CN116726151B - Daily throwing eye drops for cornea damage repair and preparation method thereof - Google Patents
Daily throwing eye drops for cornea damage repair and preparation method thereof Download PDFInfo
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- CN116726151B CN116726151B CN202310232104.4A CN202310232104A CN116726151B CN 116726151 B CN116726151 B CN 116726151B CN 202310232104 A CN202310232104 A CN 202310232104A CN 116726151 B CN116726151 B CN 116726151B
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a daily throwing eye drop for cornea damage repair and a preparation method thereof, wherein the eye drop consists of the following raw materials: the recombinant human type III collagen comprises, by mass, 0.001% -0.5% of superoxide dismutase SOD, 1:0.5% -1:50 of recombinant human type III collagen, and 1:0.5% -1:20 of sodium hyaluronate, an osmotic pressure regulator, a pH regulator and injection water. The composition has the effects of promoting cell adhesion and proliferation, repairing cornea injury, removing free radicals of eyes, stabilizing lacrimal glands, relieving dry eyes, obviously improving symptoms such as eye fatigue, redness and swelling, inflammation and the like, has a simple preparation process, does not contain stimulating components such as preservative and the like, is packaged in a sterile daily throwing mode, and is convenient to use, safe and effective.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a daily throwing eye drop for repairing cornea damage and a preparation method thereof.
Background
The cornea is a avascular transparent tissue, consisting of an epithelial layer, a stromal layer, and an endothelial layer. The causes of cornea injury include chemistry, machinery, laser and the like, the pathological mechanism of the cornea injury plays a very important role in the participation of an immune system besides the direct injury effect, and immune inflammatory cells interact with immune molecules to form a complex immune inflammatory network to mediate pathological changes of cornea injury. In recent years, due to the increase of cornea trauma, the wide application of broad-spectrum antibiotics and corticosteroids in clinic and the increase of the use of cornea contact lenses, the incidence of keratitis also tends to rise year by year. The current common treatment methods are to use antibiotic eye drops, including chloramphenicol eye drops, erythromycin eye drops and 4 th-generation quinolone eye drops represented by levofloxacin, such as CN112022801A, CN107998399A, CN106902079A and CN104147592A, but the result of abusing the antibiotic eye drops is very serious, firstly, the drug resistance of pathogenic bacteria is increased, the clinical curative effect is reduced, the disease is not cured for a long time, and secondly, the antibiotics and auxiliary components thereof can influence the normal physiological environment of eyes to a certain extent, interfere the normal metabolism of cornea epithelium, even aggravate the original condition, and can not be used for a long time. Therefore, it is important to develop an eye drop which has high biosafety, excellent efficacy and convenient use.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides an eye drop which has reasonable compatibility, convenient use, safety and effectiveness, can effectively promote the repair of cornea injury, remove eye free radicals, and improve the symptoms of eye fatigue, red swelling, inflammation and the like, and provides a preparation method thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a daily throwing eye drop for repairing cornea injury, which consists of the following raw materials: the recombinant human type III collagen comprises, by mass, 0.001% -0.5% of superoxide dismutase SOD, 1:0.5-1:50 of recombinant human type III collagen, and 1:0.5-1:20 of sodium hyaluronate, an osmotic pressure regulator, a pH regulator and injection water.
As some preferred embodiments of the invention, the mass percentage of the recombinant human type III collagen is 0.01% -0.2%, the composition ratio of the superoxide dismutase SOD to the recombinant human type III collagen is 1:0.2-1:2, and the composition ratio of the sodium hyaluronate to the recombinant human type III collagen is 1:1-1:5.
As some preferred embodiments of the present invention, the pH of the eye drops is 6.5-8.0.
As some preferred embodiments of the present invention, the osmotic pressure of the eye drops is 260 to 340m0sm/L.
As some preferred embodiments of the present invention, the pH is 6.8 to 7.6, and the pH adjustor is selected from phosphate buffer and/or boric acid buffer; the osmotic pressure of the eye drops is 280-300 mOsm/L, and the osmotic pressure regulator is selected from sodium chloride and/or potassium chloride.
As some preferred embodiments of the invention, the superoxide dismutase SOD is recombinant human Cu, zn-SOD, and the specific activity of the superoxide dismutase SOD is 20000U/mg-30000U/mg.
As some preferred embodiments of the present invention, the recombinant human type III collagen has a molecular weight in the range of 25kDa to 90kDa.
As some preferred embodiments of the present invention, the sodium hyaluronate has an average molecular weight of 70 to 140 kilodaltons.
As some preferred embodiments of the present invention, the eye drops are topical products packaged in a sterile daily disposable form.
In another aspect of the present invention, there is provided the above-mentioned method for preparing a daily-throwing eye drop for repairing corneal damage, comprising the steps of:
s1: firstly weighing the osmotic pressure regulator and the pH regulator according to the formula amount, adding the osmotic pressure regulator and the pH regulator into injection water with the final volume of 40-60%, stirring to completely dissolve, filtering with a filter membrane for sterilization, and collecting filtrate;
s2: weighing the formula amount of sodium hyaluronate, adding the sodium hyaluronate into the filtrate obtained in the step S1, and stirring at room temperature until the sodium hyaluronate is completely dissolved;
s3: weighing superoxide dismutase SOD and recombinant human III type collagen according to the formula amount, filtering and sterilizing by a filter membrane, adding the superoxide dismutase SOD and the recombinant human III type collagen into the solution obtained in the step S2, supplementing the rest injection water, and uniformly stirring;
s4: and after the pH and osmotic pressure are determined to be qualified, filtering and sterilizing by a filter membrane in a sterile environment, and filling and sealing.
As some preferred embodiments of the invention, the filter is a 0.22 μm filter.
In the invention, superoxide dismutase SOD is an antioxidant metalloenzyme existing in organisms, is widely focused on the special biophysical properties and actions, and is an ideal substance for scavenging free radicals. SOD acts on human body in enzyme mode, and has the advantages of quick response, obvious effect, small side effect and the like. SOD has a protective effect on the collagen structures in the eye, which provide tension strength and structural tightness to the eye and thereby maintain the structural integrity of the eye. SOD is easy to combine with collagen, and can be removed before free radical damages the collagen, and can repair the collagen damaged by free radical, thereby fundamentally preventing eye diseases. The research shows that the specific activity of superoxide dismutase can be effectively maintained by being matched with a certain proportion of recombinant human III type collagen and sodium hyaluronate.
The recombinant human III type collagen has stronger bioactivity and biological function, can participate in migration, differentiation and proliferation of cells, and has been widely used in the fields of medicine and health such as burn, wound, cornea disease, beauty treatment, tissue repair, wound hemostasis and the like. In addition, sodium hyaluronate is added, is the only amino glycan which does not generate sulfation, is one of extracellular matrix components of connective tissue of a human body, widely exists in the human body, and is distributed at the parts of vitreous humor, aqueous humor, skin, joint synovial fluid, umbilical cord and the like, but because of easy denaturation, a protective agent or a stabilizing agent is often needed to be added. In the research, the stability can be effectively improved and the occurrence rate of denaturation can be reduced by adjusting the proportion of superoxide dismutase and sodium hyaluronate and compounding the superoxide dismutase and the sodium hyaluronate.
Sodium hyaluronate is highly hydrophilic, can form a water film to cover the surface of the eye, is combined with protein molecules, and promotes the connection and expansion of corneal epithelial cells by regulating the activity of extracellular matrix. Experiments show that the sodium hyaluronate with the molecular weight of 70-140 ten thousand daltons is beneficial to the stability of a compound system, and the sodium hyaluronate with the small molecular weight is compounded with the recombinant human type III collagen to be separated out, so that the molecular weight is too large and is not easy to be absorbed. The research preferably selects sodium hyaluronate with the molecular weight of 50-140 ten thousand daltons, can permeate into dermis, and plays important physiological functions of water retention, extracellular space maintenance, osmotic pressure regulation, lubrication and cell repair promotion in vivo. In addition, sodium hyaluronate has the effects of slightly dilating blood capillary, increasing blood circulation, improving intermediate metabolism, and promoting skin nutrition absorption. In the research, a certain proportion of sodium hyaluronate can more effectively maintain the stability of recombinant human type III collagen and superoxide dismutase SOD.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in:
the daily throwing eye drop for cornea injury repair provided by the invention has the beneficial effects that the functional components comprise superoxide dismutase, recombinant human III type collagen and sodium hyaluronate, the compatibility is more stable, the three components are synergistic, the communication function of cornea cells is repaired by regulating the biological function of extracellular matrix, the cell proliferation adhesion and the cornea injury wound surface repair are effectively promoted, the free radicals of eyes are eliminated, the lacrimal gland is stabilized, the dry eyes are relieved, and the symptoms such as eye fatigue, red swelling and inflammation are obviously improved.
The daily throwing eye drop for cornea injury repair provided by the invention has the advantages that the recombinant human type III collagen is compatible with superoxide dismutase and sodium hyaluronate, and the stability of the recombinant human type III collagen in a solution is obviously improved through verification, so that the biocompatibility of the recombinant human type III collagen, the superoxide dismutase and the sodium hyaluronate is good.
The prescription provided by the invention has stable compatibility, adopts a sterile daily-throwing type package, does not contain stimulating components such as preservative and the like, and is convenient, safe and effective to use. The invention overcomes the defect of adding preservative into the traditional eye drops, and effectively avoids superficial irritation or damage to the cornea of a normal human eye in the use process.
Drawings
FIG. 1 example 1 an electrophoretically detected pattern of an eye drop sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be clearly and completely described in connection with the following specific embodiments.
Unless otherwise indicated, the equipment used in this embodiment is conventional, the reagents used are conventional, all commercially available, and the methods of operation used are described in textbooks in the field. In the present embodiment, the superoxide dismutase and the recombinant human type III collagen are obtained by a genetic recombination technique and a microbial fermentation method. Recombinant human type III collagen is obtained by the method of CN 112851797B patent, and superoxide dismutase SOD is Cu and Zn-SOD obtained by the method disclosed by 2022116586173 patent, and is shown in preparation example 1.
PREPARATION EXAMPLE 1 preparation of superoxide dismutase SOD
S1: natural human Cu, zn-SOD escherichia coli expression construction
The amino acid sequences of human Cu and Zn-SOD (UniProtKB/Swiss-Prot: P00441.2) are taken as templates for codon optimization, and the following gene sequences are obtained:
(SEQ ID NO: 1), entrusting Jin Weizhi to synthesize a gene sequence, connecting the gene sequence to pET30a (+) through Nde I and Kpn I enzyme cutting sites to obtain pET30a (+) -SOD, transferring the pET30a (+) -SOD into BL21 (DE 3) competence, and obtaining the escherichia coli expression strain for expressing human Cu, zn-SOD.
S2: gene mutation
Error-prone PCR was performed using pET30a (+) -SOD as template, upstream primer: 5'-TAATACGACTCACTATAGGG-3', downstream primer 5'-GCTGAGCAATAACTAGC-3'.
The PCR system is as follows: 2X Taq PCR Master Mix. Mu.l, 1. Mu.l of plasmid template, 1. Mu.l of upstream primer, 1. Mu.l of downstream primer, 1. Mu.l of manganese chloride solution, and ddH were added 2 O makes up 50. Mu.l.
Error-prone PCR cycling procedure, namely dropping PCR, pre-denaturing for 5min at 95 ℃, and cycling for 1 time; denaturation at 95℃for 30s, denaturation at 58℃for 30s, denaturation at 72℃for 100s, a decrease in annealing temperature of 1℃per cycle, cycle 5 times; denaturation at 95℃for 30s, denaturation at 55℃for 30s, denaturation at 72℃for 100s, 25 cycles; extending at 72 ℃ for 10min, and circulating for 1 time.
S3: preparation of super competence, induced expression of monoclonal and positive mutation screening
The obtained target gene containing a certain mutation rate is subjected to digestion and recovery by Nde I and Kpn I and then is connected with a vector pET30a (+), and a connection product is added into E.coli Bl21 competent cells which are taken out of ice in advance and melted, so that super competence with higher conversion efficiency is prepared according to the operation instruction of the super competence preparation kit. The resurrected bacterial solution is coated on LB solid medium containing kanamycin, cultured for 30min at 37 ℃, and the culture dish is inverted for overnight culture, so as to construct mutant library with higher transformant quantity. Colonies on the solid medium were picked one by one into 96-well bacterial plates (each well containing 200. Mu.l of LB medium to which antibiotics had been added) and cultured at 200rpm for 12-16 hours at 37 ℃; the bacterial solution in each well was then transferred to a new 96-well plate (each well containing 500. Mu.l of LB medium to which antibiotics had been added) at an inoculum size of 2%, and cultured for 6 hours, followed by addition of IPTG for induction expression. Centrifugally collecting the induced thalli, adding 40 cell lysate into each hole, and standing at 37 ℃ for 1-2h to crack the thalli; centrifuging at 12000rpm for 10min at 4deg.C, collecting supernatant, measuring enzyme activity, screening forward mutation, and determining mutant 8-F11 to obtain mutated gene sequence (SEQ ID NO: 2):
after translation, the corresponding amino acid sequence was obtained as follows (SEQ ID NO: 3):
MATKAVCVLK GDGPGQGIIN FEQKESNGPV KVWGSIKGLT EGLHGFHVHE VGDNTAGCTS 60
AGPHFNPLSR KHGGPKDEER HVGDLGNVTA DKDGVADVSI EDSVISLSGD HCIIGRTLVV 120
HEKADDLGKG GNEESTKTGN AGSRLACGVI GIAQHHHHHH 160
by comparison with the amino acid sequences of native human Cu, zn-SOD, it was found that valine at position 15 was mutated to glycine and phenylalanine at position 51 was mutated to valine as determined by sequencing.
S4, shake flask amplification culture of high-expression mutant
After activating the 8-F11 mutant strain with high enzyme activity, inoculating the strain into 30ml of LB culture medium at a ratio of 1 per mill, and culturing at 37 ℃ and 180rpm for overnight. Then, the cells were inoculated into 100ml of LB medium at a ratio of 10% and cultured for 3 hours, followed by induction with 0.5mM IPTG for 6 hours, and then the cells were collected.
S5 purification
1. Crushing and centrifuging
The cells obtained in S4 were resuspended in 20mM Tris,500mM NaCl,20mM imidazole, pH8.0 at a ratio of 1:20, and then sonicated. The supernatant was collected by centrifugation at 12000g for 10min and filtered through a 0.45 μm filter.
2. Affinity chromatography
And (3) solution A: 20mM Tris,500mM NaCl,20mM imidazole, pH8.0
And (2) liquid B: 20mM Tris,500mM NaCl,500mM imidazole, pH8.0
The supernatant in the step 1 is loaded at a flow rate of 1ml/min by using a GE Ni Sepharose 6FF 5mL pre-loaded column and balancing with A solution; then washing impurities with the solution A, wherein the volume is 25ml; eluting with 50% B solution, and collecting eluting peak to obtain the final product.
3. Ion exchange
And (3) solution A: 20mM Tris, pH8.0
And (2) liquid B: 20mM Tris,1M NaCl,pH8.0
And (3) diluting the SOD solution obtained in the step (2) by using the ion-exchanged A solution until the electric conductivity is less than 4mS/cm.
The GE Q Sepharose 6FF 5mL pre-packed column is balanced by A solution, and the diluted SOD solution is loaded at the flow rate of 1 ml/min; then washing impurities with the solution A, wherein the volume is 25ml; eluting with 25% B solution, and collecting eluting peak to obtain purified Cu, zn-SOD.
Example 1
The composition of the daily-throwing eye drops for cornea damage repair of this example is as follows (% represents mass percent):
the preparation method comprises the following steps:
s1: firstly, weighing potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride according to the formula amount, adding the mixture into 50% of injection water with the final volume in advance, stirring to completely dissolve, filtering and sterilizing the mixture by using a filter membrane with the thickness of 0.22 mu m, and collecting filtrate;
s2: collecting the filtrate into a liquid preparation tank, weighing the sodium hyaluronate with the formula amount, adding the sodium hyaluronate into the filtrate, and stirring at room temperature until the sodium hyaluronate is completely dissolved;
s3: weighing superoxide dismutase and recombinant human III type collagen stock solution with the formula amount, filtering and sterilizing with a 0.22 μm filter membrane, adding into a liquid preparation tank, supplementing the rest injection water, and stirring uniformly;
s4: and after the pH value and osmotic pressure are determined to be qualified, filtering and sterilizing by using a filter membrane with the thickness of 0.22 mu m in a sterile environment, and carrying out 0.4 mL/filling and sealing by adopting blowing-filling integrated equipment, so as to obtain the daily throwing eye drops through integrated molding.
Example 2
The composition of the daily-throwing eye drops for cornea damage repair of this example is as follows (% represents mass percent):
the preparation method is the same as in example 1.
Example 3
The composition of the daily-throwing eye drops for cornea damage repair of this example is as follows (% represents mass percent):
example 4
The composition of the daily-throwing eye drops for cornea damage repair of the examples is as follows (percent represents mass percent):
comparative example 1
The difference is that the superoxide dismutase SOD component is not contained in the same way as in example 1.
Comparative example 2
The difference from example 1 is that the recombinant human type III collagen component is absent.
Comparative example 3
The same as in example 1, except that the sodium hyaluronate component was not contained.
Comparative example 4
The difference is that the superoxide dismutase SOD and sodium hyaluronate components are not contained in the same manner as in example 1.
Comparative example 5
The difference from example 1 is that the superoxide dismutase SOD content is 0.002%.
Comparative example 6
The difference from example 1 is that the sodium hyaluronate content is 0.001%.
Comparative example 7
The difference is that the same amount of low molecular weight (< 40 kilodaltons) sodium hyaluronate is added as in example 1.
Investigation example 1 stability experiment
Taking samples of examples 1-4 and comparative examples 1-6, respectively placing the samples at room temperature for 12 months, and examining the change of specific activities of recombinant human III type collagen and superoxide dismutase SOD in each group of samples.
Table 1 results of sample stability investigation
The above results indicate that comparative example 7 is unstable in the system of adding a low molecular weight sodium hyaluronate solution, and shows turbidity delamination upon standing. The comparative example 4 sample was left at room temperature for 12 months, the recombinant human type III collagen was almost completely degraded, and the recombinant human type III collagen in the comparative example 1 and comparative example 5 samples was degraded by 49.76% and 47.52%, respectively, and the specific activities of superoxide dismutase SOD in the comparative example 3 and comparative example 6 samples were reduced by about 8%, respectively. In examples 1 to 4, the content of the recombinant human type III collagen is above 95%, and the specific activity of the superoxide dismutase SOD is above 95%, which indicates that the superoxide dismutase SOD, the recombinant human type III collagen and the sodium hyaluronate are compatible according to a certain proportion, thereby being beneficial to improving the stability of the superoxide dismutase SOD and the recombinant human type III collagen in the solution. The eye drop product developed by the invention has better compatibility, and the components are synergistic, thereby being more beneficial to promoting the repair of cornea injury.
Investigation example 2 in vitro cytotoxicity test
The concentration was set at 2X 10 5 cell/mL of corneal stromal cell suspension was added to a 96-well plate at 200. Mu.l/well at 5% CO 2 Culturing in a 37 ℃ incubator for 6 hours, and sucking out the culture medium. The culture medium containing the eye drops of example 1 (volume ratio of eye drops to serum-free medium 1:10) was changed for a further 24h. The 96-well plate was removed and CCK 810. Mu.1/well was added. Culturing in incubator for 2-4 hr according to the display condition. Clean the sample plate, measure the absorbance at 450nm on the microplate reader, calculate the cell activity: cell activity= (subject well OD value-blank well OD value)/negative control well OD value.
The cytotoxicity test results show that the cell viability of the eye drop treatment prepared in the example 1 is not significantly different from that of the matrix group, and the cell viability is above 90%, which indicates that the eye drop has no cytotoxicity.
Investigation example 3 eye irritation test
Healthy white rabbits are taken as test subjects, and are randomly divided into 2 groups, wherein 3 animals in each group have no obvious difference in age and weight. Among them, 1 group was the eye drops of example 1, and the other 1 group was the physiological saline control group. The lower eyelid of the left eye of the animal was gently pulled open, 0.1ml of the test substance was instilled into the conjunctival sac, the upper and lower eyelids were passively closed for 5s, the right eye was a blank control, and an equal amount of water for injection was instilled. 5 times daily for 21 consecutive days. The local response of each animal was observed and recorded for about (1.+ -. 0.1) h before and after each instillation. The eye irritation response scoring criteria were performed with reference to the eye irritation test judgment criteria in GB/T16886.10, specifically as shown in Table 3. If more than half of the animals in the test group showed positive results at any stage of observation (footnoted score in Table 2), the test material was considered an ocular stimulus.
TABLE 2
TABLE 3 eye irritation study results
The results of the eye irritation test show that the formulation of example 1 is non-irritating to the eyes of rabbits.
Investigation example 4 cornea damage repair test
60 SD male rats were selected, and the age and weight of the rats were randomly divided into 12 groups of control group, model group, eye drops of examples 1 to 4 and eye drops of comparative examples 1 to 6, each group being 5. The model set and each eye drop set establish a corneal uv damage model according to the following method: firstly, isoflurane is adopted to carry out anesthesia treatment on rats, compound topiramate eye drops are dripped into the right eyes after anesthesia, the right eyes are fixed by rubberized fabric to be in an open state, the cornea of the rats is irradiated by an ultraviolet lamp tube for 20min, and continuous irradiation is carried out for 4 days. The rats successfully molded were sampled, the right eyes were buffered with sterile PBS in the model group, and the right eyes were dropped with examples 1 to 4 and comparative examples 1 to 6, respectively, 5 times daily, for 5 days continuously. The degree of repair of the corneal lesion was judged by the amount of Lactate Dehydrogenase (LDH) released in tears. The LDH release amount is reduced, which indicates that the cornea damage is repaired; the increase in LDH release indicates corneal damage. The LDH release amounts of each group were measured before irradiation, after molding, after 1d, 3d, and 5d administration, and 100 μl samples were collected after local eye-spotting with 200 μl physiological saline and soaking for 60s, and the enzyme-labeled ELISA method was used to test the LDH release amounts of each group and average the results.
TABLE 4 results of LDH Release test at various times before and after ultraviolet irradiation
The results of the rat cornea damage repair test show that: after successful eye molding of a rat irradiated by ultraviolet, LDH release is rapidly increased, and the content is about 7-10 times that of the LDH before irradiation, which indicates that the cornea of the rat is damaged by ultraviolet irradiation; along with the prolonged administration time, the LDH released by the embodiments 1-4 is obviously lower than that released by the model group and the comparative examples 1-6, which shows that the eye drops prepared by combining SOD, recombinant human III type collagen and sodium hyaluronate according to a certain proportion can promote the repair of damaged cornea, wherein the repair effect of the embodiment 1 is optimal.
In conclusion, the daily throwing eye drops for cornea wound healing, which are prepared by the invention, do not contain preservative and are convenient to use. The product has good biocompatibility, can effectively promote cell adhesion and proliferation, repair cornea injury, remove eye free radicals, stabilize lacrimal gland, relieve dry eye, and obviously improve eye symptoms such as eye fatigue, red swelling, inflammation, etc.
Although the invention has been described in detail with reference to the foregoing embodiments, those skilled in the art may modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (6)
1. The daily throwing eye drop for repairing cornea injury is characterized by comprising the following raw materials: the recombinant human type III collagen comprises, by mass, 0.01% -0.2% of superoxide dismutase SOD, 1:0.5% -1:2 of recombinant human type III collagen, and 1:1% -1:5 of sodium hyaluronate, osmotic pressure regulator, pH regulator and injection water;
the superoxide dismutase SOD is recombinant human Cu, zn-SOD, and the specific activity of the superoxide dismutase SOD is 20000U/mg-30000U/mg;
the molecular weight of the recombinant human III type collagen ranges from 25kDa to 90kDa;
the average molecular weight of the sodium hyaluronate is 500 kDa-1400 kDa.
2. The daily-throwing eye drop for repairing cornea damage according to claim 1, wherein the pH value of the eye drop is 6.5-8.0; the osmotic pressure of the eye drops is 260-340 m0sm/L.
3. The daily-throwing eye drop for repairing cornea damage according to claim 1, wherein the pH value is 6.8-7.6; the pH regulator is selected from phosphate buffer and/or boric acid buffer; the osmotic pressure of the eye drops is 280-300 mOsm/L, and the osmotic pressure regulator is selected from sodium chloride and/or potassium chloride.
4. The daily disposable eye drop for repairing corneal damage according to claim 1, wherein the eye drop is a topical product and is packaged in a sterile daily disposable form.
5. A method of preparing a daily disposable eye drop for repairing corneal damage according to any one of claims 1 to 4, comprising the steps of:
s1: firstly, weighing an osmotic pressure regulator and a pH regulator according to the formula amount, adding the osmotic pressure regulator and the pH regulator into injection water with the final volume of 40-60%, stirring to completely dissolve, filtering with a filter membrane for sterilization, and collecting filtrate;
s2: weighing the formula amount of sodium hyaluronate, adding the sodium hyaluronate into the filtrate obtained in the step S1, and stirring at room temperature until the sodium hyaluronate is completely dissolved;
s3: weighing superoxide dismutase SOD and recombinant human III type collagen according to the formula amount, filtering and sterilizing by a filter membrane, adding the superoxide dismutase SOD and the recombinant human III type collagen into the solution obtained in the step S2, supplementing the rest injection water, and uniformly stirring;
s4: and after the pH and osmotic pressure are determined to be qualified, filtering and sterilizing by a filter membrane in a sterile environment, and filling and sealing.
6. The method according to claim 5, wherein the filter is a 0.22 μm filter.
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