Eye drops containing umbilical cord mesenchymal stem cell exosome and preparation and application thereof

Abstract

The invention relates to eye drops containing umbilical cord mesenchymal stem cell exosomes. The eye drops are prepared from the following raw materials: sodium hyaluronate, an L-ascorbyl palmitate / hydroxypropyl-beta-cyclodextrin inclusion compound, panthenol, a solution containing the umbilical cord mesenchymal stem cell exosomes, water and normal saline, the concentration of the sodium hyaluronate is 0.2 to 0.6 mg / mL; the concentration of the L-ascorbyl palmitate / hydroxypropyl-beta-cyclodextrin inclusion compound ranges from 0.1 mg / mL to 0.4 mg / mL; the concentration of the panthenol is 0.2 to 1.0 mg / mL; the concentration of the umbilical cord mesenchymal stem cell exosome is 0.2 * 10 < 10 >-0.6 * 10 < 10 > parts / mL. The eye drops have good effects on treating xerophthalmia and repairing corneal epithelium injury.

Description

Technical Field

[0001] This application relates to the field of ophthalmic biomedical technology, and in particular to an eye drop containing umbilical cord mesenchymal stem cell exosomes and its preparation and application. Background Technology

[0002] Artificial tears eye drops, such as sodium hyaluronate eye drops, mainly relieve dry eye symptoms by replenishing tears and lubricating the ocular surface. However, they can only temporarily relieve discomfort and cannot repair damaged ocular surface tissues. Furthermore, long-term use of products containing preservatives may aggravate ocular surface irritation.

[0003] Glucocorticoid eye drops, such as dexamethasone eye drops, can suppress ocular surface inflammation, but long-term use can lead to side effects such as increased intraocular pressure and cataracts, and has a weak effect on repairing corneal damage.

[0004] Stem cell-derived eye drops, such as bone marrow mesenchymal stem cell eye drops, although they have repair capabilities, have a long in vitro stem cell culture period (about 14-21 days), and cell activity is easily affected by storage conditions, resulting in high clinical application costs and poor stability. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an eye drop containing umbilical cord mesenchymal stem cell exosomes, its preparation, and its application. The aim is to achieve low side effects, efficient repair of the ocular surface, and long-term stable storage, while also taking into account anti-inflammatory and tissue regeneration functions, thus solving the problems of existing products that "treat the symptoms but not the root cause" and "conflict between safety and practicality".

[0006] In a first aspect, this application provides an eye drop containing umbilical cord mesenchymal stem cell exosomes, comprising the following ingredients: sodium hyaluronate, L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex, panthenol, a solution containing umbilical cord mesenchymal stem cell exosomes, water, and physiological saline; wherein the concentration of sodium hyaluronate is 0.2-0.6 mg / mL; the concentration of L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex is 0.1-0.4 mg / mL; the concentration of panthenol is 0.2-1.0 mg / mL; and the concentration of umbilical cord mesenchymal stem cell exosomes is 0.2 × 10⁻⁶ mg / mL. 10 -0.6×10 10 particles / mL.

[0007] By adopting the above technical solution, this application provides an eye drop containing umbilical cord mesenchymal stem cell exosomes. This eye drop has more comprehensive functions, significant repair effects, higher safety, and no risk of side effects. This may be because umbilical cord mesenchymal stem cell exosomes, as the core active ingredient, contain multiple growth factors such as EGF and bFGF, and anti-inflammatory factors such as IL-10, which can simultaneously achieve "anti-inflammatory + ocular surface repair + lacrimal gland function regulation". Furthermore, umbilical cord mesenchymal stem cell exosomes are derived from umbilical cord tissue and have low immunogenicity (HLA-DR negative rate >98%). Sodium hyaluronate can thicken, moisturize, and prolong the drug's residence time. L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex can scavenge free radicals on the ocular surface, promote collagen synthesis, and enhance corneal epithelial toughness. Panthenol can penetrate into the corneal epithelial layer, promote wound healing, and relieve dryness and discomfort of the ocular surface.

[0008] Optionally, the mass ratio of L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex to panthenol is 1:(2-5).

[0009] By adopting the above technical solution and adjusting the amounts of L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex and panthenol according to the above mass ratio, when the mass ratio of L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex to panthenol is 1:(2-5), the L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex scavenge free radicals on the ocular surface, promotes collagen synthesis, and enhances the toughness of the corneal epithelium; panthenol penetrates into the corneal epithelium, promotes wound healing, and relieves dryness and discomfort of the ocular surface, thus ensuring the therapeutic effect of the eye drops on dry eye syndrome and corneal epithelial damage repair.

[0010] Optionally, the sodium hyaluronate has an average molecular weight of 1.5 × 10⁻⁶. 6 Da.

[0011] Optionally, the preparation of the L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex includes the following steps: a1: Dissolve hydroxypropyl-β-cyclodextrin in water, then add L-ascorbate palmitate and stir to obtain an L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin solution; a2: Filter and dry the L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin solution prepared in step a1 to obtain the L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex.

[0012] Optionally, the umbilical cord mesenchymal stem cell exosomes are added to the eye drops in the form of lyophilized umbilical cord mesenchymal stem cell exosome powder. The preparation method of the lyophilized umbilical cord mesenchymal stem cell exosome powder includes the following preparation steps: b1: Collect the supernatant from serum-free culture of P3-P5 generation umbilical cord mesenchymal stem cells, centrifuge, filter, and obtain pretreated supernatant; b2: Concentrate the pretreated supernatant obtained in step b1 to 1 / 10-1 / 20 of its original volume to obtain TFF concentrate, replace the TFF concentrate with replacement fluid, the volume of which is 5-8 times that of the TFF concentrate, and collect the TFF concentrate; b3: Using a size exclusion chromatography column, load the TFF concentrate prepared and collected in step b2 with a loading volume of 5%-10% of the column volume, filter, and obtain a solution containing umbilical cord mesenchymal stem cell exosomes; b4: Add a freeze-drying protectant to the solution containing umbilical cord mesenchymal stem cell exosomes prepared in step b3, mix well, incubate, and freeze-dry to obtain umbilical cord mesenchymal stem cell exosome freeze-dried powder.

[0013] Optionally, in step b2, the replacement solution is sterile PBS with a pH of 7.2-7.4.

[0014] Optionally, in step b2, a polyethersulfone (PES) hollow fiber membrane module with a molecular weight cutoff of 100 kDa is selected to concentrate the pretreatment supernatant, the transmembrane pressure is set to 10-15 psi, and the tangential flow rate is 200-300 mL / min.

[0015] Optionally, in step b3, the chromatography column is a Superdex 200Increase 10 / 300GL.

[0016] Optionally, the mobile phase of the chromatography column in step b3 is sterile PBS, the flow rate is 0.4-0.6 mL / min, and the column temperature is 3-5℃.

[0017] Optionally, in step b3, the concentration of umbilical cord mesenchymal stem cell exosomes is 2 × 10⁻⁶. 10 particles / mL, 3×10 10 particles / mL, 4×10 10 particles / mL, 5×10 10 particles / mL, 6×10 10 particles / mL, 7×10 10 particles / mL or higher.

[0018] Optionally, in step b4, the freeze-drying protectant includes mannitol, trehalose, and histidine; The mass-to-volume ratio of mannitol to the solution containing umbilical cord mesenchymal stem cell exosomes prepared in step b3 is 28.5-57 mg / mL; the mass-to-volume ratio of trehalose to the solution containing umbilical cord mesenchymal stem cell exosomes prepared in step b3 is 30-90 mg / mL; and the mass-to-volume ratio of histidine to the solution containing umbilical cord mesenchymal stem cell exosomes prepared in step b3 is 1.5-3 mg / mL. Optionally, in step b4, the incubation temperature is 4°C.

[0019] Optionally, in step b4, the specific operation of lyophilization is as follows: the hUC-MSC-Exo sample containing the lyophilization protectant is dispensed into a sterile lyophilization bottle, placed on the shelf of a lyophilizer, and cooled to -40°C at a rate of 1-2°C / min, and maintained for 2-4 hours to completely freeze the sample; under a vacuum of ≤10Pa, the shelf temperature is gradually raised to -10°C and maintained for 8-12 hours to sublimate the ice crystals in the sample; then the temperature is further raised to 25°C and maintained for 4-6 hours to remove residual moisture; after lyophilization, the lyophilization bottle is sealed under vacuum to obtain hUC-MSC-Exo lyophilized powder.

[0020] In a second aspect of this application, this application provides a method for preparing eye drops containing umbilical cord mesenchymal stem cell exosomes as described in the first aspect of this application, comprising the following steps: S1: Take sodium hyaluronate, L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex and panthenol, add water, stir, filter and sterilize to obtain the base solution; S2: Reconstitute the lyophilized powder of umbilical cord mesenchymal stem cell exosomes with physiological saline, adjust the osmotic pressure, and under sterile conditions, add the reconstituted and osmotically adjusted umbilical cord mesenchymal stem cell exosome solution to the base solution prepared in step S1, stir, adjust the osmotic pressure and pH, and obtain eye drops.

[0021] Optionally, in step S2, the osmotic pressure is adjusted to 260-340 mOsm / kg.

[0022] Optionally, in step S2, the pH is adjusted to 6-7.

[0023] In a third aspect of this application, this application provides the use of the eye drops containing umbilical cord mesenchymal stem cell exosomes as described in the first aspect of this application in the preparation of eye drops for treating dry eye syndrome or repairing corneal epithelial damage.

[0024] In summary, the present invention has at least one of the following beneficial technical effects: 1. This application provides an eye drop containing umbilical cord mesenchymal stem cell exosomes. This eye drop has more comprehensive functions, significant repair effects, higher safety, and no risk of side effects. Umbilical cord mesenchymal stem cell exosomes are the core active ingredient, containing multiple growth factors such as EGF and bFGF, and anti-inflammatory factors such as IL-10, which can simultaneously achieve "anti-inflammation + ocular surface repair + lacrimal gland function regulation". Furthermore, umbilical cord mesenchymal stem cell exosomes are derived from umbilical cord tissue and have low immunogenicity (HLA-DR negative rate >98%). Sodium hyaluronate can thicken, moisturize, and prolong the drug's residence time. L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex can scavenge free radicals on the ocular surface, promote collagen synthesis, and enhance corneal epithelial toughness. Panthenol can penetrate into the corneal epithelial layer, promote wound healing, and relieve dryness and discomfort of the ocular surface.

[0025] 2. This application provides a method for preparing eye drops containing exosomes from umbilical cord mesenchymal stem cells. The resulting eye drops have better stability, greater practicality, and a shorter preparation cycle. Exosomes can be extracted from umbilical cord mesenchymal stem cells after culturing to the 3rd-5th generation, with a total cycle of only 7-10 days, which is more than 50% shorter than that of bone marrow mesenchymal stem cells. The exosomes are stable during storage; after 6 months of storage at 4℃, the exosome activity retention rate reaches 92.3%. No ultra-low temperature storage at -80℃ is required, facilitating clinical transportation and use.

[0026] 3. This application provides the application of eye drops containing umbilical cord mesenchymal stem cell exosomes in the preparation of eye drops for treating dry eye syndrome or repairing corneal epithelial damage. The cost is controllable and easy to scale up production. Umbilical cord tissue is widely available, and exosome extraction uses an "ultracentrifugation method," requiring conventional equipment and simple operation, without the need for special reagents. Production costs are 30%-40% lower than those of bone marrow mesenchymal stem cell eye drops, making it suitable for large-scale industrial production and enriching the variety of ophthalmic therapeutic products. Detailed Implementation

[0027] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] Hydroxypropyl-β-cyclodextrin was purchased from Hubei Hongyunlong Biotechnology Co., Ltd.

[0029] Preparation Example 1 Preparation of L-ascorbate palmitate / hydroxypropyl-β-cyclodextrin inclusion complex (AP / HP-β-CD): a1: Hydroxypropyl-β-cyclodextrin (HP-β-CD) was dissolved in ultrapure water at a concentration of 200 mg / mL, and then L-ascorbate palmitate (AP) was added to make the molar ratio of AP to HP-β-CD 1:1. The mixture was stirred at 45℃ and 450 r / min for 24 h to obtain an AP / HP-β-CD solution. a2: The AP / HP-β-CD solution prepared in step a1 was filtered through a 0.45 μm microporous membrane, and the filtrate was freeze-dried to obtain the AP / HP-β-CD inclusion complex.

[0030] Example 1 1. Preparation of solution containing umbilical cord mesenchymal stem cell exosomes (hUC-MSC-Exo): b1: Collect the supernatant of P3-P5 generation umbilical cord mesenchymal stem cells after serum-free culture for 48 hours, centrifuge at 3000×g for 10 minutes to remove cell debris, and then filter through a 0.45μm microporous membrane to remove subcellular structures and large particulate impurities to obtain pretreated supernatant; b2: Select a polyethersulfone (PES) hollow fiber membrane module with a molecular weight cutoff of 100 kDa, set the transmembrane pressure (TMP) to 10-15 psi and the tangential flow rate to 200-300 mL / min, concentrate the pretreatment supernatant obtained in step b1 to 1 / 15 of its original volume to obtain TFF concentrate, and desalt it online with sterile PBS (pH=7.3) as the replacement medium. The volume of the replacement medium is 6.5 times that of the TFF concentrate. Replace until the conductivity of the filtrate is ≤20 μS / cm, and collect the TFF concentrate. b3: Size exclusion chromatography (SEC) column (e.g., Superdex 200Increase 10 / 300GL) was used with sterile PBS as the mobile phase at a flow rate of 0.5 mL / min and a column temperature of 4 °C. The TFF concentrate prepared in step b2 (7.5% of the column volume) was loaded and filtered through a 0.22 μm filter membrane to obtain a solution containing umbilical cord mesenchymal stem cell exosomes (hUC-MSC-Exo). The main peak component of the exosomes was collected based on 280 nm UV detection and NTA signal. NTA verification showed a particle size of 80-150 nm, purity ≥98%, and recovery rate ≥75%.

[0031] 2. Preparation of hUC-MSC-Exo lyophilized powder c1: Take 1 mL of the hUC-MSC-Exo-containing solution prepared by the above method, wherein the concentration of hUC-MSC-Exo is 6 × 10⁻⁶. 10The particles / mL were then added, followed by the addition of a lyophilization protectant consisting of 42.75 mg mannitol, 60 mg trehalose, and 22.5 mg histidine. After gentle mixing, the mixture was incubated at 4°C for 30 minutes to obtain hUC-MSC-Exo containing the lyophilization protectant. c2: The hUC-MSC-Exo sample prepared in step c1 was aliquoted into sterile lyophilization vials and placed on a freeze dryer shelf. The sample was cooled to -1.5°C / min. The sample was frozen completely at 40℃ for 3 hours. Under a vacuum of ≤10Pa, the shelf temperature was gradually increased to -10℃ and maintained for 10 hours to sublimate the ice crystals in the sample. Then, the temperature was further increased to 25℃ and maintained for 5 hours to remove residual moisture. After freeze-drying, the freeze-drying bottle was sealed under vacuum to obtain hUC-MSC-Exo freeze-dried powder. The test showed that the hUC-MSC-Exo particle size distribution was stable after freeze-drying, the purity was ≥95%, and the recovery rate after reconstitution was ≥70%.

[0032] Example 2 Preparation process of eye drops S1: Take 6 mg of sodium hyaluronate, 3 mg of AP / HP-β-CD inclusion complex and 9 mg of panthenol, add 4 mL of deionized sterile water, stir until completely dissolved to prepare the base solution, filter through a 0.22 μm filter membrane for sterilization, and set aside for later use; S2: Take all the hUC-MSC-Exo lyophilized powder prepared in Example 1, reconstitute it with 9 mL of physiological saline, pre-adjust the osmotic pressure of the reconstituted hUC-MSC-Exo solution to 300 mOsm / kg, and under sterile conditions, slowly add the reconstituted and osmotically adjusted hUC-MSC-Exo solution to the base solution prepared in step S1, add physiological saline to make up to 15 mL, stir evenly, and test and finely adjust the osmotic pressure to 300 mOsm / kg and pH to 6.5 to obtain eye drops. Dispense into sterile preservative-free eye drop bottles, 5 mL per bottle, seal and store at 4°C.

[0033] The sodium hyaluronate used in this invention has an average molecular weight of 1.5 × 10⁻⁶. 6 Da.

[0034] Example 3 Example 3 provides a preparation process for eye drops, which differs from Example 2 in that the total mass of AP / HP-β-CD inclusion complex and panthenol remains unchanged, and the mass ratio of AP / HP-β-CD inclusion complex to panthenol is 1:2.

[0035] Example 4 Example 4 provides a preparation process for eye drops, which differs from Example 2 in that the total mass of AP / HP-β-CD inclusion complex and panthenol remains unchanged, and the mass ratio of AP / HP-β-CD inclusion complex to panthenol is 1:5.

[0036] Comparative Example 1 Comparative Example 1 provides a preparation process for eye drops, which differs from Example 2 in that panthenol is replaced by AP / HP-β-CD inclusion complex.

[0037] Comparative Example 2 Comparative Example 2 provides a preparation process for eye drops, which differs from Example 2 in that the AP / HP-β-CD inclusion complex is replaced with panthenol by an equal mass.

[0038] Comparative Example 3 Comparative Example 3 provides a preparation process for eye drops, which differs from Example 2 in that it does not contain sodium hyaluronate.

[0039] Comparative Example 4 Comparative Example 4 provides a preparation process for eye drops, which differs from Example 2 in that it does not contain hUC-MSC-Exo lyophilized powder.

[0040] Comparative Example 5 Comparative Example 5 provides a preparation process for eye drops, which differs from Example 2 in that the AP / HP-β-CD inclusion complex is replaced by an equal mass of L-ascorbic acid palmitate.

[0041] Animal experiments 1. New Zealand rabbit acute eye stimulation test (safety test) Examine both eyes of the experimental animals within 24 hours prior to the start of the experiment. Animals exhibiting eye irritation symptoms, corneal defects, or conjunctival damage should not be used in the experiment. On the day of the experiment, gently pull back the lower eyelid of one eye of the experimental animal and instill 0.1 mL of the eye drops prepared in Example 2 (denoted as the test substance) into the conjunctival sac, passively closing the upper and lower eyelids for 1 second to prevent loss of the test substance. The other eye is left untreated as a self-control. Do not rinse the eyes within 24 hours after instilling the test substance. Examine the animal's eyes at 1, 24, 48, and 72 hours after instilling the test substance. If no irritation reaction occurs after 72 hours, the experiment can be terminated. A magnifying glass, handheld slit lamp, biological microscope, or other available instruments and equipment can be used to examine the eye irritation reaction. After the 24-hour observation and recording are completed, apply sodium fluorescein to the eyes of all animals for further examination. In each inspection, the scores for eye irritation reactions should be recorded according to Section 5.1 of Chapter 6 of the "Cosmetic Safety Technical Specifications" (2015 Edition), Acute Eye Irritation / Corrosion Test, Table 1: Scoring Criteria for Eye Damage. The average score of the irritation reaction of the cornea, iris, or conjunctiva of the animal after administration of the test substance, along with the recovery time, should be used for evaluation. The intensity of eye irritation caused by the test substance should be determined according to Section 5.1 of Chapter 6 of the "Cosmetic Safety Technical Specifications" (2015 Edition), Acute Eye Irritation / Corrosion Test, Table 2: Grading of Raw Material Eye Irritation Reactions. During the observation period, no irritation reactions were observed in any of the three animals. The results are shown in Table 1.

[0042] Table 1. New Zealand rabbit eye stimulation response scores (within 3 days) Note: The mean integral refers to the mean integral of the stimulus response of the cornea, iris, or conjunctiva of the animal at 24, 48, and 72 hours after administration of the test substance.

[0043] Based on Example 2 and Table 1, the eye drops prepared in Example 2 showed a mean irritation response score of 0 for the cornea, iris, or conjunctiva of the animals at 24, 48, and 72 hours after instillation. According to Section 5.1 of Chapter 6 of the "Cosmetic Safety Technical Specifications" (2015 edition), the raw material eye irritation response grading standard for acute eye irritation / corrosiveness tests indicates that the sample is non-irritating.

[0044] 2. Experimental study on the therapeutic effect of dry eye syndrome Establishment of a mouse dry eye model: Mice were placed in a low-humidity environment (humidity <30%) and injected intraperitoneally with 0.2 mg / kg atropine daily for 14 consecutive days to establish a dry eye model. Successful modeling criteria: tear secretion decreased by ≥50% compared to the normal group, and tear film breakup time <5 seconds.

[0045] Grouping and administration: 144 mice were randomly divided into a model group and experimental groups 1-8. The model group was instilled with physiological saline, while experimental groups 1-7 were instilled with the eye drops prepared in Examples 2-4 and Comparative Examples 1-5, respectively. Each group had 16 eyes, and the mice were instilled with 1 μL of the eye drops 3 times a day for 21 consecutive days.

[0046] Testing indicators: Tear secretion: The tear secretion of nine groups of mice was measured using the phenol red cotton thread test on day 21 after modeling. Under a slit lamp, a phenol red cotton thread was placed in the lower conjunctival sac of the mouse in the outer 1 / 3 using microscopic forceps. After 15 seconds, the thread was removed, and the wet length of the cotton thread was observed under a microscope using vernier calipers. The unit is millimeters (mm). Data were recorded after each test, and the results are shown in Table 2.

[0047] Table 2. Tear secretion on day 21 Based on Example 2, the model group, and Table 2, it can be seen that after treatment with the eye drops prepared in Example 2, the tear secretion in Example 2 was significantly better than that in the model group.

[0048] As can be seen from Examples 2-4 and Table 2, adjusting the mass ratio of AP / HP-β-CD inclusion complex to panthenol can affect the therapeutic effect of eye drops on dry eye syndrome. In Example 2, when the mass ratio of AP / HP-β-CD inclusion complex to panthenol was 1:3, the prepared eye drops showed the best tear secretion after treatment.

[0049] Based on Example 2, Comparative Examples 1-2 and Table 2, it can be seen that in Comparative Examples 1-2, after panthenol and AP / HP-β-CD inclusion complex were replaced by each other in equal mass, the eye drops prepared were less effective in treating dry eye syndrome than those in Example 2.

[0050] Based on Example 2, Comparative Example 3 and Table 2, it can be seen that the eye drops prepared in Comparative Example 3 do not contain sodium hyaluronate and have a significantly worse therapeutic effect on dry eye in mice than those in Example 2.

[0051] Based on Example 2, Comparative Example 4 and Table 2, it can be seen that the eye drops prepared in Comparative Example 4 do not contain hUC-MSC-Exo lyophilized powder, and their therapeutic effect on dry eye syndrome in mice is significantly worse than that in Example 2.

[0052] Based on Example 2, Comparative Example 5, and Table 2, it can be seen that the eye drops prepared by replacing the AP / HP-β-CD inclusion complex with L-ascorbate palmitate by the same mass in Comparative Example 5 showed a worse therapeutic effect on dry eye in mice than those in Example 2. This may be because L-ascorbate palmitate, due to its oil-soluble properties, easily forms droplets on the corneal surface, resulting in low penetration efficiency; while the AP / HP-β-CD inclusion complex has better water solubility, stability, and penetration, achieving highly efficient synergistic antioxidant and corneal repair effects with panthenol.

[0053] Tear film breakup time (BUT): Tear film stability was assessed based on BUT, measured on day 21 after modeling. 1 μL of 1% sodium fluorescein was instilled into the conjunctival sac of mice, and after 10 seconds, the mice blinked three times. The blinks were observed under cobalt blue slit light. The time from the last blink to the appearance of the first dark spot was defined as the tear film breakup time (in seconds). Screening criteria: BUT (2.5 ± 0.5 s) was repeated three times per eye, and the average value was taken. The results are shown in Table 3.

[0054] Table 3. Tear film breakup on day 21 Based on Example 2, the model group, and Table 3, it can be seen that after treatment with the eye drops prepared in Example 2, the tear film breakup time of Example 2 was significantly prolonged compared with that of the model group.

[0055] As can be seen from Examples 2-4 and Table 2, adjusting the mass ratio of AP / HP-β-CD inclusion complex to panthenol can affect the therapeutic effect of eye drops on dry eye syndrome. In Example 2, when the mass ratio of AP / HP-β-CD inclusion complex to panthenol was 1:3, the tear film breakup time of the prepared eye drops was prolonged compared with that in Examples 3 and 4.

[0056] Based on Example 2, Comparative Examples 1-2 and Table 3, it can be seen that in Comparative Examples 1-2, after panthenol and AP / HP-β-CD inclusion complex were replaced by each other in equal mass, the tear film breakup time was shorter than that in Example 2, indicating that the therapeutic effect of the eye drops prepared in Comparative Examples 1-2 was worse than that in Example 2.

[0057] Based on Example 2, Comparative Example 3 and Table 3, it can be seen that the eye drops prepared in Comparative Example 3 do not contain sodium hyaluronate and the tear film breakup time is significantly shorter than that in Example 2. This indicates that the eye drops prepared in Comparative Example 3 have a significantly worse therapeutic effect on dry eye in mice than those in Example 2.

[0058] Based on Example 2, Comparative Example 4 and Table 3, it can be seen that the eye drops prepared in Comparative Example 4 do not contain hUC-MSC-Exo lyophilized powder, and the tear film breakup time is significantly shorter than that in Example 2. This indicates that the eye drops prepared in Comparative Example 4 have a significantly worse therapeutic effect on dry eye syndrome in mice than those in Example 2.

[0059] Based on Example 2, Comparative Example 5 and Table 3, it can be seen that in Comparative Example 5, replacing the AP / HP-β-CD inclusion complex with L-ascorbic acid palmitate by the same mass resulted in eye drops with a shorter tear film breakup time than in Example 2, and a worse therapeutic effect on dry eye in mice than in Example 2.

[0060] Corneal epithelial integrity: Measurements were performed on day 21 post-modeling. 1 μL of liquid fluorescein sodium (10 mg / mL) was dropped into the conjunctival sac, and the mice were assisted to blink several times. Corneal epithelial integrity and fluorescein sodium staining were observed under a slit-lamp microscope with blue light. The percentage of positively stained corneal area was calculated, and the results are shown in Table 4.

[0061] Table 4. Corneal epithelial integrity on day 21 Based on Example 2, the model group, and Table 4, it can be seen that after treatment with the eye drops prepared in Example 2, the corneal staining area of ​​Example 2 was significantly lower than that of the model group, indicating that the eye drops prepared in Example 2 can effectively improve dry eye syndrome.

[0062] As can be seen from Examples 2-4 and Table 2, adjusting the mass ratio of AP / HP-β-CD inclusion complex to panthenol can affect the therapeutic effect of eye drops on dry eye syndrome. In Example 2, when the mass ratio of AP / HP-β-CD inclusion complex to panthenol was 1:3, the corneal staining area after treatment with the prepared eye drops was lower than that in Examples 3 and 4.

[0063] Based on Example 2, Comparative Examples 1-2 and Table 4, it can be seen that in Comparative Examples 1-2, after panthenol and AP / HP-β-CD inclusion complex were replaced by each other by equal mass, the corneal staining area was higher than that in Example 2. This indicates that the eye drops prepared in Comparative Examples 1-2 were less effective in treating dry eye in mice than those in Example 2.

[0064] Based on Example 2, Comparative Example 3 and Table 4, it can be seen that the eye drops prepared in Comparative Example 3 do not contain sodium hyaluronate, and the corneal staining area is significantly higher than that in Example 2. This indicates that the eye drops prepared in Comparative Example 3 have a significantly worse therapeutic effect on dry eye in mice than those in Example 2.

[0065] Based on Example 2, Comparative Example 4 and Table 4, it can be seen that the eye drops prepared in Comparative Example 4 do not contain hUC-MSC-Exo lyophilized powder, and the corneal staining area is significantly higher than that in Example 2. This indicates that the eye drops prepared in Comparative Example 4 have a significantly worse therapeutic effect on dry eye in mice than those in Example 2.

[0066] Based on Example 2, Comparative Example 5 and Table 4, it can be seen that in Comparative Example 5, replacing the AP / HP-β-CD inclusion complex with L-ascorbic acid palmitate by the same mass resulted in an eye drop preparation with a higher corneal staining area after treatment than in Example 2, and a worse therapeutic effect on dry eye in mice than in Example 2.

[0067] 3. Corneal epithelial damage repair experiment Establishment of a mouse corneal epithelial injury model: A superficial epithelial defect was formed in the center of the mouse cornea by gently scratching with a 1 mm diameter corneal trephine. Fluorescein staining confirmed that the damage area was consistent.

[0068] Grouping and administration: 96 mice were randomly divided into a model group and experimental groups 1-5. The model group was instilled with physiological saline, while experimental groups 1-5 were instilled with the eye drops prepared in Example 2 and Comparative Examples 1-4, respectively. Each group had 16 eyes, and the mice were instilled with 1 μL of the eye drops 3 times a day.

[0069] Detection indicators: At 12h, 24h, 48h, and 72h postoperatively, 1μL of liquid fluorescein sodium (10mg / mL) was instilled into the conjunctival sac, and the staining of the corneal epithelium with fluorescein sodium was observed under blue light using a slit-lamp microscope. The healing rate of the corneal defect area was calculated, and the results are shown in Table 5.

[0070] Table 5. Corneal epithelial damage repair status Based on Example 2, the model group, and Table 5, it can be seen that after treatment with the eye drops prepared in Example 2, the corneal defect area healing rate of Example 2 was significantly higher than that of the model group, and the complete healing time was shorter than that of the model group, indicating that the eye drops prepared in Example 2 can effectively accelerate corneal epithelial repair.

[0071] Based on Example 2, Comparative Examples 1-2 and Table 5, it can be seen that in Comparative Examples 1-2, after panthenol and AP / HP-β-CD inclusion complex were replaced by each other in equal mass, the corneal defect area healing rate was lower than that in Example 2, and the time to complete healing was longer than that in Example 2. This indicates that the eye drops prepared in Comparative Examples 1-2 were less efficient in corneal epithelial repair than those in Example 2.

[0072] Based on Example 2, Comparative Example 3 and Table 5, it can be seen that the eye drops prepared in Comparative Example 3 do not contain sodium hyaluronate, and the corneal defect area healing rate is significantly lower than that in Example 2. This indicates that the eye drops prepared in Comparative Example 3 are significantly less efficient at repairing corneal epithelium than those in Example 2.

[0073] Based on Example 2, Comparative Example 4 and Table 5, it can be seen that the eye drops prepared in Comparative Example 4 do not contain hUC-MSC-Exo lyophilized powder, and the corneal defect area healing rate is significantly lower than that in Example 2. This indicates that the eye drops prepared in Comparative Example 4 are significantly less efficient at repairing corneal epithelium than those in Example 2.

[0074] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made to the products, methods and principles of this application should be covered within the scope of protection of this application.

Claims

1. An eye drop containing umbilical cord mesenchymal stem cell exosome, characterized by, The raw materials include sodium hyaluronate, L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound, panthenol, umbilical cord mesenchymal stem cell exosome-containing solution, water and physiological saline; The concentration of the sodium hyaluronate is 0.2-0.6 mg / mL; The concentration of the L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound is 0.1-0.4 mg / mL; The concentration of the panthenol is 0.2-1.0 mg / mL; The concentration of the umbilical cord mesenchymal stem cell exosome is 0.2 x 10 10 -0.6 x 10 10 particles / mL.

2. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 1, characterized in that, The mass ratio of the L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound to the panthenol is 1:(2-5).

3. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 1, characterized in that, The preparation of the L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound includes the following steps: a1: dissolving hydroxypropyl-β-cyclodextrin in water, then adding L-ascorbic acid palmitate, stirring to obtain an L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin solution; a2: filtering and drying the L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin solution prepared in step a1 to obtain an L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound.

4. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 1, characterized in that, The umbilical cord mesenchymal stem cell exosome is added to the eye drops in the form of umbilical cord mesenchymal stem cell exosome lyophilized powder, and the preparation method of the umbilical cord mesenchymal stem cell exosome lyophilized powder includes the following preparation steps: b1: collecting the serum-free culture supernatant of P3-P5 generation umbilical cord mesenchymal stem cells, centrifuging, filtering to obtain a pretreated supernatant; b2: concentrating the pretreated supernatant prepared in step b1 to 1 / 10-1 / 20 of the original volume to obtain a TFF concentrate, replacing the TFF concentrate with a replacement liquid, the volume of the replacement liquid being 5-8 times that of the TFF concentrate, and collecting the TFF concentrate; b3: using a size exclusion chromatography column, loading the TFF concentrate prepared and collected in step b2, the loading volume being 5%-10% of the column volume, filtering to obtain an umbilical cord mesenchymal stem cell exosome-containing solution; b4: adding a lyophilization protective agent to the umbilical cord mesenchymal stem cell exosome-containing solution prepared in step b3, mixing, incubating, lyophilizing to obtain umbilical cord mesenchymal stem cell exosome lyophilized powder.

5. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 4, characterized by, The replacement liquid in step b2 is sterile PBS, and the pH value is 7.2-7.

4.

6. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 4, characterized in that, The mobile phase of the chromatography column in step b3 is sterile PBS, the flow rate is 0.4-0.6 mL / min, and the column temperature is 3-5℃.

7. The eye drop comprising umbilical cord mesenchymal stem cell exosome according to claim 4, characterized in that, In step b4, the lyophilization protective agent includes mannitol, trehalose and histidine; The mass / volume ratio of the mannitol to the umbilical cord mesenchymal stem cell exosome-containing solution prepared in step b3 is 28.5-57 mg / mL; the mass / volume ratio of the trehalose to the umbilical cord mesenchymal stem cell exosome-containing solution prepared in step b3 is 30-90 mg / mL; and the mass / volume ratio of the histidine to the umbilical cord mesenchymal stem cell exosome-containing solution prepared in step b3 is 1.5-3 mg / mL.

8. A method of preparing the eye drop containing umbilical cord mesenchymal stem cell exosome according to any one of claims 1 to 7, characterized by, The method includes the following steps: S1: Take sodium hyaluronate, L-ascorbic acid palmitate / hydroxypropyl-β-cyclodextrin inclusion compound and panthenol, add water, stir, filter sterilization, get the base liquid; S2: The umbilical cord mesenchymal stem cell exosome freeze-dried powder is reconstituted with normal saline, the osmotic pressure is adjusted, and the reconstituted and adjusted umbilical cord mesenchymal stem cell exosome solution is added to the base liquid prepared in step S1, stirred, adjusted to the osmotic pressure and pH, to obtain eye drops.

9. The method of claim 8, wherein the umbilical cord mesenchymal stem cell exosome-containing eye drop is prepared by the steps of: In the step S2, the osmotic pressure is adjusted to 260-340 mOsm / kg.

10. Use of the umbilical cord mesenchymal stem cell exosome-containing eye drops of any one of claims 1-7 in the preparation of eye drops for treating dry eye or repairing corneal epithelial damage.

Images