Recombinant collagen contact lens lubricating liquid and application thereof

Abstract

The invention provides a recombinant collagen contact lens lubricating liquid and application thereof. The recombinant collagen contact lens lubricating liquid comprises recombinant III-type collagen, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, polyvinyl alcohol and water for injection, the recombinant collagen contact lens lubricating liquid can be safely and effectively used for moisturizing and lubricating contact lenses, improving and relieving dry eyes and improving the comfort level of wearing the lenses; besides, the recombinant collagen contact lens lubricating liquid can repair cornea and conjunctiva injury, can maintain long-term stability under a normal-temperature storage condition, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of medical devices and eye care, and particularly relates to a recombinant collagen contact lens lubricating liquid and application thereof. Background Art

[0002] Due to lifestyle and genetic factors, the number of people with myopia is increasing year by year, especially among adolescents, whose age is gradually decreasing and the incidence rate is rapidly increasing. Myopia has become a global medical and health issue and a hot topic in society. With the growing demand for myopia prevention and control and refractive correction, the number of contact lens wearers is increasing, with a trend of younger age and longer-term wear.

[0003] Corneal contact lenses can be divided into hard contact lenses and soft contact lenses according to their hardness or softness. A World Health Organization research report shows that in recent years, the proportion of people choosing hard contact lenses to correct various types of refractive errors has been growing rapidly worldwide. Hard contact lenses mainly include hard gas permeable contact lenses, hard gas permeable contact lenses for orthokeratology, scleral lenses and other types. Orthokeratology lenses, commonly known as OK lenses, are a special type of hard gas permeable contact lenses suitable for adolescents aged 8-18. They are customized products that temporarily change the shape of the cornea to temporarily reduce the degree of myopia and are used to prevent and delay myopia. After wearing orthokeratology lenses, the curvature of the central area of the cornea becomes flattened within a certain range, thereby temporarily reducing the degree of myopia by a certain amount. It is a reversible non-surgical physical correction treatment method. Modern orthokeratology lenses reshape the cornea by wearing them at night. This wearing method has little foreign body sensation, is easy to adapt to, and lenses are not easily lost. Wearers no longer need to wear frame glasses or soft contact lenses to correct their vision during the day, making it convenient for them to participate in sports and recreational activities. However, due to the uneven quality of fitting institutions, insufficient professional knowledge, a lack of technical personnel, and a lack of safety management and supervision, the efficacy and safety (quality) issues of contact lenses and their supporting products are particularly prominent. At the same time, orthokeratology lenses have high requirements for lens care hygiene and require the use of products such as care solutions and lubricants. If cleaning and care are not in place, it may cause eye inflammation and other problems.

[0004] The lubricating products currently on the market are mainly sodium hyaluronate and artificial tears. Although these products can greatly improve the lubricity of the ocular surface, they do not have the activity of actively repairing the cornea and conjunctiva. In recent years, studies have found that collagen has good lubricating properties and can effectively promote the repair of epithelial cells and peripheral nerve fibers in human tissues and organs, and recombinant collagen has a positive effect on the migration of human corneal epithelial cells in vitro. The prior art also reports a new type of bioengineered cornea made of collagen extracted from pig skin that has been highly purified and strictly sterilized. The researchers stabilized the loose collagen molecules to form a strong and transparent material that can withstand surgical operations during the processing and implantation of the eye. It can not only be mass-produced, but also effectively improve the corneal thickness and curvature of the visually impaired, successfully helping blind patients regain their sight, proving that natural animal-derived collagen has a repair effect on ocular surface tissue and has a high degree of biocompatibility.

[0005] Simpson's team sequentially added recombinant human collagen type III (RHCIII) aqueous solution, 2-morpholinoethanesulfonic acid, and 2-methacryloyloxyethyl phosphorylcholine (MPC) to a syringe mixing system to prepare RHCIII-MPC hydrogels at 0°C. After cross-linking, the gel was demolded and cleaned, then bottled in phosphate-buffered saline (PBS) or PBS containing 1% chloroform (C-PBS). The PBS bottles were irradiated with 17 kGy electron beams. The C-PBS and 17 kGy-irradiated implants were used in rabbits. The study showed that the implants successfully re-epithelialized and enhanced the regeneration of corneal epithelium, stroma, and nerves, demonstrating great potential for corneal treatment. Malhotra also prepared composite implants based on recombinant human type III collagen and 2-methacryloyloxyethyl phosphorylcholine by introducing KR-12-releasing silica nanoparticles, which reduced the spread of herpes simplex virus type 1 (HSV-1) and promoted the repair of HSV-1-infected corneas.

[0006] While recombinant collagen technology overcomes the poor solubility and high immunogenicity of animal-derived collagen, its long-term stability in aqueous solutions remains a challenge. During storage, recombinant collagen is easily hydrolyzed into short peptides and amino acids in aqueous solutions, compromising product efficacy. Therefore, there is a need to develop an ophthalmic lubricant with excellent ocular surface lubrication, the ability to actively repair corneal and conjunctival damage, and the ability to maintain long-term stability under ambient temperature storage conditions. Summary of the Invention

[0007] In response to some deficiencies in the prior art, the present invention provides a recombinant collagen contact lens lubricant and its application; the recombinant collagen contact lens lubricant comprises recombinant type III collagen, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, polyvinyl alcohol and water for injection; the recombinant collagen contact lens lubricant can be safely and effectively used for moisturizing and lubricating contact lenses, improving and relieving dry eyes, and enhancing the comfort of wearing lenses; in addition, the recombinant collagen contact lens lubricant can also repair corneal and conjunctival damage and maintain long-term stability under room temperature storage conditions, and has good market application prospects.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical means:

[0009] The invention first provides a recombinant collagen contact lens lubricating liquid, which comprises recombinant collagen, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, polyvinyl alcohol and water for injection.

[0010] Preferably, each liter of recombinant collagen contact lens lubricant comprises 2.5-10 g of polyvinyl alcohol, 4.25-17 g of sodium chloride, 0.03-0.12 g of disodium hydrogen phosphate, 0.7-2.8 g of sodium dihydrogen phosphate and 20-80 g of recombinant collagen, with the remainder being water for injection.

[0011] Preferably, each liter of recombinant collagen contact lens lubricant comprises 5 g of polyvinyl alcohol, 8.5 g of sodium chloride, 0.06 g of disodium hydrogen phosphate, 1.4 g of sodium dihydrogen phosphate and 40 g of recombinant collagen, with the remainder being water for injection.

[0012] Preferably, the recombinant collagen includes recombinant type III collagen, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0013] Preferably, the preparation method of the recombinant collagen contact lens lubricant comprises:

[0014] Mix polyvinyl alcohol, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate, then add water for injection, pre-mix under stirring conditions, then add recombinant collagen at room temperature, mix evenly, filter, fill, and store at room temperature.

[0015] Preferably, the premixing is carried out by stirring at 80° C. and 300 rpm for 2 to 3 hours.

[0016] Preferably, the filtration is: filtering through 0.45 μm and 0.22 μm filter membranes in sequence.

[0017] The present invention also provides the use of the recombinant collagen contact lens lubricant as a contact lens lubricant.

[0018] Preferably, the contact lens comprises any one of a soft contact lens, a hard contact lens, a colored contact lens and a silicone hydrogel contact lens.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The recombinant type III collagen used in the recombinant collagen contact lens lubricant described herein contains a large number of hydrophilic groups, which enhance the lubricating and moisturizing properties of the lubricant, moisturizing the ocular surface, reducing friction between the ocular surface and the lens, and effectively alleviating symptoms such as dry eyes. Furthermore, the recombinant collagen exhibits strong cell adhesion, guiding epithelial cell migration into the defect area, enhancing the adhesion and cell stability of epithelial cells surrounding the corneal / conjunctival injury, thereby accelerating the healing of corneal / conjunctival injuries. Furthermore, the recombinant type III collagen is sequence-stable in aqueous solution, is not easily degraded, and maintains long-term stability under room temperature storage conditions.

[0021] The present invention compounds recombinant collagen with polyvinyl alcohol to produce the recombinant collagen contact lens lubricant. The combination of recombinant collagen and polyvinyl alcohol enhances the film-forming properties and viscosity of the contact lens when used alone, forming a protective film on the corneal / conjunctival surface to protect the underlying tissues and prolong its residence time on the ocular surface.

[0022] The recombinant collagen contact lens lubricant of the present invention is non-irritating and non-cytotoxic to the eyes, can safely and effectively relieve dry eyes and discomfort caused by wearing contact lenses, and improve the comfort of wearing lenses; in addition, the recombinant collagen contact lens lubricant can also repair corneal and conjunctival damage and maintain long-term stability under room temperature storage conditions, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart of the preparation process of the recombinant collagen contact lens lubricant.

[0024] Figure 2 These are pictures of cells before and after cell migration.

[0025] Figure 3 is the moisturizing rate of different lubricants.

[0026] Figure 4 This is the result of corneal sign scoring.

[0027] Figure 5 This is a diagram showing the corneal healing rate.

[0028] Figure 6These are the results of corneal HE staining after lubricant treatment; Figure a is recombinant collagen contact lens lubricant; b is sodium hyaluronate contact lens lubricant; and c is normal saline.

[0029] Figure 7 This is the result of Masson staining of the cornea after lubricant treatment; in the figure, a is recombinant collagen contact lens lubricant; b is sodium hyaluronate contact lens lubricant; c is normal saline. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the embodiments of the present invention, all that is not explained in detail is accomplished using conventional experimental methods, and the processes involved in the embodiments are understood and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the art, and therefore will not be described in detail.

[0031] The collagen used in Examples 1 to 3 is recombinant type III collagen, which is sourced from Jiangsu Chuangjian Medical Technology Co., Ltd., and its amino acid sequence is shown in SEQ ID NO: 1; sodium chloride is sourced from Jiangsu Qinfen Pharmaceutical Co., Ltd.; sodium dihydrogen phosphate and disodium hydrogen phosphate are sourced from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd.; and polyvinyl alcohol is sourced from Sichuan Laite Juxin Pharmaceutical Excipients Co., Ltd.

[0032] SEQ ID NO: 1

[0033] GPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGE KGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESG KPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPP GPVGPAGKSGAPGPQGPRGDKGETGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGPQGVKGESGKPGANGLSGENGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGG

[0034] Example 1: Preparation of recombinant collagen contact lens lubricant

[0035] Formula for 1L recombinant collagen contact lens lubricant:

[0036]

[0037] The preparation process of 1L recombinant collagen contact lens lubricant is as follows Figure 1 As shown, the specific steps include:

[0038] The above-mentioned polyvinyl alcohol, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate are added to a beaker in sequence, and then water for injection is added to the beaker, and stirred at 80°C and 300 r / min for 2 to 3 hours for pre-mixing. After mixing, it is cooled to room temperature, and then recombinant type III collagen is added thereto, and stirred at 330 r / min for 10 to 20 minutes until mixed uniformly to obtain a mixed liquid. The mixed liquid is filtered through 0.45 μm and 0.22 μm filter membranes in sequence to obtain the recombinant collagen contact lens lubricant, which is then filled and stored at room temperature.

[0039] Formula for 1L recombinant collagen contact lens lubricant:

[0040]

[0041] Preparation of 1L recombinant collagen contact lens lubricant:

[0042] The above-mentioned polyvinyl alcohol, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate are added to a beaker in sequence, and then water for injection is added to the beaker, and stirred at 80°C and 300 r / min for 2 to 3 hours for pre-mixing. After mixing, it is cooled to room temperature, and then recombinant type III collagen is added thereto, and stirred at 330 r / min for 10 to 20 minutes until uniformly mixed to obtain a mixed liquid. The mixed liquid is filtered through 0.45 μm and 0.22 μm filter membranes in sequence to obtain the recombinant collagen contact lens lubricant, which is then filled and stored at room temperature.

[0043] Example 3: Preparation of recombinant collagen contact lens lubricant

[0044] Formula for 1L recombinant collagen contact lens lubricant:

[0045]

[0046] Preparation of 1L recombinant collagen contact lens lubricant:

[0047] The above-mentioned polyvinyl alcohol, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate are added to a beaker in sequence, and then water for injection is added to the beaker, and stirred at 80°C and 300 r / min for 2 to 3 hours for pre-mixing. After mixing, it is cooled to room temperature, and then recombinant type III collagen is added thereto, and stirred at 330 r / min for 10 to 20 minutes until uniformly mixed to obtain a mixed liquid. The mixed liquid is filtered through 0.45 μm and 0.22 μm filter membranes in sequence to obtain the recombinant collagen contact lens lubricant, which is then filled and stored at room temperature.

[0048] Example 4: Toxicity Study of Recombinant Collagen Contact Lens Lubricant

[0049] In this example, physiological saline was used as a blank control group, recombinant collagen solution was used as control group 1, polyvinyl alcohol solution was used as control group 2, and sodium hyaluronate contact lens lubricant (Xindong Biotechnology Co., Ltd.) was used as control group 3 to investigate the cytotoxicity of recombinant collagen contact lens lubricant.

[0050] The preparation method of the recombinant collagen solution is as follows: 8.5g of sodium chloride, 0.06g of disodium hydrogen phosphate and 1.4g of sodium dihydrogen phosphate are added to a beaker in sequence, and then 950.04g of water for injection is added to the beaker, and stirred at 80°C and 300r / min for 2 to 3h for pre-mixing. After the mixing is completed, it is cooled to room temperature, and then 40g of recombinant type III collagen is added thereto, and stirred at 330r / min for 10 to 20min until the mixture is uniformly mixed to obtain a mixed liquid. The mixed liquid is filtered through 0.45μm and 0.22μm filter membranes in sequence to obtain the recombinant collagen solution, which is then filled and stored at room temperature.

[0051] The preparation steps of the polyvinyl alcohol solution are as follows: 5g of polyvinyl alcohol, 8.5g of sodium chloride, 0.06g of disodium hydrogen phosphate and 1.4g of sodium dihydrogen phosphate are weighed and added to a beaker in sequence, and then 950.04g of water for injection is added to the beaker, and stirred for 2 to 3 hours at 80°C and 300r / min for premixing. After mixing, the mixture is cooled to room temperature, filtered through 0.45μm and 0.22μm filter membranes in sequence to obtain the polyvinyl alcohol solution, filled, and stored at room temperature.

[0052] Preparation of sample extracts: physiological saline, recombinant collagen solution, polyvinyl alcohol solution, sodium hyaluronate contact lens lubricant and recombinant collagen contact lens lubricant were added to DMEM culture medium (Gibco) containing 15% fetal bovine serum (Gibco) at a concentration of 0.2 g / mL, and the mixture was shaken and extracted for 24 hours. After the extraction, the sample extracts were obtained, namely, physiological saline extract, recombinant collagen solution extract, polyvinyl alcohol solution extract, sodium hyaluronate contact lens lubricant extract and recombinant collagen contact lens lubricant extract.

[0053] Mouse epithelial fibroblasts (L-929, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were cultured. The cultured cells were removed from the culture flask using trypsin (Gibco) and centrifuged at 1000 rpm for 5 min. The supernatant was removed and the cell pellet was mixed with DMEM complete medium (Gibco) to prepare a cell suspension with a concentration of 1 × 10 5 Cells were added to a 96-well plate, with 100 μL of cell suspension per well. The cell density to be tested was 1×10 4 cells / well, cultured in a 37°C incubator with 5% CO2 for 24 h.

[0054] After the incubation period, the culture medium was aspirated and 100 μL of the above-mentioned physiological saline extract, recombinant collagen solution extract, polyvinyl alcohol solution extract, sodium hyaluronate contact lens lubricant extract, and recombinant collagen contact lens lubricant extract were added to each well. After incubation in the incubator for 24 hours, the extracts in the wells were removed and 50 μL of MTT solution (Sigma, 1 mg / mL) was added to each test well. The plate was incubated in a 37°C incubator for 2 hours. The MTT solution was then discarded and 100 μL of isopropanol solution (Chinese medicine) was added to each well. The plate was shaken and the absorbance was measured on a microtiter plate photometer with a 570 nm filter.

[0055] The cell viability of the blank control group was 98.77%, that of control group 1 was 97.20%, that of control group 2 was 89.66%, that of control group 3 was 97.17%, and that of the experimental group was 98.20%. This indicates that both recombinant collagen and polyvinyl alcohol have good cell viability.

[0056] Example 5: Cell proliferation and cell migration assay

[0057] In this example, physiological saline was used as a blank control group, recombinant collagen solution was used as control group 1, polyvinyl alcohol solution was used as control group 2, and sodium hyaluronate contact lens lubricant was used as control group 3 to investigate the cell proliferation and cell migration properties of the recombinant collagen contact lens lubricant.

[0058] (1) Cell proliferation assay:

[0059] The preparation methods of the recombinant collagen solution and the polyvinyl alcohol solution are shown in Example 4.

[0060] Preparation of sample extracts: Physiological saline, recombinant collagen solution, polyvinyl alcohol solution, sodium hyaluronate contact lens lubricant and recombinant collagen contact lens lubricant were added to DMEM culture medium (Gibco) at a concentration of 0.2 g / mL, shaken and extracted for 24 hours. After the extraction, the sample extracts were obtained, namely, physiological saline extract, recombinant collagen solution extract, polyvinyl alcohol solution extract, sodium hyaluronate contact lens lubricant extract and recombinant collagen contact lens lubricant extract.

[0061] Human corneal epithelial cells (HCET, Wuhan Punosai Life Science Co., Ltd.) were cultured and removed from the culture flask using trypsin (Gibco). The cells were centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The cell pellet was mixed with DMEM-F12 basal medium (Gibco) to prepare a cell suspension, and its concentration was adjusted to 1 × 10 5Cells were added to a 96-well plate, with 100 μL of cell suspension per well. The cell density to be tested was 1×10 4 Cells / well, 5% CO2, 37 ° C incubator culture for 24h. After the culture is completed, the culture medium is aspirated, and 100 μL of physiological saline extract, recombinant collagen solution extract, polyvinyl alcohol solution extract, sodium hyaluronate contact lens lubricant extract and recombinant collagen contact lens lubricant extract are added to each well respectively. After leaching for 24h, the culture medium is removed, and 50 μL of MTT solution (Sigma, 1mg / mL) is added to each test well, and the plate is incubated in a 37 ° C incubator for 2h. The MTT solution is then discarded, and 100 μL of isopropanol solution (Chinese medicine) is added to each well. The plate is shaken and placed on a microtiter plate photometer with a 570nm filter to measure absorbance. Repeat the above operation to measure cell viability after 48h of extraction. The test results are shown in Table 1.

[0062] Table 1. Cell proliferation investigation results

[0063] Group 24h cell viability 48h cell viability Blank control group 100％ 103.67％ Control group 1 64.4％ 71.0％ Control group 2 37.7％ 40.2％ Control group 3 67.4％ 74.8％ Experimental Group 1 75.6％ 81.8％

[0064] As can be seen from Table 1, the recombinant collagen contact lens lubricant can enhance the proliferation activity of corneal epithelial cells.

[0065] (2) Cell migration assay:

[0066] First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with a ruler, about every 0.5cm to 1cm, cross the holes horizontally, and cross 3 lines in each hole. Add about 8×10 5 Human corneal epithelial cells (HCET, purchased from Wuhan Punosai Life Science Technology Co., Ltd.) were cultured for 24 h. A 10 μL pipette tip was used to align the well plate vertically with a ruler, and the longitudinal line was gently pushed downward to form a scratch. The cells were rinsed three times with PBS to remove the scratched cells. 2 mL of the physiological saline extract, recombinant collagen solution extract, polyvinyl alcohol solution extract, sodium hyaluronate contact lens lubricant extract, and recombinant collagen contact lens lubricant extract prepared in step (1) were added, respectively, and cultured in a 37°C, 5% CO2 incubator. After 0 h and 24 h, the intersection of the transverse and longitudinal lines was used as the core and photographed under a 40x microscope. Photos of 9 parts were obtained in each well, and 27 data were collected in each group.

[0067] The scratch area of each image was calculated using ImageJ image processing software, and the migration rate of each group of cells was calculated by dividing the total area of the cells migrating in the fixed scratch area by the initial area of the fixed scratch area. The time was used as the horizontal axis and the migration area ratio was used as the vertical axis (unit %). The photos of the experimental group and the control group at the initial time 0 and the end of the experiment were compared. The data differences of each experimental group were analyzed using one-way variance and chi-square test. The results are shown in Table 2 and Figure 2 shown.

[0068] Table 2. Cell migration investigation results

[0069] Group Migration area Migration Blank control group 66.29 <![CDATA[43.75% bc ]]> Control group 1 82.12 <![CDATA[51.13% b ]]> Control group 2 31.47 <![CDATA[24.64% d ]]> Control group 3 52.27 <![CDATA[35.73% c ]]> Experimental Group 1 99.28 <![CDATA[63.51% a ]]>

[0070] Combined with Table 2 and Figure 2 It can be seen that recombinant collagen contact lens lubricant can enhance the migration activity of corneal epithelial cells.

[0071] In summary, recombinant collagen contact lens lubricant has good proliferation activity and migration activity.

[0072] Example 6: Lubricity and Moisturizing Test of Recombinant Collagen Contact Lens Lubricant

[0073] (1) Lubricity test:

[0074] The recombinant collagen contact lens lubricant and sodium hyaluronate contact lens lubricant were tested according to the test conditions (condition A) in "NB / SH / T0189-2017 Determination of the anti-wear performance of lubricating oils - Four-ball method".

[0075] Note: Samples were sent to an outsourced four-ball friction testing facility, Hangzhou Yanqu Information Technology Co., Ltd. The four-ball friction testing machine uses four steel balls arranged in an equilateral tetrahedron and loaded upwards via a hydraulic system. During the test, the contact points of all four balls are immersed in lubricant. After the test, the wear scar diameter of each ball within the oil chamber is measured until an indicator representing the lubricant's load-carrying capacity is determined. The test results are shown in Table 3.

[0076] Table 3. Lubricity results of recombinant collagen contact lens lubricant

[0077] sample Friction coefficient / μ Diameter of friction spot / mm Recombinant collagen contact lens lubricant <![CDATA[0.1978 * ]]> <![CDATA[1.2117 * ]]> Sodium hyaluronate contact lens lubricant 0.3517 1.3939

[0078] As can be seen from Table 3, the friction coefficient and friction spot diameter of the recombinant collagen contact lens lubricant are smaller than those of the sodium hyaluronate contact lens lubricant (p<0.05), so the lubricating effect of the recombinant collagen contact lens lubricant is significantly better than that of the sodium hyaluronate contact lens lubricant.

[0079] (2) Moisture test:

[0080] S1. Weigh a certain amount of agar powder to prepare a 5 wt% agar solution, heat to dissolve, pour into a culture dish, let it solidify, and weigh it (W1).

[0081] S2. Evenly apply 1 mL of 0.9% saline, recombinant collagen contact lens lubricant, and sodium hyaluronate contact lens lubricant respectively on a Petri dish containing solid agar, weigh the mass (W2) using an analytical balance, and then place the dish in a 37°C oven.

[0082] S3. Place the culture dish in a constant temperature and humidity chamber at 25°C and 40% relative humidity. After 0h, 2h, 4h, 6h, and 8h, remove the culture dish and weigh the mass (W3). After weighing, immediately place the dish in the constant temperature and humidity chamber.

[0083] S4. Calculate its moisturizing rate, the results are shown in Table 4 and Figure 3 shown.

[0084] Moisturizing rate (%) = (W2-W3) / (W2-W1)*100%

[0085] W1 is the weight of agar and Petri dish (g), W2 is the weight of sample and agar plus Petri dish before the experiment (g), and W3 is the weight of sample and agar plus Petri dish after the specified time interval (g).

[0086] Table 4. Moisturizing rate of different lubricants

[0087]

[0088] Combined with Table 4 and Figure 3 It can be seen that with the extension of drying time, the moisturizing rate of each component decreased significantly, but compared in each time period, the moisturizing property of recombinant collagen contact lens lubricant was significantly better than that of sodium hyaluronate contact lens lubricant and normal saline (p<0.05).

[0089] In summary, recombinant collagen contact lens lubricant has good lubricating and moisturizing properties.

[0090] Example 7: Effectiveness of recombinant collagen contact lens lubricant

[0091] In this example, physiological saline was used as a blank control group, sodium hyaluronate contact lens lubricant (purchased from Xindong Biotech Co., Ltd.) was used as a positive control group, and the recombinant collagen contact lens lubricant prepared in Example 1 was used as an experimental group. The effectiveness of the recombinant collagen contact lens lubricant of the present invention was investigated through animal experiments. The specific steps are as follows:

[0092] Twelve healthy New Zealand white rabbits (half male and half female, weighing about 2.0-2.5 kg, purchased from Hunan Taiping Biotechnology Co., Ltd., license number: SCXK (Xiang) 2020-0005) were randomly divided into three groups: blank control group, positive control group and experimental group, with 4 rabbits in each group.

[0093] New Zealand white rabbits were adaptively reared for 7 days, and their eyes were checked for signs of inflammatory reactions and other pathological changes. A rabbit corneal injury model was prepared. The specific preparation steps were as follows: the New Zealand white rabbits were anesthetized with an intraperitoneal injection of sodium pentobarbital, and the right eye was anesthetized with 0.4% oxybuprocaine hydrochloride eye drops (Santen) twice for corneal surface anesthesia. Then, an 8mm corneal trephine was used to make a circular incision on the right cornea. The 8mm corneal trephine was used to incise the cornea, and the epithelium of the damaged cornea was carefully removed with microscope forceps and fine eye scissors. Lincomycin hydrochloride eye drops (Wuhan Wujing) were then applied to the eye for 3 days. The left eye of the rabbit was also anesthetized and instilled with lincomycin hydrochloride eye drops to obtain the rabbit corneal injury model.

[0094] Administration: New Zealand white rabbits treated with the aforementioned corneal injury model were treated with two drops of saline, sodium hyaluronate contact lens lubricant, and recombinant collagen contact lens lubricant, three times daily for 13 consecutive days. Observations were performed on days 1, 3, 5, 7, 9, 11, and 13 of treatment, and corneal signs were scored and the area of corneal epithelial damage was measured.

[0095] Among them, the grading score table is shown in Table 5, and the corneal sign scoring results are shown in Figure 4 shown.

[0096] Table 5. Grading score table

[0097] Corneal opacity Corneal fluorescent staining area 0 points The cornea is clear and has no turbidity No corneal pigmentation 1 point The cornea is slightly cloudy and the iris texture is visible Corneal staining area is less than or equal to 1 / 4 2 points Moderate corneal opacity and unclear iris texture Corneal staining area is greater than 1 / 4 and less than 1 / 2 3 points Moderate corneal opacity, pupil is not visible Corneal staining area is greater than 1 / 2 and less than 3 / 4 4 points Moderate corneal opacity and invisible pupil Corneal staining area is greater than 3 / 4 to the entire cornea

[0098] from Figure 4 As can be seen, there were no significant differences in scores between the saline group and the control group during the first five days of administration, but scores decreased on the seventh day. There were no significant differences in scores between the sodium hyaluronate contact lens lubricant group and the control group during the first three days of administration, but scores decreased significantly on the fifth (p < 0.05) and seventh (p < 0.01) days of administration. There were no significant differences in scores between the recombinant collagen contact lens lubricant group and the control group during the first three days of administration, but scores increased on the fifth (p < 0.05) day of administration and decreased significantly on the seventh (p < 0.05) day of administration. In summary, recombinant collagen contact lens lubricant is more effective in repairing corneal damage than hyaluronic acid contact lens lubricant and saline.

[0099] This example also investigated the corneal healing rate after treatment with physiological saline, sodium hyaluronate contact lens lubricant, and recombinant collagen contact lens lubricant. The results are shown in Tables 6 and Figure 5 shown.

[0100] Table 6. Corneal healing rates of different lubricants

[0101] Group Recombinant collagen contact lens lubricant Sodium hyaluronate contact lens lubricant Normal saline Healing rate 62.45％ 61.09％ 52.57％*

[0102] Combined with Table 6 and Figure 5 As can be seen, the corneal injury healing rates of both the sodium hyaluronate contact lens lubricant and recombinant collagen contact lens lubricant groups increased during the administration period, and the healing rates of each group were basically the same, with no significant differences. In summary, sodium hyaluronate contact lens lubricant and recombinant collagen contact lens lubricant are equally effective in healing rabbit corneal injuries, and both are superior to the saline group (p < 0.05).

[0103] In this embodiment, after the treatment cycle, corneal tissue was collected, embedded in paraffin, and sliced. The morphological changes of corneal tissue were observed under a light microscope using HE and Masson staining. The staining results are as follows: Figure 6 and 7 shown.

[0104] from Figure 6 As can be seen, the corneal tissue structure of the samples in the recombinant collagen contact lens lubricant group was normal, with inflammatory cell infiltration in the stroma and capillary congestion in some samples. In the sodium hyaluronate contact lens lubricant group, only a small amount of inflammatory cell infiltration was seen in some samples, and some samples showed corneal tissue disorganization, epithelial cell edema, and capillary congestion. In the normal saline group, the corneal tissue had epithelial cell edema, a disordered stroma, and a large amount of inflammatory cell infiltration. This shows that recombinant collagen contact lens lubricant and sodium hyaluronate contact lens lubricant have a certain degree of effect on corneal structural restoration.

[0105] from Figure 7 It can be seen that compared with the normal saline group, the collagen fiber area of each drug group showed a downward trend, indicating that each drug has a certain degree of effect on the treatment of corneal damage, but there is no significant difference between them.

[0106] In conclusion, recombinant collagen contact lens lubricant is effective in healing corneal injuries.

[0107] Example 8: Stability Test

[0108] In this example, the recombinant collagen contact lens lubricant prepared in Example 1 was placed in an environment of 40°C and 40% RH and tested at 0, 1, 2, 3, 6, and 9 months. The test items and requirements are shown in Table 7, and the test results are shown in Table 8.

[0109] Table 7. Stability testing items and requirements for recombinant collagen contact lens lubricant

[0110]

[0111]

[0112] Table 8. Stability test results of recombinant collagen contact lens lubricant

[0113]

[0114] As can be seen from Table 8, the main active ingredient of the recombinant collagen contact lens lubricant, recombinant collagen, is not easily degraded under high-temperature accelerated conditions, its content and purity remain stable, and other indicators are within the quality control range, proving that this product has long-term stability.

[0115] In summary, the recombinant collagen contact lens lubricant of the present invention can be safely and effectively used to moisten and lubricate contact lenses, improve and relieve dry eyes, and enhance the comfort of wearing lenses. In addition, the recombinant collagen contact lens lubricant can also actively repair corneal and conjunctival damage and maintain long-term stability under room temperature storage conditions, thus having good market application prospects.

[0116] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. A recombinant collagen contact lens lubricant, characterized in that: The recombinant collagen contact lens lubricating liquid comprises: recombinant collagen, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, polyvinyl alcohol and water for injection.

2. The recombinant collagen contact lens lubricant according to claim 1, characterized in that Each liter of recombinant collagen contact lens lubricant includes 2.5-10g of polyvinyl alcohol, 4.25-17g of sodium chloride, 0.03-0.12g of disodium hydrogen phosphate, 0.7-2.8g of sodium dihydrogen phosphate and 20-80g of recombinant collagen, and the rest is water for injection.

3. The recombinant collagen contact lens lubricant according to claim 2, characterized in that Each liter of recombinant collagen contact lens lubricant includes 5g of polyvinyl alcohol, 8.5g of sodium chloride, 0.06g of disodium hydrogen phosphate, 1.4g of sodium dihydrogen phosphate and 40g of recombinant collagen, and the rest is water for injection.

4. The recombinant collagen contact lens lubricant according to claim 1, wherein The recombinant collagen includes recombinant type III collagen, and its amino acid sequence is shown in SEQ ID NO:

1.

5. The recombinant collagen contact lens lubricant according to claim 1, wherein The preparation method of the recombinant collagen contact lens lubricating liquid comprises: Mix polyvinyl alcohol, sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate, then add water for injection, pre-mix under stirring conditions, then add recombinant collagen at room temperature, mix evenly, filter, fill, and store at room temperature.

6. The recombinant collagen contact lens lubricant according to claim 5, characterized in that The premixing step is: stirring at 80° C. and 300 r / min for 2 to 3 hours.

7. The recombinant collagen contact lens lubricant according to claim 5, characterized in that The filtration is as follows: filtering through 0.45 μm and 0.22 μm filter membranes in sequence.

8. Use of the recombinant collagen contact lens lubricant according to any one of claims 1 to 7 as a contact lens lubricant.

9. The use according to claim 8, characterized in that The contact lens includes any one of a soft contact lens, a hard contact lens, a colored contact lens and a silicone hydrogel contact lens.

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