An eye drop containing multiple trace elements and stem cell extract, its preparation method and application

Abstract

This invention relates to the field of biomedical technology, specifically to an eye drop containing multiple trace elements and stem cell extract, its preparation method, and its application. The eye drop, by weight, comprises the following components: 0.5-1.5 parts mesenchymal stem cell supernatant, 0.05-0.15 parts mesenchymal stem cell extract, and 5-15 parts sodium hyaluronate solution; the mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements. This invention has the advantages of high tear secretion, high tear film stability, and good corneal damage repair effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an eye drop containing multiple trace elements and stem cell extracts, its preparation method, and its application. Background Technology

[0002] Common symptoms of dry eye syndrome include dryness, redness, pain, foreign body sensation, blurred vision, photophobia, and tearing. Among various eye diseases, dry eye syndrome is a common condition that seriously affects patients' quality of life. Currently, traditional eye drops for dry eye syndrome often contain various antibiotics and chemical drugs, or are simply single-medium aqueous solutions that only serve a moisturizing function. Their effects on repairing corneal and conjunctival damage and restoring tear film stability are not significant. Therefore, there is an urgent need for a new type of eye drop to solve these problems.

[0003] Trace elements play a vital role in eye development. For example, Zn, Se, and Cu are crucial for retinal health, participating in the formation and maintenance of photoreceptor cells and helping to protect the retina from oxidative and photodamage. Ga and Mg are involved in nerve conduction, playing a vital role in visual transmission and information processing, and helping to maintain normal nerve conduction function in the eye. Cu, Zn, and Fe are important for the health of ocular blood vessels, helping to maintain their elasticity and stability and helping to prevent ocular vascular diseases.

[0004] Sodium hyaluronate is a mucopolysaccharide widely found in the intercellular matrix of animal tissues. It is currently the best medium in ophthalmic preparations, as it can increase drug bioavailability, reduce drug irritation to the eyes, decrease adverse reactions of preservatives in preparations, promote the healing of corneal damage, and quickly relieve eye discomfort symptoms.

[0005] The prior art (Huang Shouqiang. Study on the efficacy of human umbilical cord mesenchymal stem cells in the treatment of dry eye syndrome in mice [D]. Anhui Medical University, 2016.) discloses the transplantation of human umbilical cord mesenchymal stem cells into mice with dry eye disease through three transplantation treatment methods: tail vein injection, lacrimal gland injection and eye drops.

[0006] Chinese Patent Publication No. CN111658607A discloses a sustained-release eye drop for umbilical cord mesenchymal stem cells, comprising separately packaged powder A and separately packaged solvent B. Each part of the sustained-release eye drop for umbilical cord mesenchymal stem cells includes 0.1mg-0.5mg of powder A and 10g of solvent B. Powder A is a dried powder of umbilical cord mesenchymal stem cell culture supernatant. By weight, solvent B includes 0.2-0.6 parts of sodium hyaluronate, 0.15-0.5 parts of taurine, 0.1-0.3 parts of sodium alginate, and 98.6-99.55 parts of sterile water.

[0007] However, the aforementioned eye drops containing umbilical cord mesenchymal stem cells or umbilical cord mesenchymal stem cell supernatant and sodium hyaluronate can only replenish tear volume in the short term and temporarily alleviate corneal epithelial damage. They cannot repair the eyes or improve tear film stability.

[0008] Therefore, it is essential to develop an eye drop containing multiple trace elements and stem cell extracts that can solve the above-mentioned technical problems, as well as its preparation method and application. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide an eye drop containing multiple trace elements and stem cell extracts, which has high tear secretion, high tear film stability, and good corneal damage repair effect, as well as its preparation method and application.

[0010] This invention is achieved through the following technical solutions:

[0011] The first aspect of the present invention provides an eye drop containing multiple trace elements and stem cell extract, comprising the following components by weight: 0.5-1.5 parts of mesenchymal stem cell supernatant, 0.05-0.15 parts of mesenchymal stem cell extract and 5-15 parts of sodium hyaluronate solution;

[0012] The mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements.

[0013] As one embodiment of the present invention, the mesenchymal stem cells include one or more of the following: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, skin mesenchymal stem cells, dental pulp mesenchymal stem cells, urinary mesenchymal stem cells, and mesenchymal stem cells derived from induced pluripotent stem cells.

[0014] In one embodiment of the present invention, the multi-element trace elements include Al, Mg, Ba, Mn, Ca, Mo, Cd, Ni, Co, Se, Cr, Sr, Cu, Ti, Fe, V and Zn.

[0015] Preferably, the specific composition of the multi-element trace elements is as follows: Al content 500-5000 mg / L, Mg content 500-5000 mg / L, Ba content 0.01-0.1 mg / L, Mn content 50-500 mg / L, Ca content 50-500 mg / L, Mo content 1-10 mg / L, Cd content 0.001-0.01 mg / L, Ni content 1-10 mg / L, Co content 0.5-5 mg / L, Se content 10-100 mg / L, Cr content 1-10 mg / L, Sr content 0.5-5 mg / L, Cu content 0.5-5 mg / L, Ti content 50-500 mg / L, Fe content 500-5000 mg / L, V content 1-10 mg / L, and Zn content 100-1000 mg / L.

[0016] In one embodiment of the present invention, the mesenchymal stem cell supernatant is obtained by centrifugation and filtration after adding multiple trace elements to the mesenchymal stem cell culture system.

[0017] In one embodiment of the present invention, the mesenchymal stem cell extract is: mesenchymal stem cells obtained by adding multiple trace elements to a mesenchymal stem cell culture system, and then extracting the mesenchymal stem cells by ultrasonic lysis extraction.

[0018] This invention adds a certain amount of multiple trace elements to the mesenchymal stem cell culture system, which can promote cell proliferation, maintain youthful cell morphology, and enhance cell adhesion. After being taken up by cells, the multiple trace elements can exist in the cell matrix and various organelles. At the same time, they can be encapsulated during intracellular vesicle formation and then excreted into the cell culture medium to form exosomes loaded with multiple trace elements. These exosomes can then cross the membrane to deliver the loaded multiple trace elements into the target cells to exert their effects.

[0019] The supernatant of mesenchymal stem cells is rich in various cytokines secreted by the cells. The cell extract obtained after cell lysis also contains various cytokines, active proteases, mitochondria, and other components. All of these components play a positive role in repairing eye tissue damage and promoting optic nerve development.

[0020] Preferably, the ultrasonic lysis extraction method specifically includes the following steps: centrifuging and washing the obtained mesenchymal stem cells with physiological saline, resuspending them in the supernatant of the mesenchymal stem cells, ultrasonically lysing them, and filtering them to obtain the final product.

[0021] The present invention preferably uses mesenchymal stem cell supernatant to resuspend mesenchymal stem cells. As an alternative, physiological saline, 1‰ sodium hyaluronate solution, or water for injection can also be used for resuscitation.

[0022] More preferably, the density of the mesenchymal stem cell supernatant resuspended is 1×10⁻⁶. 6 -1×10 7 / mL; the ultrasonic lysis time is 20-60s, wherein each lysis lasts 1-3s and the interval is 3-9s; the filtration is first filtered with a 2000-mesh sterile filter and then filtered with a 0.22μm sterile filter membrane.

[0023] Preferably, the preparation process of the mesenchymal stem cell culture system is as follows: stem cells are passaged to P3 using serum-free stem cell culture medium, seed cells are cryopreserved, P3 seed cells are thawed in batches using serum-free stem cell culture medium, multiple trace elements are added during the thaw process, and the cells are passaged to P5-P8 to obtain the final product.

[0024] Preferably, the addition ratio of each multi-element micronutrient should be screened through a cell proliferation experiment, as follows:

[0025] The multi-element stock solution was diluted with serum-free stem cell culture medium at dilution ratios of 1000, 1500, 2000, 2500, and 3000. Fourth-generation umbilical cord stem cells were resuscitated, and when cell confluence reached approximately 80%, the cells were digested and counted. The cells were divided into six equal portions, centrifuged, and resuspended in the culture medium supplemented with the five groups of multi-element solutions and the control group (without added trace elements), respectively, with the density adjusted to 15000 / mL. 200 μL of the resuspended umbilical cord stem cells from each of the six groups were added to each well of a 96-well plate. Cell proliferation was assessed using the MTT assay when confluence reached approximately 80%. MTT assay procedure: 10 μL of 5 mg / mL MTT solvent (thiazolyl blue) was added to each well of a 96-well plate under light-protected conditions. The plate was incubated at 37°C for 4 hours, after which the culture medium was discarded. Add 200 μL of DMSO to each well, shake thoroughly to mix, and after complete color development (20-30 min), measure the absorbance (OD) at 490 nm using a microplate reader, and calculate the cell proliferation rate. Cell proliferation (relative activity) rate (%) = (OD value of trace element group / OD value of control group) × 100%. Based on the experimental results, select a multi-element dilution factor X where the cell proliferation (relative activity) rate is >1, especially the optimal cell proliferation rate. max (The group with the highest cell proliferation rate). Under normal circumstances, the cell proliferation (relative activity) rate is >1, and the dilution factor is 2000-3000 times.

[0026] In the preparation of the mesenchymal stem cell culture system, the present invention does not have special requirements for serum-free stem cell culture medium, and commercially available serum-free mesenchymal stem cell culture medium can meet the requirements of the present invention.

[0027] More preferably, the last generation is cultured using an in vitro 3D culture system that simulates the in vivo microenvironment.

[0028] Depending on the production scale and cell quantity, the cells need to be passaged to P5, P6, P7, or P8. The appropriate passage number is selected based on the production scale and cell quantity. Within this range, the passage number has virtually no impact on the efficacy of the eye drops. Only in the last generation is an in vitro 3D culture system that simulates the in vivo microenvironment used to maximize the harvest of cells and supernatant.

[0029] In this invention, the multi-element trace elements are continuously added in each generation after revival.

[0030] More preferably, the stem cells are extracted from tissues such as the umbilical cord of a newborn, placenta, amnion, or abdominal fat, skin, and dental pulp of a healthy adult.

[0031] More preferably, during the resuscitation process, the proportion of multiple trace elements added to the serum-free stem cell culture medium is X. max :1 (refers to the dilution factor of a multi-element trace element, i.e., X) max (mL of serum-free stem cell culture medium contains 1mL of multi-element trace elements).

[0032] In one embodiment of the present invention, the concentration of the sodium hyaluronate solution is 0.05-0.5 wt%.

[0033] Preferably, the preparation process of the sodium hyaluronate solution is as follows: weigh 0.5-5g of sodium hyaluronate, measure 1000ml of physiological saline to completely dissolve the sodium hyaluronate, and filter it through a 0.22μm sterile filter membrane to obtain the solution.

[0034] In one embodiment of the present invention, the eye drops further include at least one of a pH adjuster and an osmotic pressure adjuster.

[0035] Preferably, the amount of the pH adjuster is 0.01-0.5 parts, and the amount of the osmotic pressure adjuster is 0.01-0.5 parts.

[0036] Preferably, the pH adjuster includes one or more of the following: sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, and phosphoric acid.

[0037] Preferably, the osmotic pressure regulator comprises one or more of sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, and glucose.

[0038] As one embodiment of the present invention, the eye drops do not contain preservatives.

[0039] A second aspect of the present invention provides a method for preparing the above-mentioned eye drops, comprising the following steps: mixing the mesenchymal stem cell supernatant, mesenchymal stem cell extract, and sodium hyaluronate solution evenly, and adding a pH adjuster and an osmotic pressure adjuster as needed to obtain the eye drops.

[0040] Preferably, the eye drops are stored at 2-8°C.

[0041] A third aspect of the present invention provides the use of the above-described eye drops or the eye drops prepared by the above-described preparation method in the preparation of a medicament for treating dry eye syndrome.

[0042] The beneficial effects of this invention are:

[0043] (1) By adding a certain proportion of multi-element trace elements, the present invention can promote the proliferation of mesenchymal stem cells, thereby harvesting more stem cells.

[0044] (2) The eye drops of the present invention contain a variety of anti-inflammatory factors and growth factors secreted by mesenchymal stem cells, which can relieve ocular inflammation and promote the repair of ocular tissue damage. In addition, the eye drops contain a variety of trace elements, some of which are in a free state and can directly act on various tissues of the ocular surface, while others exist in the cell matrix components and are encapsulated in exosomes, and are delivered to deeper ocular tissues to promote local metabolism and maintain normal biological functions, such as promoting the proliferation of lacrimal gland cells and promoting tear secretion.

[0045] (3) The present invention simultaneously adds mesenchymal stem cell supernatant containing multiple trace elements and mesenchymal stem cell extract containing multiple trace elements. Compared with adding only one of them, the two have a significant synergistic effect in increasing tear secretion, improving tear film stability and improving corneal damage repair.

[0046] (4) In the process of stem cell culture, the present invention adds multiple trace elements. Compared with the direct addition of multiple trace elements, some trace elements that are not easily absorbed by the human body can exist in the cell matrix components and are encapsulated in exosomes, which are more conducive to absorption, thereby improving tear secretion, tear film stability and corneal damage repair effect.

[0047] (5) The present invention simultaneously adds mesenchymal stem cell supernatant, mesenchymal stem cell extract and multiple trace elements. Compared with adding only mesenchymal stem cell supernatant and mesenchymal stem cell extract, or adding only multiple trace elements, the three have a significant synergistic effect in increasing tear secretion, improving tear film stability and improving corneal damage repair. Attached Figure Description

[0048] Figure 1This is a diagram showing the growth status of umbilical cord mesenchymal stem cells in Example 1 of the present invention; (a) in the diagram represents seed cells of generation P3, and (b) represents stem cells cultured in a 3D system of generation P6.

[0049] Figure 2 This is a graph showing the effect of different proportions of various trace elements added on the proliferation rate of umbilical cord mesenchymal stem cells in Example 1 of the present invention. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention. Unless otherwise specified, all reagents used in the embodiments of the present invention are obtained through conventional commercial means.

[0051] The specific composition of the multi-element trace elements used in the various embodiments of the present invention is as follows: Al content is 1460 mg / L, Mg content is 1790 mg / L, Ba content is 0.032 mg / L, Mn content is 154 mg / L, Ca content is 85.2 mg / L, Mo content is 3.9 mg / L, Cd content is 0.0074 mg / L, Ni content is 4.25 mg / L, Co content is 1.39 mg / L, Se content is 21.1 mg / L, Cr content is 6.87 mg / L, Sr content is 0.89 mg / L, Cu content is 0.82 mg / L, Ti content is 104 mg / L, Fe content is 1920 mg / L, V content is 6.28 mg / L, and Zn content is 606 mg / L.

[0052] Example 1

[0053] An eye drop containing multiple trace elements and stem cell extract comprises the following components, by weight: 1 part of umbilical cord stem cell supernatant containing multiple trace elements, 0.1 part of umbilical cord stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution.

[0054] The preparation method of umbilical cord stem cell supernatant containing multiple trace elements is as follows: stem cells are extracted from neonatal umbilical cord tissue and passaged in serum-free stem cell culture medium (prepared by mixing Gibco's MEM Alpha medium and EliteCell's EliteGro serum substitute at a volume ratio of 20:1) at a cell seeding density of 10,000 cells / cm³. 2The cells were cultured to passage P3, cryopreserved, and then thawed in batches to passage P6. During the thawing of P3 stem cells, a multi-element micronutrient was added to the culture medium at a ratio of 2500:1 (dilution factor of the multi-element micronutrient). The supernatant and umbilical cord stem cells containing the multi-element micronutrient were harvested. The supernatant was centrifuged and filtered through a 0.45 μm filter to obtain the umbilical cord stem cell supernatant containing the multi-element micronutrient.

[0055] The preparation method of umbilical cord stem cell extract containing multiple trace elements is as follows: umbilical cord stem cells are washed three times by centrifugation with physiological saline, counted, and the harvested umbilical cord stem cell supernatant containing multiple trace elements is centrifuged at 5×10⁻⁶. 6 The solution was resuspended at a density of 1 / mL, sonicated for 40 seconds (2 seconds per cycle, 6 seconds interval), passed through a 2000-mesh multilayer sterile filter, and then through a 0.22μm sterile filter membrane to obtain an umbilical cord stem cell extract containing multiple trace elements.

[0056] The growth status diagram of umbilical cord mesenchymal stem cells in this embodiment is shown below. Figure 1 As shown. This embodiment also investigated the effects of different proportions of added multi-element trace elements on the proliferation rate of umbilical cord mesenchymal stem cells, such as... Figure 2 As shown in the figure. Among them, the "* control" group indicates that no trace elements were added.

[0057] The preparation method of sodium hyaluronate solution is as follows: Weigh 1g of sodium hyaluronate, measure 1000ml of physiological saline to completely dissolve the sodium hyaluronate, the solution has uniform density and no lumps, and filter it through a 0.22μm sterile filter membrane.

[0058] The preparation method of the eye drops includes the following steps: mixing the supernatant of umbilical cord stem cells containing multiple trace elements, the extract of umbilical cord stem cells containing multiple trace elements, and the sodium hyaluronate solution evenly to obtain the eye drops.

[0059] Example 2

[0060] The difference between Example 2 and Example 1 lies in the different proportions of the components. In this example, the following components are included by weight: 0.5 parts of umbilical cord stem cell supernatant containing multiple trace elements, 0.05 parts of umbilical cord stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as in Example 1.

[0061] Example 3

[0062] The difference between Example 3 and Example 1 lies in the different proportions of the components. In this example, the following components are included by weight: 1.5 parts of umbilical cord stem cell supernatant containing multiple trace elements, 0.15 parts of umbilical cord stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as in Example 1.

[0063] Example 4

[0064] An eye drop containing multiple trace elements and stem cell extract comprises the following components, by weight: 1 part of adipose stem cell supernatant containing multiple trace elements, 0.1 part of adipose stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution.

[0065] The preparation method of adipose-derived stem cell supernatant containing multiple trace elements is as follows: stem cells are extracted from abdominal adipose tissue of healthy adults and passaged in serum-free stem cell culture medium (prepared by mixing Gibco's MEM Alpha medium and EliteCell's EliteGro serum substitute at a volume ratio of 20:1) at a cell seeding density of 10,000 cells / cm³. 2 The cells were cultured to passage P3, cryopreserved, and then thawed in batches to passage P6. During the thawing of P3 stem cells, a multi-element micronutrient was added to the culture medium at a ratio of 2500:1 (dilution factor of the multi-element micronutrient). The supernatant and adipose-derived stem cells containing the multi-element micronutrient were harvested. The supernatant was centrifuged and filtered through a 0.45 μm filter to obtain the adipose-derived stem cell supernatant containing the multi-element micronutrient.

[0066] The preparation method of adipose-derived stem cell extract containing multiple trace elements is as follows: Adipose-derived stem cells are washed three times by centrifugation with physiological saline, counted, and the harvested adipose-derived stem cell supernatant containing multiple trace elements is centrifuged at 5×10⁻⁶. 6 The solution was resuspended at a density of 1 / mL, sonicated for 40 seconds (2 seconds per cycle, 6 seconds interval), passed through a 2000-mesh multilayer sterile filter, and then through a 0.22μm sterile filter membrane to obtain an adipose-derived stem cell extract containing multiple trace elements.

[0067] The preparation method of sodium hyaluronate solution is as follows: Weigh 1g of sodium hyaluronate, measure 1000ml of physiological saline to completely dissolve the sodium hyaluronate, the solution has uniform density and no lumps, and filter it through a 0.22μm sterile filter membrane.

[0068] The preparation method of the eye drops includes the following steps: mixing the supernatant of adipose stem cells containing multiple trace elements, the extract of adipose stem cells containing multiple trace elements, and the sodium hyaluronate solution evenly to obtain the eye drops.

[0069] Example 5

[0070] The difference between Example 5 and Example 4 lies in the different proportions of the components. In this example, the following components are included by weight: 0.5 parts of adipose-derived stem cell supernatant containing multiple trace elements, 0.05 parts of adipose-derived stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as in Example 4.

[0071] Example 6

[0072] The difference between Example 6 and Example 4 lies in the different proportions of the components. In this example, the following components are included by weight: 1.5 parts of adipose-derived stem cell supernatant containing multiple trace elements, 0.15 parts of adipose-derived stem cell extract containing multiple trace elements, and 10 parts of sodium hyaluronate solution. The rest is the same as in Example 4.

[0073] Example 7

[0074] The difference between Example 7 and Example 1 lies in the content of the added trace elements. Specifically, the content of Al is 500 mg / L, Mg is 500 mg / L, Ba is 0.01 mg / L, Mn is 50 mg / L, Ca is 50 mg / L, Mo is 1 mg / L, Cd is 0.001 mg / L, Ni is 1 mg / L, Co is 0.5 mg / L, Se is 10 mg / L, Cr is 1 mg / L, Sr is 0.5 mg / L, Cu is 0.5 mg / L, Ti is 50 mg / L, Fe is 500 mg / L, V is 1 mg / L, and Zn is 100 mg / L.

[0075] Example 8

[0076] The difference between Example 8 and Example 1 lies in the content of the added trace elements. Specifically, the content of Al is 5000 mg / L, Mg is 5000 mg / L, Ba is 0.1 mg / L, Mn is 500 mg / L, Ca is 500 mg / L, Mo is 10 mg / L, Cd is 0.01 mg / L, Ni is 10 mg / L, Co is 5 mg / L, Se is 100 mg / L, Cr is 10 mg / L, Sr is 5 mg / L, Cu is 5 mg / L, Ti is 500 mg / L, Fe is 5000 mg / L, V is 10 mg / L, and Zn is 1000 mg / L.

[0077] Comparative Example 1

[0078] Based on Example 1, the addition of multiple trace elements was omitted during the resuscitation and culture of P3 generation stem cells.

[0079] Comparative Example 2

[0080] Based on Example 4, the addition of multiple trace elements was removed during the resuscitation and culture of P3 generation stem cells.

[0081] Comparative Example 3

[0082] Based on Examples 1 and 4, the eye drop formula omits the addition of stem cell supernatant and stem cell extract, and only contains sodium hyaluronate solution.

[0083] Comparative Example 4

[0084] Based on Example 1, the umbilical cord stem cell supernatant containing multiple trace elements was adjusted to 1.1 parts, and no umbilical cord stem cell extract containing multiple trace elements was added.

[0085] Comparative Example 5

[0086] Based on Example 1, the umbilical cord stem cell supernatant containing multiple trace elements was not added, and the umbilical cord stem cell extract containing multiple trace elements was adjusted to 1.1 parts.

[0087] Comparative Example 6

[0088] Based on Example 1, the multi-element trace elements were not added during the P3 generation stem cell resuscitation culture, but were added directly during the preparation of the eye drops. The specific steps are as follows:

[0089] The mesenchymal stem cell supernatant, mesenchymal stem cell extract, sodium hyaluronate solution, and various trace elements were mixed evenly.

[0090] Comparative Example 7

[0091] Based on Examples 1 and 4, the addition of stem cell supernatant and stem cell extract was omitted from the formulation. Instead, multiple trace elements were directly added to the sodium hyaluronate solution to obtain the eye drops. The specific steps are as follows:

[0092] Weigh 1g of sodium hyaluronate, dissolve it completely in 1000ml of physiological saline, and filter through a 0.22μm sterile membrane to obtain a 1‰ sodium hyaluronate solution. Dilute the multi-element trace elements 2500 times with the 1‰ sodium hyaluronate solution to obtain the eye drops.

[0093] Experimental Example

[0094] I. The protein concentration, osmotic pressure, and pH of the eye drops prepared in Examples 1-8 and Comparative Examples 1-7 were measured respectively. The specific results are shown in Table 1.

[0095] II. Irritation tests were conducted on the eye drops prepared in Examples 1-8 and Comparative Examples 1-7 respectively for comparison:

[0096] Experimental Methods: BALB / c mice were used to establish a dry eye model by topical application of benzalkonium chloride solution. Animals were randomly divided into a control group and an experimental group. Moderate to severe dry eye models were established by applying 2 g / L benzalkonium chloride solution to both eyes twice daily for two weeks. Treatment began after successful model establishment. The control group received no treatment. The experimental group received eye drops as described in Examples 1-8 and Comparative Examples 1-7, with 20 μL added to each eye three times daily for seven days. Changes before and after drug intervention were recorded, including observing and recording tear film function-related indicators before treatment and on days 1, 4, and 7 after treatment.

[0097] Schirmer I test (SIT): A phenol red cotton thread is placed at the outer third of the right canthus of the mouse eye, and the duration is 60 seconds. The length of the red portion of the phenol red cotton thread is measured using calipers, and the amount of tear secretion is calculated. Care should be taken to control variables during each test; the test should be performed by the same person at the same time, location, lighting conditions, humidity, and temperature.

[0098] Tear film breakup time (BUT): 1 μL of 10 g / L sodium fluorescein eye drops was instilled into the conjunctival sac of experimental mice, inducing blinking. The time to the appearance of the first tear film breakup point in the corneal staining area was observed and recorded under cobalt blue light using a slit-lamp microscope. Care should be taken to control variables during each examination; the procedure should be performed by the same person at the same time, location, lighting conditions, humidity, and temperature.

[0099] Corneal FL scoring: 1 μL of 10 g / L sodium fluorescein eye drops was instilled into the conjunctival sac of experimental mice to induce blinking. The extent and location of corneal epithelial defects in each group of mice were observed under a slit-lamp microscope. Scoring criteria: The cornea was divided into 4 quadrants, with each quadrant scored from 0 to 3 points. The scoring criteria for each quadrant were as follows: (1) 0 points: no staining; (2) 1 point: mild staining < 5 spots; (3) 2 points: moderate staining ≥ 5 spots; (4) 3 points: severe staining ≥ 5 spots, with filamentous staining or ulceration. The maximum total score was 12 points.

[0100] The specific results are shown in Tables 2, 3, and 4.

[0101] Experimental results:

[0102] Table 1 Comparison of Physicochemical Indicators

[0103]

[0104]

[0105] Table 2 Results of the tear secretion test (SIT)

[0106]

[0107] Note: Compared with the control group, each group...* P < 0.05 ** P < 0.01, *** P < 0.001.

[0108] Compared with Example 1, Comparative Examples 1 and 3-7 # P < 0.05 ## P < 0.01, ### P < 0.001.

[0109] Compared with Example 4, Comparative Examples 2, 3, and 7 △ P < 0.05 △△ P < 0.01, △△△ P < 0.001.

[0110] The SIT value can indirectly reflect the basal tear secretion of mice. Compared with the comparative examples, the tear secretion of mice in each example was significantly higher than that of mice in the comparative examples.

[0111] Table 3 Tear film breakup time (BUT)

[0112]

[0113]

[0114] Note: Compared with the control group, each group... * P < 0.05 ** P < 0.01, *** P < 0.001.

[0115] Compared with Example 1, Comparative Examples 1 and 3-7 # P < 0.05 ## P < 0.01, ### P < 0.001.

[0116] Compared with Example 4, Comparative Examples 2, 3, and 7 △ P < 0.05 △△ P < 0.01, △△△ P < 0.001.

[0117] Tear film breakup time is a direct and sensitive method for assessing tear film stability. Compared to the comparative examples, the tear film breakup time of mice in each example was longer than that of mice in the comparative examples.

[0118] Table 4 Corneal FL Score

[0119]

[0120]

[0121] Note: Compared with the control group, each group... * P < 0.05 ** P < 0.01, *** P < 0.001.

[0122] Compared with Example 1, Comparative Examples 1 and 3-7 # P < 0.05 ## P < 0.01, ### P < 0.001.

[0123] Compared with Example 4, Comparative Examples 2, 3, and 7 △ P < 0.05 △△ P < 0.01, △△△ P < 0.001.

[0124] The corneal FL score is used to assess the health of corneal epithelial tissue through corneal fluorescein staining. A higher score indicates more severe damage to the corneal epithelium. Compared to the control groups, the FL scores of mice in each example were significantly lower than those in the control groups.

[0125] As shown in Tables 2-4, Example 1 of the present invention simultaneously added mesenchymal stem cell supernatant containing multiple trace elements and mesenchymal stem cell extract containing multiple trace elements. Compared with Comparative Example 4 or Comparative Example 5, which only added one of them, the two had a significant synergistic effect in increasing tear secretion, improving tear film stability, and improving corneal damage repair.

[0126] In Example 1 of this invention, multiple trace elements are added during the stem cell culture process. Compared with Comparative Example 6, which directly adds multiple trace elements during the preparation of eye drops, some trace elements that are not easily absorbed by the human body can exist in the cell matrix components and are encapsulated in exosomes, which are more conducive to absorption and improve tear secretion, tear film stability and corneal damage repair effect.

[0127] Examples 1 and 4 of this invention simultaneously added mesenchymal stem cell supernatant, mesenchymal stem cell extract, and multiple trace elements. Compared with Comparative Example 1 or Comparative Example 2, which only added mesenchymal stem cell supernatant and mesenchymal stem cell extract, or Comparative Example 7, which only added multiple trace elements, the three components showed a significant synergistic effect in increasing tear secretion, improving tear film stability, and enhancing corneal damage repair.

[0128] When the ratio of the added multi-element trace elements in Example 1 was 1000:1, compared with the ratio of 2500:1, the SIT, BUT, and FL scores of the eye drops were comparable, with no significant difference.

[0129] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. An eye drop containing multiple trace elements and stem cell extract, characterized in that, The product comprises, by weight, the following components: 0.5-1.5 parts mesenchymal stem cell supernatant, 0.05-0.15 parts mesenchymal stem cell extract, and 5-15 parts sodium hyaluronate solution; wherein the mesenchymal stem cells include umbilical cord mesenchymal stem cells. The mesenchymal stem cell supernatant and the mesenchymal stem cell extract contain multiple trace elements; The mesenchymal stem cell supernatant is obtained by centrifugation and filtration after adding multiple trace elements to the mesenchymal stem cell culture system. The mesenchymal stem cell extract is obtained by adding multiple trace elements to the mesenchymal stem cell culture system and then extracting the mesenchymal stem cells by ultrasonic lysis extraction. The multi-element trace elements include Al, Mg, Ba, Mn, Ca, Mo, Cd, Ni, Co, Se, Cr, Sr, Cu, Ti, Fe, V, and Zn.

2. The eye drops according to claim 1, characterized in that, The specific composition of the multi-element trace elements is as follows: Al content is 500-5000 mg / L, Mg content is 500-5000 mg / L, Ba content is 0.01-0.1 mg / L, Mn content is 50-500 mg / L, Ca content is 50-500 mg / L, Mo content is 1-10 mg / L, Cd content is 0.001-0.01 mg / L, Ni content is 1-10 mg / L, Co content is 0.5-5 mg / L, Se content is 10-100 mg / L, Cr content is 1-10 mg / L, Sr content is 0.5-5 mg / L, Cu content is 0.5-5 mg / L, Ti content is 50-500 mg / L, Fe content is 500-5000 mg / L, V content is 1-10 mg / L, and Zn content is 100-1000 mg / L.

3. The eye drops according to claim 1, characterized in that, The concentration of the sodium hyaluronate solution is 0.05-0.5 wt%.

4. The eye drops according to claim 1, characterized in that, The eye drops also include at least one of a pH adjuster and an osmotic pressure adjuster.

5. The eye drops according to claim 4, characterized in that, The pH adjuster is used in an amount of 0.01-0.5 parts by weight, and the osmotic pressure adjuster is used in an amount of 0.01-0.5 parts; the pH adjuster includes one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, boric acid, borax, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, sodium hydroxide, potassium hydroxide, hydrochloric acid, and phosphoric acid; the osmotic pressure adjuster includes one or more of sodium chloride, potassium chloride, boric acid, borax, sodium sulfate, potassium sulfate, sodium nitrate, potassium nitrate, sodium acetate, mannitol, glycerol, propylene glycol, and glucose.

6. The eye drops according to claim 1, characterized in that, The preparation process of the mesenchymal stem cell culture system is as follows: stem cells are passaged to P3 using serum-free stem cell culture medium, seed cells are cryopreserved, P3 seed cells are thawed in batches using serum-free stem cell culture medium, and multiple trace elements are added during the thaw process. The cells are then passaged to P5-P8 to obtain the final product.

7. The eye drops according to claim 6, characterized in that, During the resuscitation process, the dilution ratio of the multi-trace elements in the serum-free stem cell culture medium was 2000-3000:

1.

8. The eye drops according to claim 1, characterized in that, The ultrasonic lysis extraction method specifically includes the following steps: centrifuging and washing the obtained mesenchymal stem cells with physiological saline, resuspending them, ultrasonically lysing them, and filtering them to obtain the final product; the resuspension solution is at least one of the following: mesenchymal stem cell supernatant, physiological saline, 1‰ sodium hyaluronate solution, and water for injection.

9. The eye drops according to claim 8, characterized in that, The density of the mesenchymal stem cell supernatant resuspended at 1×10⁻⁶ 6 -1×10 7 / mL; the ultrasonic lysis time is 20-60s, wherein each lysis lasts 1-3s and the interval is 3-9s; the filtration is first filtered with a 2000-mesh sterile filter and then filtered with a 0.22μm sterile filter membrane.

10. A method for preparing the eye drops according to any one of claims 1-9, characterized in that, The process includes the following steps: mixing the mesenchymal stem cell supernatant, mesenchymal stem cell extract, and sodium hyaluronate solution evenly to obtain the eye drops.

11. The use of an eye drop according to any one of claims 1-9 or an eye drop prepared by the preparation method according to claim 10 in the preparation of a medicament for treating dry eye syndrome.

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