Water-soluble cellulose derivative-silk fibroin artificial tear with long-acting retention and non-irritating characteristics and preparation method of water-soluble cellulose derivative-silk fibroin artificial tear

Abstract

The invention discloses a water-soluble cellulose derivative-silk fibroin artificial tear with the characteristics of long-acting retention and no irritation and a preparation method thereof. The artificial tear comprises a water-soluble cellulose derivative, silk fibroin, an osmotic pressure regulator, a pH regulator and water. After the water-soluble cellulose derivative is dissolved, a transparent viscous colloid can be formed, a protective film can be formed on the ocular surface, a tear film is stabilized, tear evaporation is delayed, friction is reduced, and dryness and foreign body sensation are rapidly relieved. The polar group of the silk fibroin and the silk fibroin form a stable network through a hydrogen bond, eyelid shearing and tear erosion are resisted, the bioavailability of the silk fibroin is improved, and the silk fibroin has the effects of resisting inflammation and promoting corneal epithelium repair. The osmotic pressure regulator and the pH regulator are cooperated, so that the osmotic pressure and the pH value of the artificial tear approach to those of human tears, the ocular surface is prevented from being stimulated, and the medication comfort and compliance are improved. The artificial tear can quickly relieve the dry eye symptom, realizes long-acting cornea repair, and provides a new strategy for development of a high-efficiency long-acting eye drug delivery system.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation, specifically relating to a water-soluble cellulose derivative—silk fibroin artificial tear—with long-lasting retention and non-irritating properties, and its preparation method. Background Technology

[0002] Dry eye syndrome is a group of diseases caused by insufficient tear secretion or excessive evaporation, resulting in abnormal tear quality or quantity, decreased tear film stability, and secondary chronic ocular surface inflammation and various uncomfortable symptoms (dryness, foreign body sensation, burning pain, visual fluctuations, etc.). Epidemiological data shows that in recent years, the prevalence of dry eye syndrome in urban populations has been rising steadily at a rate of 10% to 20% per year, with the incidence rate reaching as high as 30% in some areas, seriously affecting patients' daily life and work efficiency.

[0003] Currently, clinical treatment mainly employs non-pharmacological interventions (such as warm compresses, eye cleaning, and meibomian gland massage). While these methods can improve symptoms to some extent, they are cumbersome to perform, slow to take effect, and have limited duration of action. Therefore, given that non-pharmacological interventions cannot meet the long-term needs of patients, developing a drug treatment strategy that is both highly effective and safe, with long-term retention on the ocular surface, has become an urgent need to improve the treatment efficacy of dry eye syndrome. Artificial tears can directly replenish tears, stabilize the tear film, lubricate the ocular surface, and synergistically promote epithelial repair and alleviate inflammatory responses, thus relieving the discomfort symptoms caused by dry eye syndrome in the short term. Patent ZL 202410910722.4 discloses a tear eye drop composition containing stem cell exosomes. This method involves culturing mesenchymal stem cells in a culture medium containing prostaglandin E2 and IL-4, collecting the culture supernatant, and isolating exosomes from it as bioactive components for use in eye drops. However, exosomes have poor in vitro stability and are easily cleared by eyelid movement and tear flushing, resulting in low bioavailability. For example, patent ZL202411207258.9 discloses a moistening boric acid eye drop, which uses a liposome shell made by compounding collagen with carboxymethyl chitosan. Subsequently, this is compounded with calcium disodium EDTA and hydroxypropyl methylcellulose in the boric acid complex to form a stable network structure. Finally, the combination of carboxymethyl chitosan and ethylparaben prolongs the antibacterial effect, thereby effectively protecting eye health. However, patients with dry eye syndrome need to use artificial tears for a long time to maintain the therapeutic effect. The preservatives such as ethylparaben added to this formulation are very likely to cause eye irritation, affecting long-term patient compliance and safety.

[0004] Research has found that the core pathology of dry eye lies in the hyperosmolar state of the ocular surface caused by tear film instability and its vicious cycle. Therefore, an ideal treatment strategy needs to effectively restore and maintain tear film stability. Although existing artificial tears can form a protective film on the ocular surface to alleviate hyperosmolarity, they are limited by issues such as retention time, stability, or preservatives, making it difficult to fundamentally break this vicious cycle. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a water-soluble cellulose derivative-silk fibroin artificial tear with long-lasting retention and non-irritating properties, and its preparation method. The artificial tear comprises a water-soluble cellulose derivative, silk fibroin, an osmotic pressure regulator, a pH regulator, and water. The water-soluble cellulose derivative exhibits significant shear-thinning rheological properties, which are highly compatible with the application requirements of artificial tears: it maintains a high apparent viscosity at rest, slowing the rate of drainage from the ocular surface to the lacrimal duct; during blinking, the viscosity drops sharply due to shearing, significantly reducing eyelid movement resistance, avoiding stickiness and momentary blurred vision, and significantly improving patient subjective comfort and medication adherence. By precisely controlling the concentration and compound composition of the water-soluble cellulose derivative, the overall rheological properties of the tear substitute can be controlled within a predetermined threshold range slightly lower than the viscosity of natural human tears. While ensuring eye comfort, this maximizes the adhesion and retention capacity of the material on the corneal surface, fully utilizing its inherent moisturizing and lubricating functions to quickly relieve dry eye symptoms. The abundant hydroxyl and carboxyl groups in the silk fibroin molecule can form a stable hydrogen bond network with the polar groups of water-soluble cellulose derivatives, effectively resisting the shear forces generated by eyelid movement and the washing effect of tears. This significantly improves the bioavailability of silk fibroin itself, promotes corneal epithelial cell migration, and exerts anti-inflammatory effects. Simultaneously, the excellent biocompatibility of silk fibroin supports cell migration and proliferation, enhancing ocular tolerance. Osmotic pressure regulators effectively replenish electrolyte losses caused by tear film instability and rapid tear evaporation, while pH regulators ensure that the pH of artificial tears is close to that of human tears, minimizing eye irritation. In terms of safety, the use of ultraviolet sterilization combined with single-dose packaging completely eliminates traditional preservatives, minimizing eye irritation and damage for long-term users and further ensuring medication safety. Compared to traditional artificial tears, the product of this invention can not only break the vicious cycle of dry eye and quickly relieve symptoms, but also reduce the frequency of administration through its long-lasting retention properties, thereby improving patient compliance with long-term treatment. At the same time, it minimizes the irritation caused by long-term use, providing an innovative approach to developing artificial tears with "high retention capacity, no irritation, and rapid relief" and has the potential for long-term use in the treatment of dry eye.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A method for preparing a water-soluble cellulose derivative-silk fibroin artificial tear with long-lasting retention and non-irritating properties includes the following steps: (1) Add anhydrous sodium carbonate to boiling deionized water and stir evenly, then add silk and boil to degumme; wash and dry the degummed silk, then dissolve it in lithium bromide aqueous solution or calcium chloride / anhydrous ethanol / water ternary solution, then dialyze in deionized water, and obtain silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add the osmotic pressure regulator to ultrapure water, stir to dissolve, and obtain solution S2; (3) Add the water-soluble cellulose derivative to hot ultrapure water, stir to dissolve, and then cool to obtain solution S3; (4) Mix solutions S1, S2 and S3, add pH adjuster, add ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation to obtain water-soluble cellulose derivative-silk fibroin artificial tears with long-lasting retention and non-irritating properties.

[0007] Preferably, in step (1), the concentration of the lithium bromide aqueous solution is 6.0~12.0 mol / L, and the molar ratio of calcium chloride, anhydrous ethanol and water in the calcium chloride / anhydrous ethanol / water ternary solution is (0~3):(0~4):(6~12).

[0008] Preferably, in step (2), the osmotic pressure regulator is composed of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and boric acid.

[0009] Preferably, in step (2), the osmotic pressure regulator is composed of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and boric acid in a mass ratio of (1~6):(1~8):(1~20):(1~20):(1~1600000).

[0010] Preferably, in step (3), the water-soluble cellulose derivative is sodium carboxymethyl cellulose.

[0011] Preferably, in step (3), the temperature of the ultrapure water is 40~80℃, and the cooling temperature is 15~30℃.

[0012] Preferably, in step (4), the pH adjuster is sodium hydroxide.

[0013] Preferably, in step (5), the duration of ultraviolet irradiation sterilization is 0.5 to 6.0 hours.

[0014] The innovation of this invention compared to existing technologies lies in: 1. The molecular structure of water-soluble cellulose derivatives is rich in polar groups, enabling them to efficiently adsorb and retain water through hydrogen bonding, with a water-holding capacity significantly exceeding their own weight. This property allows them to form a stable hydrating gel film on the ocular surface, continuously replenishing moisture to the dry cornea and effectively relieving dry eye symptoms. Furthermore, by precisely optimizing the concentration and ratio of water-soluble cellulose derivatives, the viscosity of the solution can be controlled within a critical range slightly lower than the viscosity of natural tears. This strategy maximizes ocular comfort while significantly enhancing the adhesion of the formulation to the ocular surface, prolonging residence time, and reducing the frequency of administration, thereby effectively improving patient adherence to long-term treatment.

[0015] 2. The numerous hydroxyl and carboxyl groups in the silk fibroin molecule can form a stable hydrogen bond network with the polar groups of water-soluble cellulose derivatives. This intermolecular interaction effectively resists the shear forces generated by eyelid movement and the washing effect of tears, significantly improving the bioavailability of the drug and facilitating its effects in promoting corneal epithelial cell migration and anti-inflammation.

[0016] 3. This invention employs an integrated process system combining ultraviolet sterilization technology with unit-dose packaging, effectively avoiding adverse reactions such as corneal epithelial damage, conjunctival inflammation, and goblet cell apoptosis caused by long-term use of preservative-containing eye drops. Furthermore, the sterile single-dose packaging fundamentally eliminates the risk of secondary contamination and microbial proliferation after opening, significantly reducing the product's potential irritation and chemical damage to the ocular surface, thereby greatly improving the safety of clinical medication. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a photograph of the sample prepared successfully and after automatic filling and sterilization, as described in Example 6 of this invention.

[0019] Figure 2 These are the shear viscosity curves of the samples described in Examples 1 and 4 of this invention.

[0020] Figure 3 This is a comparison chart of the physicochemical properties of the sample described in Example 6 of this invention and commercially available artificial tears products.

[0021] Figure 4This is an MTT result graph of the extract of the sample described in Example 6 of this invention after co-culturing with mouse fibroblasts (L929) for 1 day.

[0022] Figure 5 This is a CCK8 result image of the extracts of the samples described in Examples 1, 2, 3, 6 and 9 of this invention after co-culturing with human umbilical vein endothelial cells (HUVEC) for 1 day.

[0023] Figure 6 These are live / dead staining images of the extracts of the samples described in Examples 1, 2, 3, 6 and 9 of this invention after co-culturing with HUVECs for 1 day.

[0024] Figure 7 This is a graph showing the changes in guinea pig weight after the sample and control group were treated with eye drops for 27 consecutive days, as described in Example 6 of this invention.

[0025] Figure 8 The images show the tear secretion results after the samples described in Examples 1-9 of this invention were used on the eyes of mice with dry eye syndrome. Detailed Implementation

[0026] To better understand the present invention, preferred embodiments will be described below in conjunction with examples. It should be noted that these descriptions are intended to further illustrate the features and advantages of the present invention and do not constitute a limitation on the scope of the claims. Furthermore, it should be understood that after reading the disclosure of this invention, those skilled in the art can make various alterations or modifications to the invention, and equivalent forms of these alterations or modifications also fall within the scope defined by the appended claims.

[0027] The present invention discloses a method for preparing a water-soluble cellulose derivative-silk fibroin artificial tear with long-lasting retention and non-irritating properties, comprising the following steps: (1) Add anhydrous sodium carbonate to boiling deionized water and stir evenly, then add silk and boil to degumme; wash and dry the degummed silk, then dissolve it in lithium bromide aqueous solution or calcium chloride / anhydrous ethanol / water ternary solution, then dialyze in deionized water, and obtain silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add the osmotic pressure regulator to ultrapure water, stir to dissolve, and obtain solution S2; (3) Add the water-soluble cellulose derivative to hot ultrapure water, stir to dissolve, and then cool to obtain solution S3; (4) Mix solutions S1, S2 and S3, add pH adjuster, add ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation to obtain water-soluble cellulose derivative-silk fibroin artificial tears with long-lasting retention and non-irritating properties.

[0028] The MTT assay for cytotoxicity involved in this embodiment of the invention comprises the following steps: Artificial tear samples were accurately weighed and extracted using 0.9% sodium chloride injection (SC) as the extraction medium at a sample-to-medium ratio of 0.2 g / mL. The extraction process was carried out at a constant temperature of 37°C for 72 hours. After extraction, the resulting stock solution (i.e., 100% concentration extract) was serially diluted with SC as the diluent to prepare sample extracts of 100%, 75%, 50%, and 25% concentrations, respectively. Mouse fibroblast L-929 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 IU / mL, streptomycin 100 µg / mL) and in an incubator (5% CO2, 37°C, >90% humidity). Cells were digested with 0.25% trypsin (containing EDTA) to prepare single-cell suspensions. After centrifugation at 200g for 3 min, the cells were resuspended in the culture medium, and the cell density was adjusted to 1 × 10⁶ cells / mL. 5 Cells / mL. 100 µL of cell suspension was seeded into each well of a 96-well plate and incubated for 24 h in an incubator (5% CO2, 37°C, >90% humidity). After cell adhesion and monolayer formation, the original culture medium was discarded, and 100 µL of different concentrations of experimental sample extract (100%, 75%, 50%, 25%), blank control, positive control (100%), and negative control (100%) were added respectively. The plates were incubated at 37°C, 5% CO2, and >90% humidity for 24 h, with six replicates per group. After incubation, the 96-well plates were removed for cell morphology observation. The culture medium was then discarded, and 50 µL of MTT solution (1 mg / mL) was added to each well, with further incubation for 2 h. The supernatant was then discarded, and 100 µL of isopropanol was added to each well to dissolve the crystals. The absorbance was measured using a microplate reader at 570 nm as the dominant absorption wavelength and 650 nm as the reference wavelength. Cell viability was calculated.

[0029] The CCK8 cytotoxicity assay involved in this embodiment of the invention comprises the following steps: HUVEC cells were cultured in RPM I640 medium containing 10% FBS and 1% penicillin-streptomycin, under the same conditions as the L-929 cells described above. HUVEC cells in the logarithmic growth phase were harvested and cultured at 8 × 10⁻⁶ cells / cells. 3Cells were seeded at a density of [number] cells / well in 96-well plates, with 100 μL of culture medium added to each well. Cells were cultured for 24 h to allow adherence. The original culture medium was then discarded. Experimental groups were treated with 100 μL of culture medium containing extracts from samples 1, 2, 3, 6, and 9, respectively, while the control group received blank culture medium. Each group had six replicates. Cells were cultured at 37°C and 5% CO2 for another 24 h, followed by the addition of 10 μL of CCK8 reagent to each well for an additional 2 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated.

[0030] The live / dead staining detection test involved in the embodiments of the present invention comprises the following steps: Artificial tear samples were accurately weighed and extracted using 0.9% sodium chloride injection (SC) at a sample-to-medium ratio of 0.2 g / mL. The extraction process was carried out at a constant temperature of 37°C for 72 hours. After extraction, the resulting stock solution (i.e., 100% concentration extract) was serially diluted with SC to prepare sample extracts of 100%, 75%, 50%, and 25% concentrations. HUVEC cells were then subjected to a 2×10⁻⁶ m² / mL extraction. 4 Cells were seeded at a density of cells / well in 24-well plates (with sterile coverslips), and 500 μL of culture medium was added to each well. Cells were cultured for 24 h to allow adherence. The original culture medium was then discarded. 500 μL of culture medium containing the extracts (stock solutions) of samples from Examples 1, 2, 3, 6, and 9 were added to the experimental groups, while blank culture medium was added to the control group. Each group was divided into three replicates, and cells were cultured for another 24 h. Before staining, the staining working solution was prepared according to the instructions of the live / dead cell staining kit: Calcein-AM (live cell dye) was diluted to 2 μmol / L and PI (dead cell dye) was diluted to 4 μmol / L with PBS buffer. During staining, the culture medium in the wells was discarded, and the cells were washed twice with PBS. 500 μL of the staining working solution was added to each well, and the cells were incubated at 37°C in the dark for 15 min. After incubation, the cells were washed twice with PBS to remove unbound dye. The coverslips were removed, inverted, and placed on a slide for observation under a fluorescence microscope: live cells showed green fluorescence, and dead cells showed red fluorescence. The results were recorded by photographing and analyzing the proportion of live cells.

[0031] The guinea pig eye safety test (weight monitoring) involved in this embodiment of the invention comprises the following steps: Ten healthy guinea pigs, weighing 250-300g, half male and half female, were selected. After acclimatization for 3 days in an environment with a temperature of 22-25℃ and a humidity of 50%-60%, they were randomly divided into an experimental group (n=5) and a control group (n=5). The experimental group received the sample described in Example 6 in both eyes, while the control group received an equal volume of physiological saline. The administration method was as follows: at 9:00 AM daily, one drop (approximately 50 μL) of the sample or physiological saline was instilled into the conjunctival sac of each guinea pig's eyes using a sterile dropper, for 27 consecutive days. After instillation, the guinea pig's eyelids were gently closed for 10 seconds to prevent leakage. The guinea pigs' weight was measured before administration (day 0) and after 27 days, serving as initial and final weights, respectively. During the experiment, the guinea pigs' mental state, food and water intake, and any adverse reactions such as redness, swelling, or abnormal discharge from the eyes were observed daily.

[0032] The test for detecting tear secretion in a mouse model of dry eye involved in this invention includes the following steps: Mice were randomly divided into groups of five, including a control group. Daily, 10 μL of 0.2% benzalkonium chloride (BAC) solution was instilled into the conjunctival sacs of both eyes of mice in the experimental group using a 10 μL pipette. After instillation, the eyelids were gently pressed for 10 seconds to prevent drug spillage. The control group mice received an equal volume of sterile PBS. This treatment was repeated for 7 consecutive days. During the modeling period, the mice's behavior was observed. If mice in the experimental group exhibited dry eye-related symptoms such as frequent eye rubbing, eyelid partial closure, and increased ocular surface secretions, this was verified using a tear secretion test: one end of a 5 mm × 30 mm filter paper strip (folded at 5 mm) was inserted into the conjunctival sac of the lower eyelid of the mouse, and the wetted length of the filter paper was measured after 1 minute. If the wetted length in the experimental group was less than 5 mm, while the control group did not exhibit the above symptoms and the wetted length was greater than 5 mm, the dry eye model was considered successfully established.

[0033] Tear secretion was measured using the phenol red cotton thread method. A 5mm segment of sterile phenol red cotton thread was cut. One end of the thread was held with ophthalmic forceps, and the effective end was vertically inserted into the conjunctival sac of the lower eyelid of the mouse (avoiding contact with the cornea), while the other end hung down naturally. After 1 minute, the thread was gently removed with ophthalmic forceps, and the length of the tear-infiltrating segment was immediately measured with a ruler. Measurements were performed on both eyes of each mouse, with three repetitions for each eye. The average of the three measurements was taken as the tear secretion of that eye, and the average of both eyes was taken as the final tear secretion of a single mouse.

[0034] Example 1: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1g of sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 1 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 6.25 mg of pH adjuster NaOH, then add 4 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0035] Example 2: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1g of sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 2 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 12.5 mg of pH adjuster NaOH, then add 3 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0036] Example 3: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1g of sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 3 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 25 mg of pH adjuster NaOH, then add 2 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0037] Example 4: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.25g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 2 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 6.25 mg of pH adjuster NaOH, then add 3 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0038] Example 5: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.25g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 1 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 25 mg of pH adjuster NaOH, then add 4 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0039] Example 6: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.25g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 3 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 12.5 mg of pH adjuster NaOH, then add 2 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0040] Example 7: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.5g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 3 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 6.25 mg of pH adjuster NaOH, then add 2 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0041] Example 8: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.5g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 1 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 12.5 mg of pH adjuster NaOH, then add 4 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0042] Example 9: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.5g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (4) Mix 2 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 25 mg of pH adjuster NaOH, then add 3 mL of ultrapure water, stir well to obtain solution S4; (5) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0043] Comparative Example 1: (1) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (2) Add 1g of sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and then let it cool naturally to 25℃ to obtain solution S3; (3) Mix 5 mL of solution S2 and 15 mL of solution S3, add 6.25 mg of pH adjuster NaOH, then add 5 mL of ultrapure water and stir well to obtain solution S4; (4) Solution S4 is filled by fully automated aseptic filling and sterilized by ultraviolet irradiation for 2 hours to obtain artificial tears.

[0044] Comparative Example 2: (1) Add 17g of anhydrous sodium carbonate to 8L of boiling deionized water and stir evenly. Then add 17.125g of silk and boil to degumme for 30min. Wash the degummed silk with deionized water, dry it at 60℃ for 4h, dissolve it in 9.3mol / L lithium bromide aqueous solution, dialyze it in deionized water for 2d, and obtain a 10wt% silk fibroin aqueous solution, i.e., solution S1, after centrifugation and filtration. (2) Add 0.794 μg sodium chloride, 1.015 μg potassium chloride, 2.2 μg magnesium chloride, 1.85 μg calcium chloride and 0.39 g boric acid to 5 mL of ultrapure water, stir to dissolve, and obtain solution S2; (3) Add 1.25g sodium carboxymethyl cellulose to 15mL of ultrapure water at 60℃, stir to dissolve, and let cool naturally to 25℃ to obtain solution S3; (4) Mix 3 mL of solution S1, 5 mL of solution S2 and 15 mL of solution S3, add 6.25 mg of pH adjuster NaOH, then add 2 mL of ultrapure water, stir well to obtain solution S4; (5) The solution S4 is directly encapsulated to obtain artificial tears.

[0045] Table 1. Artificial Tear Performance Test Table

[0046] The data from Examples 1, 2, 3, 4, and 7 show that artificial tears containing different concentrations of sodium carboxymethyl cellulose exhibit significantly different viscosity characteristics. Viscosity is a key factor determining the retention time of artificial tears on the ocular surface. The viscosities of the artificial tears in Examples 1, 2, and 3 were 16.92 cP, 17.12 cP, and 17.23 cP, respectively, while the viscosity of the artificial tear in Example 4 significantly increased to 26.98 cP. This difference indicates that sodium carboxymethyl cellulose is the main component affecting the viscosity of artificial tears. Notably, the viscosity of the artificial tear in Example 7 was as high as 42.32 cP, far exceeding the Rheological behavior of commercial artificial tear solutions (…). J Cataract Refract Surg The 30 cP fuzzy threshold reported in (2021, 1;47:649−654) highlights the dominant role of sodium carboxymethyl cellulose concentration in the rheological properties of artificial tears and provides a key reference for formulation design.

[0047] Analysis of the data from Examples 3, 4, 5, 6, and 9 shows that the content of the osmotic pressure regulator is a key factor affecting the osmotic pressure of artificial tears. The osmotic pressure values ​​of the artificial tears in Examples 3, 6, and 9 are 297 mOsmol / kg, 304 mOsmol / kg, and 315 mOsmol / kg, respectively, all within the physiologically tolerable range for human use and suitable for practical applications. However, the osmotic pressures of the artificial tears in Examples 4 and 5 are 108 mOsmol / kg and 189 mOsmol / kg, respectively, which are significantly lower. Although hypotonic solutions can temporarily relieve dryness of the ocular surface, long-term or frequent use may cause edema or even rupture of ocular epithelial cells, thus adversely affecting the repair of ocular tissues.

[0048] Data analysis from Example 6 and Comparative Example 1 shows that silk fibroin significantly promotes cell proliferation and effectively enhances the treatment effect of dry eye syndrome. In the repair of ocular damage in a dry eye model mouse, the artificial tear repair score of Example 6 containing silk fibroin reached 9 points, while the artificial tear repair score of Comparative Example 1 without silk fibroin was only 7 points (ocular damage scoring criteria are shown in Table 2). The clear difference in scores indicates that silk fibroin plays a crucial role in ocular surface repair and is an important functional component for improving the therapeutic effect of artificial tears.

[0049] Table 2 Ocular Injury Scoring System

[0050]

[0051] Analysis of the data from Example 6 and Comparative Example 2 shows that artificial tears without UV sterilization treatment have significantly limited therapeutic efficacy. Mouse eye injury repair experiments revealed a significantly reduced repair effect in this group of products, indicating that UV sterilization is crucial for ensuring the final therapeutic effect of artificial tears.

[0052] Figure 1 This is a schematic diagram of the artificial tears described in Example 6 after automatic filling and sterilization. As shown in the figure, the artificial tears described in Example 6 remain clear and transparent before and after packaging and sterilization. Through standardized production process control, the sample contains no visible impurities, particles, or suspended matter, and the solution system is pure and clear. The excellent stability exhibited by the sample ensures a gentle, safe, and comfortable experience for the eye tissue during eye drop use, further meeting the safety requirements for long-term use of artificial tears.

[0053] Figure 2 The figures show the shear viscosity curves of the artificial tears described in Examples 1 and 4. Sodium carboxymethyl cellulose exhibits excellent shear-thinning properties; as shown, the viscosity of the artificial tears decreases rapidly with increasing shear rate. This property not only helps the artificial tears form a firmly adhered and highly stable protective film on the ocular surface, effectively resisting tear washout and clearing effects caused by blinking, but also significantly prolongs the drug's retention time on the ocular surface. Furthermore, it avoids stickiness and blurred vision in the initial application stage, greatly improving immediate comfort during use.

[0054] Figure 3This is a comparison chart of the physicochemical properties of the artificial tears described in Example 6 and commercially available artificial tears. As shown in the figure, the light transmittance of the artificial tears in Example 6 is comparable to that of the commercially available product, while its viscosity is significantly better. Regarding other key performance indicators, the artificial tears in Example 6 have a pH value of 7.35 and an osmotic pressure of 300 mOsmol / kg; the commercially available product has a pH value of 7.3 and an osmotic pressure of 296 mOsmol / kg. Both pH values ​​and osmotic pressures are within the physiologically acceptable range for the human eye, meeting the requirements for the use of artificial tears.

[0055] Figure 4 This is a graph showing the MTT assay results of the artificial tear extract described in Example 6 after co-culturing mouse fibroblasts (L929) for one day. The results show that the cell survival rate in the negative control group exceeded 90%, indicating that the experimental system was effective; while the cell survival rate in the 100% concentration extract treatment group was 73.8%, which was not lower than the cytotoxicity threshold (usually requiring a cell survival rate of not less than 70%), indicating that the sample had no significant cytotoxicity to L929 cells.

[0056] Figure 5 This is a graph showing the CCK8 assay results of the artificial tears extracts described in Examples 1, 2, 3, 6, and 9 after co-culturing human umbilical vein endothelial cells (HUVECs) for one day. As can be seen from the graph, all groups showed good cell proliferation. Among them, the groups with osmotic pressure within the human isotonic range (280~320 mOsmol / kg) showed even better cell proliferation.

[0057] Figure 6 These are live / dead staining results of the extracts of artificial tears described in Examples 1, 2, 3, 6, and 9 after co-culturing with HUVECs for one day. The staining images show that, consistent with the CCK-8 quantitative results, most HUVECs are stained green (live cells), indicating good biocompatibility of the sample material.

[0058] Figure 7 This graph shows the weight changes of guinea pigs after 27 consecutive days of applying the artificial tears described in Example 6 of this invention to their eyes during a guinea pig eye safety test. As can be seen from the graph, the weight change trend of the experimental group (with artificial tears applied) and the blank control group was basically the same. Throughout the experiment, the guinea pigs were observed for clinical symptoms daily, and no abnormalities were observed; all guinea pigs grew healthily. The above results indicate that the artificial tears had no adverse effects on the eyes or the whole body of guinea pigs and were non-irritating.

[0059] Figure 8The figures show the tear secretion results after the artificial tears described in Examples 1-9 were applied to the eyes of mice with dry eye syndrome. As can be seen from the figures, the baseline tear secretion of the mice with dry eye syndrome was only 5.21±0.046 mm. However, after applying the artificial tears from each example, the tear secretion of the mice was significantly increased, indicating that the artificial tears from the examples can effectively promote tear secretion and relieve dry eye symptoms.

[0060] The above embodiments are provided only to help understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make further improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0061] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a water-soluble cellulose derivative-silk fibroin artificial tear having long-lasting retention and non-irritation properties, characterized by: It comprises the following steps: (1) adding anhydrous sodium carbonate into boiling deionized water and stirring uniformly, then adding silk and boiling for degumming; after washing and drying the degummed silk, dissolving it in lithium bromide aqueous solution or calcium chloride / absolute ethanol / water ternary solution, then dialyzing in deionized water, centrifuging and filtering to obtain a silk fibroin aqueous solution, namely solution S1; (2) adding an osmotic pressure regulator into ultrapure water and stirring to dissolve, to obtain solution S2; (3) adding a water-soluble cellulose derivative into hot ultrapure water and stirring to dissolve, then cooling to obtain solution S3; (4) mixing solution S1, solution S2 and solution S3, adding a pH regulator, and then adding ultrapure water and stirring uniformly to obtain solution S4; (5) filling solution S4 through a full-automatic sterile filling device and sterilizing by ultraviolet irradiation to obtain a water-soluble cellulose derivative-silk fibroin artificial tear with long retention and non-irritation characteristics.

2. The method of claim 1, wherein: In the water-soluble cellulose derivative-silk fibroin artificial tear with long retention and non-irritation characteristics, the mass ratio of silk fibroin: osmotic pressure regulator: water-soluble cellulose derivative: pH regulator is 0.10-4.00: 0.15-4.00: 0.50-5.00: 0.05-0.

50.

3. The method of claim 1, wherein: In step (1), the concentration of the lithium bromide aqueous solution is 6.0-12.0 mol / L, and the molar ratio of calcium chloride, absolute ethanol and water in the calcium chloride / absolute ethanol / water ternary solution is 0-3: 0-4: 6-12.

4. The method of claim 1, wherein: In step (2), the osmotic pressure regulator is composed of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and boric acid.

5. The method of claim 4, wherein: The mass ratio of sodium chloride, potassium chloride, magnesium chloride, calcium chloride and boric acid is 1-6: 1-8: 1-20: 1-20: 1-1600000.

6. The method of claim 1, wherein: In step (3), the water-soluble cellulose derivative is sodium carboxymethyl cellulose.

7. The method of claim 1, wherein: In step (4), the pH regulator is sodium hydroxide.

8. The method of claim 1, wherein: In step (5), the duration of ultraviolet irradiation sterilization is 0.5-6.0 hours.

9. A water-soluble cellulose derivative-silk fibroin artificial tear with long retention and non-irritation characteristics prepared by the preparation method of any one of claims 1-8.

10. Use of the water-soluble cellulose derivative-silk fibroin artificial tear with long retention and non-irritation characteristics of claim 9 in the preparation of a medicament for treating eye diseases.

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