solvent, 12 g of a condensing agent and 5 g of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 5-30° C. for 20-40 minutes to obtain a mixed solution, which was then added dropwise to 750 g of a carboxymethyl chitosan aqueous solution at a dropping rate of 1-5 ml / min. After the addition was complete, the mixture was stirred at 20-40° C. for 8-24 hours, dialyzed using a dialysis bag with a molecular weight of 3500D, and freeze-dried to obtain tannic acid-modified carboxymethyl chitosan;

[0059] The mass concentration of the carboxymethyl chitosan aqueous solution is 8%;

[0060] ② Add 2 g of hyaluronic acid and 2 g of tannic acid-modified carboxymethyl chitosan obtained in step ① to 9000 g of normal saline, stir and mix for 0.5 to 2 hours, add 0.4 g of adipose-derived mesenchymal stem cell exosome freeze-dried powder, and stir for 10 to 20 minutes to obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops.

[0061] Cytotoxicity evaluation

[0062] Cytotoxicity testing of each ingredient in the eye drop solution was performed on human corneal epithelial cells (HCE-T). HCE-T cells were cultured in DMEM / F12 medium containing 15% fetal bovine serum, 5 μg / mL insulin, 10 ng / mL human epidermal growth factor, and 1% double-stranded antibody.

[0063] To prepare the culture medium solution: 1.5 mg of tannic acid-modified carboxymethyl chitosan, 1.5 mg of hyaluronic acid, and 0.3 mg of lyophilized exosome powder from adipose-derived mesenchymal stem cells were sterilized by UV irradiation for 1 hour. Add 1.5 mg of tannic acid-modified carboxymethyl chitosan and 1.5 mg of hyaluronic acid to 33 mL of DMEM / F12 culture medium and mix thoroughly. Then, add 0.3 mg of lyophilized exosome powder from adipose-derived mesenchymal stem cells to achieve a total sample concentration of 1 mg / mL. The culture medium solution was then diluted with DMEM / F12 culture medium to obtain total sample concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively.

[0064] HCE-T cells were seeded in 96-well plates and the cell density was controlled at 5×10 3 Cells were cultured at 37°C in a 5% CO2 environment for 12 hours. The original DMEM / F12 culture medium was removed and a culture medium solution with a total sample concentration of 200 μg / mL was added. After culturing for 48 hours, the cell survival status was tested using a Calcein AM / PI live / dead cell assay kit and photographed using a fluorescence inverted microscope. The results were as follows: Figure 1As shown in the figure, Calcein AM reagent stains live cells (green fluorescence), and PI reagent stains dead cells (red fluorescence). It can be seen that HCE-T cells have normal morphology and high survival rate, with only very few dead cells, proving that 200 μg / mL eye drops (HSE) are non-toxic to cells.

[0065] The cell viability was further detected using CCK8 kit. HCE-T cells were seeded in 96-well plates and the cell density was controlled at 5×10 3 After 12 hours of culture, the original culture medium was removed and culture medium solutions with total sample concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL were added. After 24 and 48 hours of culture, CCK-8 solution (10%) was added and incubated for another hour. The absorbance of each well was measured at 450 nm using a microplate reader to calculate the cell survival rate. The results are as follows: Figure 2 and 3 As shown in the figure, after HCE-T cells were cultured with culture medium solutions containing 50 μg / mL, 100 μg / mL, and 200 μg / mL of sample mass concentrations for 24 and 48 hours, the cell viability remained around 100%, with no significant decrease, further demonstrating that the raw materials of the eye drops are non-toxic to cells.

[0066] The following tests used the carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops obtained in Example 5 as the eye drops (HSE) sample. The remaining examples also had similar effects and will not be described in detail.

[0067] Biocompatibility studies

[0068] The irritation of eye drops to the eyes was tested using the chorioallantoic membrane test on fertilized chicken eggs. Freshly fertilized eggs were cultured in an incubator for 9 days at a temperature of 37.5°C. The large part of the egg was then opened with a shell cracker to expose the chorioallantoic membrane. 300 μL of eye drops (HSE) sample was added to the chorioallantoic membrane in the experimental group, 300 μL of normal saline was added to the blank group, and 300 μL of 0.1M sodium hydroxide aqueous solution was added to the control group. The chorioallantoic membrane was observed after 5 minutes, and the results were as follows: Figure 4 As shown in the figure, the chorioallantoic membrane showed no noticeable changes under the action of physiological saline, while sodium hydroxide, a potential irritant, can rapidly cause bleeding, dissolution, and coagulation of blood vessels in the chorioallantoic membrane. In contrast, the eye drops of the present invention (HSE) showed no noticeable changes in the blood vessels in the chorioallantoic membrane, demonstrating that the eye drops are non-irritating to the eyes.

[0069] Evaluation of the therapeutic effect on dry eye disease

[0070] The therapeutic effect of eye drops on dry eye disease was evaluated in male C57BL / 6 mice (6-7 weeks old, 20g). A 0.2% benzalkonium chloride PBS solution was dripped into the eyes of the mice twice daily for 14 consecutive days. A blank control group was treated with normal saline. On the 15th day, tear volume was measured using tear secretion test filter paper. The results are as follows: Figure 5 As shown in the figure, compared with the blank group, the tear volume of the model group decreased significantly, which was about 1 / 2 of the blank group, proving that the mouse dry eye model was successfully established.

[0071] Mice with dry eye were divided into four groups: dry eye group, HSE group, HSE / 10 group, and HSE / 100 group. The treatments of each group were as follows: mice in the dry eye group received no treatment; mice in the HSE group were treated with eye drops (HSE) samples; mice in the HSE / 10 group were treated with eye drops (HSE) samples diluted 10 times; and mice in the HSE / 100 group were treated with eye drops (HSE) samples diluted 100 times. Each mouse received 2 drops of eye drops per day for 14 consecutive days. Tear volume was measured using tear secretion test filter paper. The results are shown in the figure. Figure 6 After treatment with the eye drops, the tear volume of mice was significantly higher than that of the dry eye group. The eye drops were not very effective at low concentrations (HSE / 100 group, treated with 100-fold diluted eye drops), but had a better therapeutic effect on dry eye at high concentrations (HSE and HSE / 10 groups).

[0072] Sodium fluorescein staining to observe ocular surface abnormalities: On the 7th and 14th days after administration, one drop of 1% sodium fluorescein solution was dripped into the mouse eyes, and then the mouse eyes were observed under a microscope and photographed. The results are as follows: Figure 7 As shown. On the 7th and 14th days, the green fluorescence of sodium fluorescein could be clearly observed in the model group mice, indicating that there was obvious conjunctival epithelial damage on the mouse ocular surface. Compared with the model group, the green fluorescence in the mouse eyes was significantly reduced after treatment with eye drops. On the 7th day, a small amount of green fluorescence was observed in the HSE group mice, and on the 14th day, the green fluorescence almost disappeared, indicating that the conjunctival epithelial damage in the mouse eyes was significantly alleviated, indicating that HSE eye drops have a good therapeutic effect on dry eye disease. Low concentrations of eye drops (HSE / 10 group and HSE / 100 group) can also reduce the green fluorescence in the mouse eyes, but the effect gradually decreases with decreasing concentration.

[0073] Evaluation of cell migration promotion ability

[0074] HE staining was used to observe the morphology of the mouse cornea. Benzalkonium chloride solution with a mass concentration of 0.2% was dripped into the mouse eyes for 14 days to obtain dry eye model mice. Normal mice and dry eye mice were killed (recorded as mice on day 0), and the eyeball tissue was taken, embedded in paraffin, dehydrated, and stained with HE after tissue sectioning. The results are as follows Figure 8As shown. The corneal epithelial cells of the blank group mice showed normal morphology and normal corneal thickness. The corneal epithelial cells of the model group showed obvious morphological abnormalities and the corneal thickness was significantly thinner, indicating that the dry eye disease model was successfully established. On the 7th day, the eyeball tissues of the mice in the model group and the drug-treated group were taken, embedded in paraffin, dehydrated, and stained with HE after tissue sectioning. The results showed that the corneal epithelial cells of the mice in the model group still had obvious morphological abnormalities and the corneal thickness of the mice was thickened, proving that the dry eye disease in the mice in the model group had partially recovered after the addition of benzalkonium chloride modeling drugs was stopped. In comparison, after the HSE group was treated with eye drops, the morphology of the corneal epithelial cells returned to normal and the corneal thickness was normal, proving that the eye drops have a good therapeutic effect on dry eye disease.

[0075] At the same time, the mouse conjunctival goblet cells were stained using the PAS staining kit. Figure 9 As shown in Figure 2, on day 0, the conjunctiva of the blank group mice contained a large number of goblet cells, while the conjunctiva goblet cells of the model group mice were significantly reduced, and their number was about half of that of the blank group (e.g. Figure 10 After 7 days of administration, the number of conjunctival goblet cells in the drug group gradually returned to normal, while the number in the model group did not increase significantly (as shown in Figure 2). Figure 11 This indicates that the number of conjunctival goblet cells in the HSE group mice was significantly restored after treatment with eye drops.

[0076] Immunosilver staining to observe the expression of MMP-3

[0077] MMP-3 is an inflammation-related matrix metalloproteinase, and its expression level in the eyes of mice with dry eye disease is higher. Immunofluorescence staining was used to observe the expression level of MMP-3 in mouse eye tissue. On day 0 and day 7, normal mice and mice with dry eye disease were killed (recorded as day 0 mice), and the eyeball tissue was taken, embedded in paraffin, dehydrated, and sliced. The slices were fixed with 2% FBS and permeabilized with 0.2% Triton X-100, and further incubated with primary antibody (anti-MMP-3) at 4°C overnight, and then incubated with secondary antibody (ALEXA FLUOR488 DONKEY ANTI-MOU) at room temperature for 1 hour. The pictures were taken under a fluorescence confocal microscope. The results are as follows: Figure 12 On day 0, no green fluorescence was observed in the cornea of ​​the blank group mice, while obvious green fluorescence was observed in the model group, proving that the cornea of ​​dry eye mice expressed a large amount of inflammation-related MMP-3 ( Figure 12 A). On the 7th day, obvious green fluorescence was observed in the model group, while the green fluorescence disappeared in the drug-treated group ( Figure 12 B). This indicates that on day 7, there was still an obvious inflammatory reaction in the cornea of ​​the model group mice, while the inflammatory reaction disappeared after the HSE group mice were treated with the eye drops, which once again proves that the eye drops of the present invention have a good therapeutic effect on dry eye disease.

Claims

1. A carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, characterized in that: It contains tannic acid-modified carboxymethyl chitosan and adipose-derived mesenchymal stem cell exosome freeze-dried powder.

2. The carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 1, characterized in that: The tannic acid modified carboxymethyl chitosan is prepared according to the following steps: Add tannic acid and n-dodecanoic acid to an organic solvent, add a condensing agent and N-hydroxysuccinimide thereto, stir at 5-30° C. for 20-40 minutes to obtain a mixed solution, dropwise add the obtained mixed solution to a carboxymethyl chitosan aqueous solution at a dropping speed of 1-5 ml / min, stir at 20-40° C. for 8-24 hours after the dropwise addition, dialyze with a dialysis bag with a molecular weight of 3500D, and freeze-dry to obtain tannic acid-modified carboxymethyl chitosan; The mass concentration of the carboxymethyl chitosan aqueous solution is 5-10%; The mass ratio of tannic acid, n-dodecanoic acid, organic solvent, condensing agent, N-hydroxysuccinimide and carboxymethyl chitosan aqueous solution is 2-4: 3-5: 30-50: 8-16: 4-6: 500-1000.

3. The method for preparing the carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 2, characterized in that: The following steps are involved: ① Preparation of tannic acid modified carboxymethyl chitosan; ② Add hyaluronic acid and the tannic acid-modified carboxymethyl chitosan obtained in step ① to normal saline, stir and mix for 0.5 to 2 hours, add adipose mesenchymal stem cell exosome freeze-dried powder, stir for 10 to 20 minutes, and obtain carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops; The mass ratio of hyaluronic acid, tannic acid-modified carboxymethyl chitosan obtained in step ①, normal saline, and adipose-derived mesenchymal stem cell exosome freeze-dried powder is 1-3: 1-3: 8900-9100: 0.2-0.

5.

4. The method for preparing a carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 3, characterized in that: The organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

5. The method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 3, characterized in that: The molecular weight of the hyaluronic acid is 40-100KD.

6. The method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 3, characterized in that: The condensing agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N-dicyclohexylcarbodiimide.

7. The method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 3, characterized in that: The carboxymethyl chitosan aqueous solution is obtained by dissolving carboxymethyl chitosan with a substitution degree of ≥90% in water.

8. The method for preparing carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops according to claim 3, characterized in that: The mass concentration of the carboxymethyl chitosan aqueous solution is 6-8%.

9. The use of any one of claims 1 to 8 of the carboxymethyl chitosan-hyaluronic acid-stem cell exosome eye drops, characterized in that: Application in the treatment of dry eye disease.

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