An eye wash solution, its preparation and use

Abstract

The embodiment of the application discloses an eye wash liquid and a preparation method and application thereof. According to the weight percentage, the eye wash liquid comprises the following components: 0.01-0.1% of silk fibroin, 0.01-0.05% of sodium hyaluronate, 0.01-0.04% of vitamin B6, 0.002-0.008% of borneol, 0.1-0.4% of dihydric alcohol, 0.5-1% of sodium chloride, and the rest is purified water. The eye wash liquid can efficiently relieve the dryness, foreign body sensation and other discomfort caused by dry eye syndrome. The product has excellent safety, no cytotoxicity, no irritation to the ocular surface, good stability, and can meet the long-term use requirements of patients with dry eye syndrome. In addition, the preparation process is simple, the reaction conditions are mild and easy to control, and has the advantages of industrialized batch production.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an eye wash solution, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome is an ocular surface disease caused by multiple factors. Its core pathological features are tear film instability and abnormal ocular surface microenvironment. Clinical manifestations include dryness, foreign body sensation, burning sensation, blurred vision, and fatigue. In severe cases, it can lead to corneal epithelial damage and decreased vision, affecting the patient's quality of life. Currently, commonly used clinical interventions include artificial tears, ocular gels, and meibomian gland care, with artificial tears being the most common due to their convenience and rapid onset of action.

[0003] Sodium hyaluronate eye drops are a commonly used type of artificial tear in clinical practice, primarily used to relieve various discomfort symptoms caused by dry eye syndrome. Its main component, sodium hyaluronate, is a naturally occurring substance found in the human eye and other tissues. It shares similarities with mucin, a component inherent to tears, in terms of chemical structure, molecular weight, and rheological properties. This component can form a regular, stable, and long-lasting moisture film on the ocular surface, protecting it and relieving dryness and irritation. However, long-term clinical application has shown that sodium hyaluronate, when used alone, lacks sufficient moisturizing and long-lasting effects, making it difficult to achieve sustained water retention and repair, thus limiting its effectiveness in curing and managing dry eye syndrome. Summary of the Invention

[0004] Therefore, embodiments of the present invention provide an eye wash solution, its preparation method, and its application.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of the present invention, the present invention provides an eye wash solution, which, by weight percentage, comprises: 0.01-0.1% silk fibroin, 0.01-0.05% sodium hyaluronate, 0.01-0.04% vitamin B6, 0.002-0.008% borneol, 0.1-0.4% diol, 0.5-1% sodium chloride, and the balance being purified water.

[0007] Silk fibroin molecules contain a large number of hydrophilic groups (such as hydroxyl and amino groups), which can combine with water molecules to form a stable moisturizing film, reduce the evaporation of moisture from the ocular surface, and relieve dryness and foreign body sensation caused by dry eye syndrome. At the same time, it can form a gentle protective film on the ocular surface, reduce the damage of external stimuli to the ocular mucosa, assist in the repair of damaged ocular surfaces (such as minor corneal epithelial damage), and improve eye comfort.

[0008] To synergistically enhance the efficacy of silk fibroin and sodium hyaluronate, the applicant discovered during research that directly combining the two resulted in a composite film prone to microscopic phase separation due to differences in their hydrophilic and hydrophobic properties, leading to a loose structure and low mechanical strength. This film was easily broken and detached under frequent eyelid friction, and its moisturizing durability was insufficient. Through extensive research, the applicant unexpectedly discovered that adding a specific ratio of diol to the compound system, leveraging its amphiphilic properties, allows its molecules to embed between the hydrophobic domains of silk fibroin and the hydrophilic chains of sodium hyaluronate. This method effectively reduces interfacial tension, promotes molecular chain entanglement and interpenetration, and constructs a denser, more resilient three-dimensional hydrogel protective film. This significantly prolongs the retention time of the medication on the ocular surface, and the resulting eye wash effectively improves tear production and tear film breakup time, maintaining long-lasting eye moisture and thus enhancing the treatment effect of dry eye syndrome.

[0009] Further research revealed that when the mass ratio of silk fibroin, sodium hyaluronate, and diol is 4-6:2-4:20, the three components exhibit a better synergistic effect, which is more conducive to improving the therapeutic effect of the eye wash. Further optimized, the eye wash comprises: 0.05% silk fibroin, 0.03% sodium hyaluronate, 0.02% vitamin B6, 0.005% borneol, 0.2% diol, 0.7% sodium chloride, and the balance being purified water.

[0010] Furthermore, the diol is a C3-C6 aliphatic saturated diol.

[0011] Furthermore, the diol is selected from one or more of propylene glycol, butanediol, pentanediol, and hexanediol.

[0012] Furthermore, the molecular weight of the sodium hyaluronate is 500-1500 kDa.

[0013] According to a second aspect of the present invention, the present invention provides a method for preparing an eye wash solution as described in any of the preceding claims, the method comprising the following steps:

[0014] (1) Mix some purified water and sodium hyaluronate evenly, add vitamin B6 and sodium chloride to obtain the first mixture;

[0015] (2) Mix the diol and borneol to obtain a second mixture;

[0016] (3) Mix the first mixture and the second mixture, add silk fibroin, add purified water, stir, filter through a 0.22 μm filter membrane to obtain the eye wash solution.

[0017] Furthermore, the method for preparing the silk fibroin includes:

[0018] (1) Take degummed filaments and add lithium bromide solution with a concentration of 40-60% w / w at a solid-liquid ratio of 1g:3-6mL. Heat and stir at 120±5℃ for 4-6 hours.

[0019] (2) The solution from step (1) is subjected to ultrafiltration desalting, with the ultrafiltration column having a molecular weight cutoff of 10-15 kDa;

[0020] (3) Add the ultrafiltration solution from step (2) to phosphate buffer. The final concentration of phosphate buffer is 1-10 mM and the solid content of silk fibroin is 2-4% w / w.

[0021] (4) Heat the solution from step (3) at 120±5℃ for 4-6 hours, freeze-dry the solution, and obtain the silk fibroin.

[0022] Furthermore, in step (1), the mass of the purified water is 50-75% of the total mass of purified water.

[0023] According to a third aspect of the present invention, the present invention provides the use of the eye wash solution as described in any of the preceding claims in the preparation of a medicament for treating dry eye syndrome.

[0024] The embodiments of the present invention have the following advantages:

[0025] This invention combines silk fibroin, sodium hyaluronate, and diol in a specific ratio. The components work synergistically, with the silk fibroin and sodium hyaluronate forming a dense and resilient three-dimensional hydrogel protective film mediated by the diol. Combined with the nutritional support of vitamin B6, the osmotic pressure regulation of sodium chloride, and the soothing effect of borneol, this comprehensively improves the condition of the ocular surface. The resulting eye wash effectively relieves discomfort such as dryness and foreign body sensation caused by dry eye syndrome. The product has been proven to have excellent safety, is non-cytotoxic, non-irritating to the ocular surface, and exhibits excellent stability, meeting the long-term use needs of dry eye patients. Furthermore, the preparation process of this invention is simple, with mild and easily controllable reaction conditions, offering advantages for industrial-scale mass production. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The preparation method of silk fibroin includes the following steps:

[0028] (1) Take the degummed filaments and add them to a lithium bromide solution with a concentration of 50% w / w at a solid-liquid ratio of 1g:4mL. Heat and stir at 120±5℃ for 6 hours.

[0029] (2) The solution from step (1) was subjected to ultrafiltration desalting, with the ultrafiltration column having a molecular weight cutoff of 15 kDa;

[0030] (3) Add the ultrafiltration solution from step (2) to phosphate buffer. The final concentration of phosphate buffer is 5 mM and the solid content of silk fibroin is 3% w / w.

[0031] (4) Heat the solution from step (3) at 120±5℃ for 4 hours, and freeze-dry the solution to obtain the silk fibroin.

[0032] Sodium hyaluronate: molecular weight 500-1500kDa, Bloomage Biotechnology.

[0033] Example 1

[0034] This embodiment provides an eye wash solution, the preparation method of which includes the following steps:

[0035] (1) Add 600g of purified water to a clean container and place it at room temperature. Turn on the magnetic stirrer and adjust the speed to 600rpm. Add 0.3g of sodium hyaluronate and stir continuously for 20min until there are no visible insoluble substances. Then add 0.2g of vitamin B6 and 7g of sodium chloride in sequence and stir at 600rpm until completely dissolved to obtain mixture A.

[0036] (2) Take another container, weigh 2g of hexanediol (2-methyl-2,4-pentanediol) and 0.05g of borneol, mix them, and let stand for 20 minutes to ensure that the borneol is completely dissolved to obtain mixture B.

[0037] (3) Add mixture B to mixture A, stir evenly, then add 0.5g silk fibroin, let stand for 10min, add purified water to make up to 1000g, stir at 600rpm for 5min until the system is homogeneous.

[0038] (4) The solution is sterilized by filtration through a 0.22μm filter membrane, and the filtrate is collected to obtain the eye wash solution.

[0039] Example 2

[0040] This embodiment provides an eye wash solution, the preparation method of which includes the following steps:

[0041] (1) Add 600g of purified water to a clean container and place it at room temperature. Turn on the magnetic stirrer and adjust the speed to 600rpm. Add 0.2g of sodium hyaluronate and stir continuously for 20min until there are no visible insoluble substances. Then add 0.2g of vitamin B6 and 7g of sodium chloride in sequence and stir at 600rpm until completely dissolved to obtain mixture A.

[0042] (2) Take another container, weigh 2g of 1,2-propanediol and 0.05g of borneol, mix them, and let stand for 20 minutes to ensure that the borneol is completely dissolved to obtain mixture B.

[0043] (3) Add mixture B to mixture A, stir evenly, then add 0.4g silk fibroin, let stand for 10min, add purified water to make up to 1000g, stir at 600rpm for 5min until the system is homogeneous.

[0044] (4) The solution is sterilized by filtration through a 0.22μm filter membrane, and the filtrate is collected to obtain the eye wash solution.

[0045] Example 3

[0046] This embodiment provides an eye wash solution, the preparation method of which includes the following steps:

[0047] (1) Add 600g of purified water to a clean container and place it at room temperature. Turn on the magnetic stirrer and adjust the speed to 600rpm. Add 0.4g of sodium hyaluronate and stir continuously for 20min until there are no visible insoluble substances. Then add 0.2g of vitamin B6 and 7g of sodium chloride in sequence and stir at 600rpm until completely dissolved to obtain mixture A.

[0048] (2) Take another container, weigh 2g of 1,3-butanediol and 0.05g of borneol, mix them, and let stand for 20 minutes to ensure that the borneol is completely dissolved to obtain mixture B.

[0049] (3) Add mixture B to mixture A, stir evenly, then add 0.4g silk fibroin, let stand for 10min, add purified water to make up to 1000g, stir at 600rpm for 5min until the system is homogeneous.

[0050] (4) The solution is sterilized by filtration through a 0.22μm filter membrane, and the filtrate is collected to obtain the eye wash solution.

[0051] Example 4

[0052] This embodiment provides an eye wash solution, the preparation method of which is as follows:

[0053] (1) Add 600g of purified water to a clean container and place it at room temperature. Turn on the magnetic stirrer and adjust the speed to 600rpm. Add 0.2g of sodium hyaluronate and stir continuously for 20min until there are no visible insoluble substances. Then add 0.2g of vitamin B6 and 7g of sodium chloride in sequence and stir at 600rpm until completely dissolved to obtain mixture A.

[0054] (2) Take another container, weigh 2g of 1,2-hexanediol and 0.05g of borneol, mix them, and let stand for 20 minutes to ensure that the borneol is completely dissolved to obtain mixture B.

[0055] (3) Add mixture B to mixture A, stir evenly, then add 0.6g silk fibroin, let stand for 10min, add purified water to make up to 1000g, stir at 600rpm for 5min until the system is homogeneous.

[0056] (4) The solution is sterilized by filtration through a 0.22μm filter membrane, and the filtrate is collected to obtain the eye wash solution.

[0057] Example 5

[0058] This embodiment provides an eye wash solution, the preparation method of which is as follows:

[0059] (1) Add 600g of purified water to a clean container and place it at room temperature. Turn on the magnetic stirrer and adjust the speed to 600rpm. Add 0.1g of sodium hyaluronate and stir continuously for 20min until there are no visible insoluble substances. Then add 0.2g of vitamin B6 and 7g of sodium chloride in sequence and stir at 600rpm until completely dissolved to obtain mixture A.

[0060] (2) Take another container, weigh 2g of hexanediol (2-methyl-2,4-pentanediol) and 0.05g of borneol, mix them, and let stand for 20 minutes to ensure that the borneol is completely dissolved to obtain mixture B.

[0061] (3) Add mixture B to mixture A, stir evenly, then add 0.8g silk fibroin, let stand for 10min, add purified water to make up to 1000g, stir at 600rpm for 5min until the system is homogeneous.

[0062] (4) The solution is sterilized by filtration through a 0.22μm filter membrane, and the filtrate is collected to obtain the eye wash solution.

[0063] Comparative Example 1

[0064] This embodiment provides an eye wash solution, which differs from Example 1 only in that it does not contain the diol 1,6-hexanediol.

[0065] Test Example 1: Cytotoxicity Assay (MTT Method)

[0066] Human corneal endothelial cells (HCECs) in the logarithmic growth phase were harvested and their concentration adjusted to 2 × 10⁻⁶ cells / year. 4 Cells / mL were seeded into 96-well plates, 100 μL per well. Incubation was carried out at 37°C, 5% CO2 for 24 hours until cell attachment. Old culture medium was aspirated; eye wash solution was added to the experimental groups, and complete culture medium was added to the blank control group. Six replicates were made for each group, 100 μL per well. Incubation continued for another 24 hours. 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 hours. The liquid in the wells was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well, shaken for 10 minutes to dissolve any crystals. The absorbance (OD) value of each well was measured at 490 nm using a microplate reader. Cell viability was calculated.

[0067] The results showed that the cell survival rate of the eye wash solutions in Examples 1-5 was 101.2%~108.6% after 24 hours. The above results indicate that the eye wash solutions provided by the present invention are non-toxic to cells.

[0068] Test Example 2: Security Detection

[0069] Methods: Ten New Zealand rabbits with normal eyes, weighing 1.5-2.5 kg each, were selected. The rabbits were randomly divided into experimental groups 1-5, with two rabbits in each group. In experimental groups 1-5, the left eye was treated with eye wash solutions from Examples 1-5, and the right eye was treated with physiological saline drops, 3 drops three times a day for 7 consecutive days. Changes in the conjunctiva were observed daily, and the conjunctiva was observed after the last administration. The animals' irritant responses to each eye wash solution were observed on days 1, 7, and 15 after drug withdrawal, including conjunctival hyperemia, secretions, and corneal staining, and the degree of reaction was scored.

[0070] The results showed that the left conjunctiva of all animals was free of congestion, edema, and discharge; the iris was normal; and there was no corneal opacity, with no significant difference compared to the right eye. According to the irritation response scoring criteria, the eye wash solution provided by this invention is a non-irritating substance.

[0071] Test Example 3: Stability Test

[0072] After sealing the eye wash solution from Example 1, store it in a refrigerator at 4°C and a constant temperature water bath at 40°C for 3 months, observing its appearance daily.

[0073] The results showed that the samples were clear and transparent in appearance, without turbidity, precipitation, or stratification, and their properties remained largely unchanged. Samples from Examples 2-5, tested under the same conditions, showed no significant difference in appearance stability compared to Example 1. Furthermore, the microbial limit tests of all the above-mentioned sample samples at the end of the tests met the relevant standard requirements. These results demonstrate that the eyewash solution provided by this invention exhibits good physical and chemical stability under different temperature conditions, and that the preservative system is effective, ensuring controllable quality within the shelf life.

[0074] Test Example 4: Validity Test

[0075] General information: 108 adult patients with dry eye were selected and randomly divided into experimental groups 1-5 and control group 1, with 18 patients in each group. There were no significant differences among the groups in terms of gender, age, and disease duration, making them comparable.

[0076] Inclusion criteria: Patients clinically diagnosed with dry eye syndrome, exhibiting symptoms such as eye fatigue, foreign body sensation, dryness, burning, eye distension, and eye pain. Tear secretion test ≤5mm / 5min, tear film breakup time ≤10s. Exclusion criteria: Patients with severe liver or kidney dysfunction; patients with cognitive impairment, confusion, or emotional apathy; pregnant or lactating patients.

[0077] Treatment method: Patients in experimental groups 1-5 and control group 1 used the eye wash solution of Examples 1-5 and Comparative Example 1, respectively, twice a day for 14 consecutive days.

[0078] Observation indicators: tear secretion and tear film breakup time.

[0079] Efficacy assessment: Cured: Symptoms such as foreign body sensation, dryness, and burning in the eyes disappear, and tear secretion test (Schirmer I test without anesthesia) >10mm / 5min; Improved: Symptoms such as foreign body sensation, dryness, and burning in the eyes are partially relieved or improved, and tear secretion is increased compared with before treatment; Ineffective: The patient cannot reach the above criteria after treatment.

[0080] The results of tear secretion and tear film breakup time after 14 days of treatment are shown in Table 1 and Table 2 below.

[0081] Table 1

[0082]

[0083] Table 2

[0084]

[0085] Note: Compared with control group 1, **p<0.01, *p<0.05.

[0086] The results showed that before treatment, there were no significant differences in tear secretion and tear film breakup time among the groups, making them comparable. After treatment, the tear secretion and tear film breakup time in experimental groups 1-5 were significantly better than those in control group 1, indicating that the eye wash provided by this invention can effectively improve patients' dry eye symptoms. Among them, the mass ratio of silk fibroin, sodium hyaluronate, and diol in the eye wash used in experimental groups 1-4 was within the range of 4-6:2-4:20, which resulted in a better synergistic effect, significantly improving patients' tear secretion and tear film breakup time, and achieving better results.

[0087] The therapeutic effects are shown in Table 3 below.

[0088] Table 3

[0089]

[0090] The results showed that the total effective rate of experimental groups 1-5 was significantly higher than that of control group 1. Among them, the eye wash used in experimental groups 1-4 had better therapeutic effects.

[0091] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An eye wash solution, characterized in that, The eye wash solution comprises, by weight percentage: 0.01-0.1% silk fibroin, 0.01-0.05% sodium hyaluronate, 0.01-0.04% vitamin B6, 0.002-0.008% borneol, 0.1-0.4% diol, 0.5-1% sodium chloride, with the balance being purified water.

2. The eye wash solution according to claim 1, characterized in that, The diol is a C3-C6 aliphatic saturated diol.

3. The eye wash solution according to claim 1, characterized in that, The diol is selected from one or more of propylene glycol, butanediol, pentanediol, and hexanediol.

4. The eye wash solution according to claim 1, characterized in that, The mass ratio of silk fibroin, sodium hyaluronate and diol is 4-6:2-4:

20.

5. The eye wash solution according to claim 1, characterized in that, The eye wash solution comprises: 0.05% silk fibroin, 0.03% sodium hyaluronate, 0.02% vitamin B6, 0.005% borneol, 0.2% diol, 0.7% sodium chloride, and the remainder is purified water.

6. The eye wash solution according to claim 1, characterized in that, The molecular weight of the sodium hyaluronate is 500-1500 kDa.

7. The method for preparing the eye wash solution according to any one of claims 1-6, characterized in that, The method includes the following steps: (1) Mix some purified water and sodium hyaluronate evenly, add vitamin B6 and sodium chloride to obtain the first mixture; (2) Mix the diol and borneol to obtain a second mixture; (3) Mix the first mixture and the second mixture, add silk fibroin, add purified water, stir, filter through a 0.22 μm filter membrane to obtain the eye wash solution.

8. The eye wash solution according to claim 7, characterized in that, The method for preparing the silk fibroin includes: (1) Take degummed filaments and add lithium bromide solution with a concentration of 40-60% w / w at a solid-liquid ratio of 1g:3-6mL. Heat and stir at 120±5℃ for 4-6 hours. (2) The solution from step (1) is subjected to ultrafiltration desalting, with the ultrafiltration column having a molecular weight cutoff of 10-15 kDa; (3) Add the ultrafiltration solution from step (2) to phosphate buffer. The final concentration of phosphate buffer is 1-10 mM and the solid content of silk fibroin is 2-4% w / w. (4) Heat the solution from step (3) at 120±5℃ for 4-6 hours, freeze-dry the solution, and obtain the silk fibroin.

9. The method for preparing the eye wash solution according to claim 7, characterized in that, In step (1), the mass of the purified water is 50-75% of the total mass of purified water.

10. The use of the eye wash solution according to any one of claims 1-6 in the preparation of a medicament for treating dry eye syndrome.

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