Temperature-sensitive eye drops containing recombinant I-type human collagen as well as preparation method and application of temperature-sensitive eye drops

Abstract

The invention relates to the field of preparation of temperature-sensitive eye drops, in particular to a temperature-sensitive eye drop containing recombinant I-type human collagen as well as a preparation method and application of the temperature-sensitive eye drop. The solution A and the solution B are stored in different bottles, and the solution A and the solution B are mixed during use. Wherein a solution in the bottle A consists of recombinant I-type human collagen and hydrochloric acid, so that the collagen is ensured to be stored in an acid environment, the performance is more stable, and the collagen is not easy to separate out; and the solution in the bottle B consists of sodium hyaluronate, poloxamer, sodium hydroxide, sodium chloride and a phosphate solution. Poloxamer is added into the bottle B, and the flowability of the eye drops can be changed along with the change of temperature, so that the retention time of the eye drops in cornea is prolonged, and a longer-acting protection effect is achieved.

Description

Technical Field

[0001] The invention relates to the field of preparation of temperature-sensitive eye drops, and in particular to a temperature-sensitive eye drop containing recombinant type I human collagen, a preparation method and an application thereof. Background Art

[0002] There is a precorneal tear film with a thickness of 7 to 10 microns on the surface of the cornea. The main function of the tear film is to keep the cornea and conjunctiva moist; improve the refractive system of the eye; fill in some small scratches on the surface of the cornea to make the cornea smoother. When the tear film is not good, there are often some symptoms: the tear film is unstable, interfering with the normal refraction of light, causing blurred vision; dry eyes, insufficient tear secretion, excessive evaporation or abnormal tear composition, leading to eye fatigue, foreign body sensation, dryness, and in severe cases, there will be eye burning, soreness, redness, eye pain, photophobia, etc.; tears reduce the protection of the cornea, the corneal epithelium is damaged, and the damaged cornea affects the normal refraction of light, which leads to decreased vision; triggering eye inflammation, such as conjunctivitis, keratitis, etc. When the tear film is not good and problems such as dry eyes and fatigue occur, doctors generally recommend the use of eye drops to relieve such symptoms.

[0003] Most of the eye drops on the market at present mainly relieve dry eyes and eye fatigue. Generally, phosphate buffer solution and the like are used as solvents, and components such as cellulose and sodium hyaluronate are added to achieve the moisturizing effect. In some studies, the effect of eye drops on corneal repair has also been studied. For example, Patent 1 (Application No.: 200510098424.7) uses 0.5% glucose as an isotonic solution and mixes it with collagen extracted from animals to prepare a collagen eye drop product, which has good effects and no toxic side effects in the treatment of human corneal injuries. Patent 2 (Application No.: 201110116905.1) mixes collagen extracted from rat tails with glucose solution and injection water to obtain an eye drop product, and relevant research on corneal injury repair has been carried out. In Patent 3 (Application No.: 201110147418.1), collagen, retinal growth promoter, vitamins, vasoconstrictors, antihistamines, preservatives, etc. are mixed with sodium chloride isotonic solution, etc. to obtain an eye drop product, and an anti-fatigue experiment is carried out. In Patent 4 (Application No.: 202110960230.2), a pH regulator, an osmotic pressure regulator, a stabilizer and a protective agent are dissolved in water and sterilized (115 °C, 30 min); recombinant type III humanized collagen filtered through a filter membrane is added in proportion and mixed evenly to obtain the product. The sample is subjected to relevant corneal repair experiments. In Patent 5 (Application No.: 202310232104.4), the sample consists of superoxide dismutase SOD, recombinant human type III collagen, sodium hyaluronate, osmotic pressure regulator, pH regulator and injection water, and corneal injury repair experiments have been carried out. Although these materials have been studied through relevant animal experiments, there are still some problems. Using raw materials from animals is more likely to cause allergic reactions; adding components such as preservatives causes greater irritation to the eyes after long-term use; general eye drops have the problems of good fluidity and short tear film protection time; collagen is sensitive to pH and temperature, and collagen precipitation is likely to occur during long-term storage under neutral conditions.

[0004] Therefore, it is very necessary to invent a temperature-sensitive eye drop product that has no allergic reaction, does not add preservatives, is non-irritating to the cornea, has a long retention time on the cornea, and is easier to store. This will bring great benefits to manufacturers, doctors and patients. Summary of the Invention

[0005] Aiming at the above-mentioned existing technical problems, the present invention aims to invent a temperature-sensitive eye drop containing recombinant type I human collagen, its preparation method and application. The preparation method of this eye drop is simple, has no allergic reaction, does not add preservatives, is non-irritating to the cornea, has a long retention time on the cornea, overcomes the shortcoming that recombinant type I human collagen is prone to precipitation during storage, and has more stable properties.

[0006] The present invention discloses a temperature-sensitive eye drop containing recombinant type I human collagen, which includes the following components:

[0007] Vial A: Recombinant human type I collagen, hydrochloric acid;

[0008] Vial B: Sodium hyaluronate, poloxamer, sodium hydroxide, sodium chloride, phosphate solution.

[0009] Preferably, the content of recombinant human type I collagen in Vial A is 0.02 - 1.5 mg / mL.

[0010] Preferably, the concentration of hydrochloric acid in Vial A is 0.001 - 0.1 M.

[0011] Preferably, the concentration of sodium hyaluronate in Vial B is 1 - 10 mg / ml; the molecular weight of sodium hyaluronate is 600,000 - 1,200,000.

[0012] Preferably, the content of poloxamer in Vial B is 10 - 100 mg / ml.

[0013] Preferably, the content of sodium hydroxide in Vial B is 0.04 - 4.9 mg / ml.

[0014] Preferably, the content of sodium chloride in Vial B is 3.2 - 13.2 mg / ml.

[0015] Preferably, the volume ratio of the solution in Vial A to the solution in Vial B is 1:1.

[0016] A preparation method of a thermosensitive eye drop containing recombinant human type I collagen comprises the following steps:

[0017] S1: Prepare hydrochloric acid solution;

[0018] S2: Dissolve recombinant human type I collagen in the hydrochloric acid solution prepared in step S1 to obtain mixed solution 1;

[0019] S3: Under a hundred - class laminar flow hood, filter the mixed solution 1 prepared in step S2 successively through 0.45 μm and 0.22 μm filters to obtain Solution A;

[0020] S4: Prepare phosphate solution;

[0021] S5: Dissolve sodium hyaluronate, poloxamer, sodium hydroxide, and sodium chloride in the phosphate solution prepared in step S4 to obtain mixed solution 2;

[0022] S6: Under a hundred - class laminar flow hood, filter the mixed solution 2 prepared in step S5 successively through 0.45 μm and 0.22 μm filters to obtain Solution B;

[0023] S7: Under a hundred - class laminar flow hood, fill Solution A obtained in step S3 and Solution B obtained in step S6 into two containers according to a volume ratio of 1:1 to obtain the final eye drop sample.

[0024] Application of a thermosensitive eye drop containing recombinant human type I collagen: After mixing solution A and solution B, drop them onto the ocular surface.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The purpose of the present invention is to provide a thermosensitive eye drop containing recombinant human type I collagen, its preparation method and application. Compared with the prior art, the eye drop product prepared by the present invention has the following advantages:

[0027] (1) The eye drop consists of bottle A and bottle B. The solution in bottle A is composed of recombinant human type I collagen and hydrochloric acid. The collagen is stored under acidic conditions and at 2 - 8°C, which is more stable and not prone to precipitate collagen fibers. After mixing the solutions in bottle A and bottle B, it can be used immediately, and the operation is simple;

[0028] (2) Compared with animal - derived collagen, recombinant human type I collagen has a lower allergic reaction and reduces the risk of introducing animal - derived diseases;

[0029] (3) Poloxamer in solution B has thermosensitivity. When in use, when the temperature reaches body temperature, the viscosity of the solution increases and the fluidity decreases. Therefore, it stays on the corneal surface for a longer time and provides more persistent protection to the cornea;

[0030] (4) The eye drop does not contain preservative components, and long - term use can reduce the irritation to the eyes. Description of the Drawings

[0031] Figure 1 : Schematic diagram of the eye drop sample. The left - most one is solution A, the middle one is solution B, and the right - most one is the mixture of solution A and solution B.

[0032] Figure 2 : Detection results of the thermosensitivity of the eye drop sample prepared in Example 1. The temperature on the left is 37°C, and the temperature on the right is 2 - 8°C.

[0033] Figure 3 : Detection results of the thermosensitivity of the eye drop sample prepared in Example 2. The temperature on the left is 37°C, and the temperature on the right is 2 - 8°C.

[0034] Figure 4 : Detection results of the thermosensitivity of the eye drop sample prepared in Example 3. The temperature on the left is 37°C, and the temperature on the right is 2 - 8°C.

[0035] Figure 5 : Detection results of the thermosensitivity of the eye drop sample prepared in Example 4. The temperature on the left is 37°C, and the temperature on the right is 2 - 8°C.

[0036] Figure 6: Thermosensitivity test results of the eye drop sample prepared in Example 5. The temperature on the left is 37 °C, and the temperature on the right is 2 - 8 °C.

[0037] Figure 7 : Detection results of recombinant type I human collagen raw material by electrophoresis.

[0038] Figure 8 : High performance liquid chromatography detection chart of recombinant type I human collagen raw material. Detailed implementation manners

[0039] The following examples are provided to better understand the present invention further. They are not limited to the described optimal implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0040] For those not specifying specific experimental steps or conditions in the examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0041] Examples 1 - 5 all take the preparation of 500 mL of Solution A and 500 mL of Solution B as examples, and can be scaled up or down proportionally according to the actual production demand.

[0042] Figure 7 and Figure 8 These are the electrophoresis detection results and high performance liquid chromatography detection charts of the recombinant type I human collagen raw materials used in Examples 1 - 5. When the purity reaches that shown in the figures, it can be used to prepare this thermosensitive eye drop containing recombinant type I human collagen.

[0043] Example 1: First, prepare hydrochloric acid with a concentration of 0.001 M. Then, take about 400 mL of hydrochloric acid, dissolve 10 mg of recombinant type I human collagen in the hydrochloric acid, and add hydrochloric acid to make the solution 500 mL to form a recombinant type I human collagen solution with a concentration of 0.02 mg / mL. Under a hundred - level laminar flow hood, sequentially filter the formed recombinant type I human collagen solution through 0.45 μm and 0.22 μm filters to obtain Solution A.

[0044] Weigh sodium dihydrogen phosphate and disodium hydrogen phosphate, dissolve them in about 400 mL of injection water, and make up the injection water to 500 mL to obtain a phosphate solution. The content of sodium dihydrogen phosphate in the solution is 4.35 mg / mL, and the content of disodium hydrogen phosphate is 6.75 mg / mL. Then, take about 400 mL of the phosphate solution, dissolve sodium hyaluronate, poloxamer, sodium chloride and sodium hydroxide in the phosphate solution, and make up the phosphate solution to 500 mL. The content of sodium hyaluronate in the solution is 5 mg / mL, the content of poloxamer is 100 mg / mL, the content of sodium chloride is 13.2 mg / mL, and the content of sodium hydroxide is 0.04 mg / mL. Finally, under a hundred-class laminar flow hood, filter the solution successively through 0.45 μm and 0.22 μm filters to obtain Solution B.

[0045] Under a hundred-class laminar flow hood, fill Solution A and Solution B into two containers in a volume ratio of 1:1 to obtain the final eye drop sample.

[0046] Example 2: First, prepare hydrochloric acid with a concentration of 0.1 M. Then, take about 400 mL of hydrochloric acid, dissolve 750 mg of recombinant human type I collagen in the hydrochloric acid, and make up the hydrochloric acid to 500 mL to form a recombinant human type I collagen solution with a concentration of 1.5 mg / mL. Under a hundred-class laminar flow hood, filter the formed recombinant human type I collagen solution successively through 0.45 μm and 0.22 μm filters to obtain Solution A.

[0047] Weigh potassium dihydrogen phosphate, dissolve it in about 400 mL of injection water, and make up the injection water to 500 mL to obtain a phosphate solution. The content of potassium dihydrogen phosphate in the solution is 6.8 mg / mL. Then, take about 400 mL of the phosphate solution, dissolve sodium hyaluronate, poloxamer, sodium chloride and sodium hydroxide in the phosphate solution, and make up the phosphate solution to 500 mL. The content of sodium hyaluronate in the solution is 1 mg / mL, the content of poloxamer is 50 mg / mL, the content of sodium chloride is 3.2 mg / mL, and the content of sodium hydroxide is 4.9 mg / mL. Finally, under a hundred-class laminar flow hood, filter the solution successively through 0.45 μm and 0.22 μm filters to obtain Solution B.

[0048] Under a hundred-class laminar flow hood, fill Solution A and Solution B into two containers in a volume ratio of 1:1 to obtain the final eye drop sample.

[0049] Example 3: First, prepare hydrochloric acid with a concentration of 0.01 M. Then, take about 400 mL of hydrochloric acid, dissolve 0.5 g of recombinant type I human collagen in the hydrochloric acid, and add hydrochloric acid to make the solution volume reach 500 mL, forming a recombinant type I human collagen solution with a concentration of 1 mg / mL. Under a hundred-level laminar flow hood, sequentially filter the formed recombinant type I human collagen solution through 0.45 μm and 0.22 μm filters to obtain Solution A.

[0050] Weigh potassium dihydrogen phosphate and dissolve it in about 400 mL of injection water, and add injection water to make the solution volume reach 500 mL to obtain a phosphate solution, where the content of potassium dihydrogen phosphate in the solution is 6.8 mg / mL. Then, take about 400 mL of the phosphate solution, dissolve sodium hyaluronate, poloxamer, sodium chloride, and sodium hydroxide in the phosphate solution, and add phosphate solution to make the solution volume reach 500 mL. The content of sodium hyaluronate in the solution is 2 mg / mL, the content of poloxamer is 60 mg / mL, the content of sodium chloride is 8.42 mg / mL, and the content of sodium hydroxide is 1.6 mg / mL. Finally, under a hundred-level laminar flow hood, sequentially filter the solution through 0.45 μm and 0.22 μm filters to obtain Solution B.

[0051] Under a hundred-level laminar flow hood, fill Solution A and Solution B into two containers in a volume ratio of 1:1 to obtain the final eye drop sample.

[0052] Example 4: First, prepare hydrochloric acid with a concentration of 0.03 M. Then, take about 400 mL of hydrochloric acid, dissolve 0.1 g of recombinant type I human collagen in the hydrochloric acid, and add hydrochloric acid to make the solution volume reach 500 mL, forming a recombinant type I human collagen solution with a concentration of 0.2 mg / mL. Under a hundred-level laminar flow hood, sequentially filter the formed recombinant type I human collagen solution through 0.45 μm and 0.22 μm filters to obtain Solution A.

[0053] Weigh potassium dihydrogen phosphate and dissolve it in about 400 mL of injection water, and add injection water to make the solution volume reach 500 mL to obtain a phosphate solution, where the content of potassium dihydrogen phosphate in the solution is 6.8 mg / mL. Then, take about 400 mL of the phosphate solution, dissolve sodium hyaluronate, poloxamer, sodium chloride, and sodium hydroxide in the phosphate solution, and add phosphate solution to make the solution volume reach 500 mL. The content of sodium hyaluronate in the solution is 6 mg / mL, the content of poloxamer is 10 mg / mL, the content of sodium chloride is 7.2 mg / mL, and the content of sodium hydroxide is 2.6 mg / mL. Finally, under a hundred-level laminar flow hood, sequentially filter the solution through 0.45 μm and 0.22 μm filters to obtain Solution B.

[0054] Under the hundred-class laminar flow hood, liquid A and liquid B were filled into two containers in a volume ratio of 1:1 to obtain the final eye drop sample.

[0055] Example 5: First, hydrochloric acid with a concentration of 0.05 M was prepared. Then, about 400 mL of hydrochloric acid was taken, and 0.25 g of recombinant type I human collagen was dissolved in the hydrochloric acid, and hydrochloric acid was added to make the solution 500 mL, forming a recombinant type I human collagen solution with a concentration of 0.5 mg / mL. Under the hundred-class laminar flow hood, the formed recombinant type I human collagen solution was filtered through filters with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain liquid A.

[0056] Potassium dihydrogen phosphate was weighed and dissolved in about 400 mL of injection water, and injection water was added to make the solution 500 mL to obtain a phosphate solution, and the content of potassium dihydrogen phosphate in the solution was 6.8 mg / mL. Then, about 400 mL of the phosphate solution was taken, and sodium hyaluronate, poloxamer, sodium chloride, and sodium hydroxide were dissolved in the phosphate solution, and the phosphate solution was added to make the solution 500 mL. The content of sodium hyaluronate in the solution was 10 mg / mL, the content of poloxamer was 30 mg / mL, the content of sodium chloride was 6.1 mg / mL, and the content of sodium hydroxide was 3.6 mg / mL. Finally, under the hundred-class laminar flow hood, the solution was filtered through filters with pore sizes of 0.45 μm and 0.22 μm in sequence to obtain liquid B.

[0057] Under the hundred-class laminar flow hood, liquid A and liquid B were filled into two containers in a volume ratio of 1:1 to obtain the final eye drop sample.

[0058] As Figure 1 shown, the left one is liquid A, the middle one is liquid B, and the right one is the mixture of liquid A and liquid B.

[0059] The liquid A samples, liquid B samples, and the mixed samples prepared in Examples 1 to 5 are all Figure 1 shown.

[0060] The eye drop samples prepared in Examples 1 to 5 were subjected to tests on appearance and properties, pH value, osmotic pressure, and sterility:

[0061] (1) Appearance and properties: The appearance and properties of the eye drop samples were determined by visual inspection and nasal olfaction.

[0062] (2) pH value: It was detected by the "pH Value Determination Method (0631)" in "Pharmacopoeia of the People's Republic of China: 2020 Edition, Volume IV".

[0063] (3) Osmotic pressure: It was detected by the "Osmolality Determination Method (0632)" in "Pharmacopoeia of the People's Republic of China: 2020 Edition, Volume IV".

[0064] (4) Sterile, detected by the "1101 Sterility Test" in "Pharmacopoeia of the People's Republic of China: Edition 2020. Volume IV".

[0065] The test results are shown in the following table:

[0066]

[0067]

[0068] From the data in the above table, it can be seen that the samples prepared in Examples 1-5 are all clear and transparent liquids, and their pH values and osmotic pressures meet the requirements for human use (general requirements for marketed eye drops: pH = 6.5 - 7.5, osmotic pressure is 260 - 340 mOsm / KgHO), and all can meet the requirements for sterile use.

[0069] The thermosensitivity of the eye drop samples prepared in Examples 1 - 5 was detected, and the detection method is as follows:

[0070] The fluidity of the sample was tested using an inclined plate. One drop of the eye drops stored at 2 - 8°C and 37°C was respectively added to an inclined plate with an inclination angle of 45°, and the time was recorded for 5 s. After taking pictures, the flow conditions of the eye drops at different temperatures were compared. At 37°C, for the eye drops of Examples 1 - 5, the flow distance on the inclined plate was less than that of the samples stored at 2 - 8°C. The results show that for the eye drops of Examples 1 - 5, at 37°C (close to human body temperature), their fluidity becomes worse, indicating that they can be retained on the corneal surface for a longer time and protect the cornea for a longer time.

[0071] The test results are as Figures 2 to 6 shown.

[0072] The efficacy experiments of the eye drop samples prepared in Examples 1 - 5 were carried out, and the experimental methods and results are as follows:

[0073] 1. Dry eye symptom relief experiment:

[0074] (1) Eighteen female rats aged 6 - 8 weeks were selected and divided into 6 groups. One group was the normal saline control group, and the experimental groups used the samples prepared in Examples 1 - 5, with a total of 6 groups, 3 rats in each group.

[0075] (2) Rats in the control group and the experimental groups were respectively subcutaneously injected with 0.5 mL of scopolamine hydrobromide at a concentration of 3 mg / mL, 4 times a day, at 8:00, 12:00, 16:00, and 20:00, for 14 consecutive days to establish a rat dry eye model. At the same time, rats in the control group and the experimental groups were instilled with normal saline or the composition of the prescription example into the eyes 5 times a day, at 8:00, 11:00, 14:00, 17:00, and 20:00, for 28 consecutive days.

[0076] (3) Before modeling and on days 0, 7, 14, 21, and 28 after medication, the tear secretion test and tear film break-up time test were performed on rats in each group, and the data were recorded. The methods of the tear secretion test and tear film break-up time test are as follows:

[0077] Tear secretion test: The tear secretion test was performed using phenol red cotton thread at the same time period before modeling and on days 0, 7, 14, 21, and 28 after medication to measure the tear secretion volume of both eyes of the rats. No anesthetic drug was used on the ocular surface of the rats (to test the secretion function of the main lacrimal gland). The phenol red cotton thread was clamped with forceps and placed at the outer 1 / 3 of the lower eyelid conjunctival sac of the rats for 30 s and then taken out. The wet length of the cotton thread was measured and recorded.

[0078] Tear film break-up time test: 1 μL of 10 g / L sodium fluorescein was dropped into the conjunctival sac, and the eyelids were closed. Observation was performed under the cobalt blue light of a slit lamp microscope. After 3 blinks, timing started from the last blink, and the time until the first black spot appeared on the cornea was the tear film break-up time.

[0079] Table 1: Results of the tear film secretion test

[0080]

[0081] Table 2: Results of the tear film break-up time (s) test

[0082]

[0083] From the data recorded in Table 1 and Table 2, it can be seen that after administration, the tear secretion of the rats gradually returned to normal, and the tear secretion volume was close to that before modeling at about 28 d. At the same time, after administration, the tear film break-up time was significantly prolonged compared with the control group and was close to the data before modeling. There was no significant difference in the data after administration among different experimental groups, indicating that there was no significant difference in their therapeutic effects. The above results show that the eye drops sample prepared by the present invention can effectively treat dry eye symptoms and promote tear secretion.

[0084] 2. Corneal defect repair experiment:

[0085] Twenty-one male SD rats, with no significant differences in age and body weight, were randomly divided into a blank control group, a model group, and an experimental group (samples prepared in Examples 1-5), with 3 rats in each group. The model group and the experimental group established a corneal ultraviolet injury model according to the following method: First, the rats were anesthetized with isoflurane. After anesthesia, compound tropicamide eye drops were instilled into the right eye, and the right eye was fixed with adhesive tape to keep it open. The cornea was irradiated with an ultraviolet lamp for 20 minutes, and the irradiation was continued for 4 days. For the rats with successful model establishment, the right eye of the model group was instilled with normal saline, and the right eye of the experimental group was instilled with eye drops, 5 times a day for 5 consecutive days. The degree of corneal injury repair was judged by the release amount of lactate dehydrogenase (LDH) in the tear fluid. A decrease in the LDH release amount indicated that the corneal injury was repaired; an increase in the LDH release amount indicated corneal injury. Before irradiation, after model establishment, 1 day, 3 days, and 5 days after administration, 200 μl of normal saline was locally instilled into the eye and infiltrated for 60 s, and then 100 μl of the sample was collected. The LDH release amount of each group was measured by ELISA using an enzyme-labeled instrument and the average value was calculated.

[0086] Table 3: Test results of LDH release at different times before and after ultraviolet irradiation

[0087]

[0088] From the data recorded in Table 3, it can be seen that after successful establishment of the model by irradiating the eyes of rats with ultraviolet light, the release of LDH increased sharply, indicating that this method damaged the cornea of the rats; the data of the blank control group were always low and stable, indicating that the eyes of the rats were not damaged and the cornea was normal; the data of the model group decreased slowly, indicating that normal saline had a slower effect on the recovery of corneal injury; after corneal injury in the experimental group, the data were higher, indicating that the cornea was damaged. As time went by, the data decreased, indicating that the sample could promote the repair of corneal injury.

[0089] Obviously, the above embodiments are only examples clearly described and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A temperature-sensitive eye drop containing recombinant type I human collagen, characterized in that, It comprises the following components: Vial A: Recombinant human type I collagen, hydrochloric acid; Vial B: Sodium hyaluronate, poloxamer, sodium hydroxide, sodium chloride, phosphate solution.

2. The thermosensitive eye drops containing recombinant type I human collagen according to claim 1, characterized in that, The content of recombinant human type I collagen in Vial A is 0.02 - 1.5 mg / ml.

3. A thermosensitive eye drop containing recombinant type I human collagen according to claim 1, characterized in that, The concentration of hydrochloric acid in Vial A is 0.001 - 0.1 M.

4. A thermosensitive eye drop containing recombinant type I human collagen according to claim 1, characterized in that, The concentration of sodium hyaluronate in Vial B is 1 - 10 mg / ml.

5. A thermosensitive eye drop containing recombinant type I human collagen according to claim 1, characterized in that, The content of poloxamer in Vial B is 10 - 100 mg / ml.

6. The thermosensitive eye drops containing recombinant type I human collagen according to claim 1, characterized in that, The content of sodium hydroxide in Vial B is 0.04 - 4.9 mg / ml.

7. The thermosensitive eye drops containing recombinant type I human collagen according to claim 1, characterized in that, The content of sodium chloride in Vial B is 3.2 - 13.2 mg / ml.

8. A thermosensitive eye drop containing recombinant type I human collagen according to claim 1, characterized in that, The volume ratio of the solution in Vial A to the solution in Vial B is 1:

1.

9. The preparation method of a temperature-sensitive eye drop containing recombinant type I human collagen according to any one of claims 1 to 8, characterized in that, It comprises the following steps: S1: Prepare hydrochloric acid solution; S2: Dissolve recombinant human type I collagen in the hydrochloric acid solution prepared in step S1 to obtain mixed solution 1; S3: Under a hundred - class laminar flow hood, filter the mixed solution 1 prepared in step S2 successively through 0.45 - μm and 0.22 - μm filters to obtain Solution A; S4: Prepare phosphate solution; S5: Dissolve sodium hyaluronate, poloxamer, sodium hydroxide, and sodium chloride in the phosphate solution prepared in step S4 to obtain mixed solution 2; S6: Under a hundred - class laminar flow hood, filter the mixed solution 2 prepared in step S5 successively through 0.45 - μm and 0.22 - μm filters to obtain Solution B; S7: Under a hundred - class laminar flow hood, fill Solution A obtained in step S3 and Solution B obtained in step S6 into two containers in a volume ratio of 1:1 to obtain the final eye - drop sample.

10. Use of a thermosensitive eye drop containing recombinant type I human collagen, characterized in that, After mixing Solution A and Solution B, drop them onto the ocular surface.

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