Amniotic membrane loaded with procyanidine and mecobalamin as well as preparation method and application of amniotic membrane

Abstract

The invention discloses a preparation method of an amniotic membrane loaded with procyanidine and mecobalamine, which comprises the following steps: soaking an amniotic membrane in a solution containing procyanidine, mecobalamine and N-hydroxysuccinimide, and reacting to obtain the amniotic membrane loaded with procyanidine and mecobalamine. The amniotic membrane is selectively loaded with procyanidine and mecobalamin, has the functions of resisting inflammation and bacteria and promoting corneal epithelium and nerve repair, and solves the problem that the repair promoting function of the treated amniotic membrane is reduced.

Description

Technical Field

[0001] The present invention generally relates to the field of biomedical materials, and in particular, to an amniotic membrane loaded with proanthocyanidins and methylcobalamin, and a preparation method and application thereof. Background Art

[0002] Corneal epithelial defects are common clinical problems in ophthalmology, often caused by a variety of non-infectious causes, such as mechanical trauma, chemical burns, dry eyes, neurotrophic keratitis, autoimmune diseases, etc. If corneal epithelial defects are not effectively repaired for a long time, it may cause a series of serious complications, including corneal ulcers and corneal perforations, which will not only seriously affect vision, but may even cause blindness.

[0003] The amniotic membrane is the innermost layer of the placenta. Its structure is similar to that of human conjunctival tissue and is rich in a variety of growth factors, which can provide a growth environment for corneal epithelial cells similar to that of ocular surface tissue. It also reduces immune rejection and inflammatory reactions after transplantation due to its avascular, neural and lymphatic characteristics. Therefore, the amniotic membrane has become an important means to promote the recovery of corneal epithelial damage in ophthalmology clinics.

[0004] However, the natural collagen fiber structure of the amniotic membrane and the activity of the growth factors that originally promote the migration of epithelial cell adhesion agents will be damaged to varying degrees during repeated cleaning, strict disinfection, improper storage, and long-term storage, resulting in reduced anti-inflammatory and epithelial repair capabilities. How to maintain and enhance the repair-promoting function of the amniotic membrane is a research focus in this field. Summary of the invention

[0005] The amniotic membrane provided by the present invention is selectively loaded with proanthocyanidins and methylcobalamin, has the functions of anti-inflammatory, antibacterial, and promoting corneal epithelial and nerve repair, and solves the problem that the repair-promoting function of the existing amniotic membrane decreases after being treated.

[0006] In a first aspect of the present invention, a method for preparing an amniotic membrane loaded with proanthocyanidins and methylcobalamin is provided, comprising: immersing the amniotic membrane in a solution containing proanthocyanidins, methylcobalamin and N-hydroxysuccinimide, and obtaining the amniotic membrane loaded with proanthocyanidins and methylcobalamin after reaction.

[0007] In the present invention, the concentration ratio of methylcobalamin to proanthocyanidins is (1-10):(30-100), preferably (2-8):(50-100), and more preferably (5-8):(50-80).

[0008] Preferably, the concentration of proanthocyanidins in the solution is 3-10 mg / mL, more preferably 5-8 mg / mL.

[0009] Preferably, the concentration of methylcobalamin in the solution is 0.1-0.8 mg / mL, more preferably 0.4-0.8 mg / mL, more preferably 0.5 mg / mL.

[0010] Preferably, the concentration of NHS in the solution is 8-20 mg / mL, more preferably 10-15 mg / mL.

[0011] Preferably, the pH of the solution is 6.5-7.5.

[0012] Preferably, the reaction is carried out at room temperature for 30 minutes to 3 hours.

[0013] In a second aspect of the present invention, an amniotic membrane loaded with proanthocyanidins and methylcobalamin is provided.

[0014] Preferably, the loading concentration of methylcobalamin on the amniotic membrane is 1.5-2.3 ug / mL, and the loading concentration of proanthocyanidins is 3-4 ug / mL.

[0015] More preferably, the loading concentration of methylcobalamin on the amniotic membrane is 2.3 ug / mL; the loading concentration of proanthocyanidins is 3.6 ug / mL.

[0016] In the third aspect of the present invention, provided is a method for preparing the amniotic membrane loaded with proanthocyanidins and methylcobalamin and the use of the prepared amniotic membrane loaded with proanthocyanidins and methylcobalamin in preparing medical devices for treating or alleviating corneal epithelial defects and promoting corneal epithelial cell repair.

[0017] It should be understood that the contents described in the summary of the invention are not intended to limit the key or important features of the embodiments of the present invention, nor are they intended to limit the scope of the present invention. Other features of the present invention will become easily understood through the following description.

[0018] The amniotic membrane provided by the present invention has the following beneficial effects:

[0019] 1) The amniotic membrane of the present invention is loaded with proanthocyanidins and methylcobalamin, and the drugs are slowly released in the amniotic membrane, thereby increasing the anti-inflammatory, antibacterial, epithelial and nerve repair-promoting functions of the amniotic membrane, shortening the course of disease, reducing the frequency of postoperative drug use, and alleviating the patient's pain and economic burden.

[0020] 2) The amniotic membrane material of the present invention has good biocompatibility and is conducive to cell adhesion and proliferation.

[0021] 3) The amniotic membrane of the present invention can be stored for a long time after drying, and is convenient to use, store and transport, and can be widely used in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .Sodium fluorescein staining image of the cornea in Comparative Example 1.

[0023] Figure 2 .Epithelial healing rate in comparative example 1.

[0024] Figure 3 .Confocal microscopy imaging of corneal epithelial defect nerve repair in Comparative Example 1.

[0025] Figure 4 .Gross image of the anterior segment of the eye with epithelial defect in Comparative Example 2. DETAILED DESCRIPTION

[0026] In order to reduce the economic burden on patients, the present invention selects proanthocyanidins and methylcobalamin as loading components, thereby reducing costs while ensuring high safety and low side effects, and also simplifies the loading method as much as possible to adapt to mass production.

[0027] The invention provides a method for preparing an amniotic membrane loaded with proanthocyanidins and methylcobalamin, comprising: immersing the amniotic membrane in a solution containing proanthocyanidins, methylcobalamin and N-hydroxysuccinimide (NHS), and obtaining the amniotic membrane loaded with proanthocyanidins and methylcobalamin after reaction.

[0028] In the present invention, the concentration ratio of methylcobalamin to proanthocyanidins is (2-10):(30-100), preferably (2-8):(50-100), and more preferably (5-8):(50-80).

[0029] Preferably, the concentration of proanthocyanidins in the solution is 3-10 mg / mL, more preferably 5-8 mg / mL.

[0030] Preferably, the concentration of methylcobalamin in the solution is 0.1-0.8 mg / mL, more preferably 0.4-0.8 mg / mL, more preferably 0.5 mg / mL.

[0031] Preferably, the concentration of NHS in the solution is 8-20 mg / mL, more preferably 10-15 mg / mL.

[0032] In the present invention, there is no limitation on the liquid used to dissolve proanthocyanidins and methylcobalamin, and those skilled in the art can select a suitable solvent according to actual conditions, including PBS buffer, sterile water for injection, physiological saline, 2-methyl-2-propylene sulfonic acid (MES), etc., preferably PBS buffer.

[0033] Furthermore, the method further comprises the step of adjusting the pH value of the solution, preferably the pH value of the solution is 6-8, more preferably 6.5-7.5. Because the pH value of tears is usually between 6.5 and 7.5, solutions with similar pH can reduce irritation to the eyes and increase comfort.

[0034] In the present invention, there is no limitation on the pH adjuster, and those skilled in the art can select a suitable solvent according to actual conditions, including citric acid, sodium citrate, boric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid, sodium acetate, sodium hydroxide and hydrochloric acid.

[0035] In the present invention, there is no limitation on the soaking method, and it can be done according to conventional operations in the art.

[0036] In the present invention, there is no limitation on the reaction conditions and reaction time, and those skilled in the art can select according to the actual situation. For example, the reaction is carried out at 20-37° C. for 1-10 hours.

[0037] Preferably, the reaction is carried out at room temperature (15-25°C) for 0.5-8 hours, for example, at 20°C for 4-6 hours; more preferably, the reaction is carried out in the dark at 22-25°C for 30 minutes to 3 hours, for example, at 25°C for 1-2 hours.

[0038] In the present invention, in order to dissolve proanthocyanidins, methylcobalamin and NHS in the solution, ultrasound, vacuum or stirring can be used to accelerate the dissolution process, for example, in a 37° C. water bath; and / or 200-400W ultrasound for 20-30 min.

[0039] In the present invention, the above method further includes a pretreatment step, namely, the preparation of amniotic membrane: taking fresh amniotic membrane, washing it with sterile saline and then rinsing it in antibiotic saline; scraping off the sponge layer, part of the fibroblast layer and serous exudate under a microscope; attaching a sterile nitrocellulose membrane (NC membrane) thereon, with the amniotic epithelial surface facing upward; and trimming it to a suitable size according to actual needs.

[0040] In the present invention, the above method further comprises a post-treatment step, namely, washing the amniotic membrane obtained after the reaction: taking the amniotic membrane out of the solution and washing it in a buffer solution to remove unreacted reagents and by-products.

[0041] In the present invention, there is no limitation on the buffer used for washing the amniotic membrane obtained after the reaction, and those skilled in the art can select, for example, PBS buffer according to actual conditions.

[0042] In the present invention, there is no limitation on the number and time of cleaning, and those skilled in the art can determine it according to the actual situation, for example, cleaning 3-10 times, each time for 10-60 minutes.

[0043] The principle of the above method is: N-hydroxysuccinimide (NHS) is used as an activator to assist cross-linking and is used to activate the carboxyl group. When NHS reacts with the carboxyl group on the amniotic membrane, an active ester intermediate is formed. This intermediate can then react with molecules containing amino groups to form a stable amide bond, which effectively increases the reactivity of the carboxyl group. Methylcobalamin is a derivative of vitamin B12 with hydroxyl groups and other functional groups; proanthocyanidins are a class of polyphenolic compounds containing multiple hydroxyl groups. Therefore, methylcobalamin and proanthocyanidins react with amino groups and (NHS-activated) carboxyl groups on the amniotic membrane through their hydroxyl groups to form multiple covalent bonds, including condensation reactions between hydroxyl groups and amino groups to form ether bonds and ester bonds, and amidation reactions between hydroxyl groups and NHS-activated carboxyl groups to form amide bonds. These covalent bonds more stably and firmly cross-link the methylcobalamin and proanthocyanidin molecules to the amniotic membrane.

[0044] The concentration of NHS directly affects the activation efficiency of the reaction. Too low a concentration of NHS may not fully activate the carboxyl groups of the amniotic membrane, resulting in reduced reaction efficiency, thus affecting the overall effect. Only when the loading concentration of methylcobalamin and proanthocyanidins reaches an effective concentration can they exert an effective therapeutic effect.

[0045] During the reaction, the appropriate mass ratio between methylcobalamin and proanthocyanidins can ensure effective contact between the reactants and promote the reaction. Too high an amount of methylcobalamin may lead to competitive inhibition of proanthocyanidins, while too low an amount may not be sufficient to support an effective reaction. As a bioactive molecule, methylcobalamin has multiple physiological functions that promote cell metabolism and repair, and potential toxicity or precipitation problems can be avoided within a suitable concentration range. Proanthocyanidins have strong antioxidant properties and biological activity, and can effectively provide cell protection and biological activity within a suitable concentration range without causing cytotoxicity. The combination of the two works together to enhance the anti-inflammatory, antibacterial, and epithelial and neural repair functions of the amniotic membrane.

[0046] The cross-linked amniotic membrane has improved mechanical properties and its biodegradation is also delayed. At the same time, because it is loaded with proanthocyanidins and methylcobalamin, the anti-inflammatory, antibacterial, and epithelial and nerve repair functions of the amniotic membrane are enhanced, solving the problem of the damage of the active ingredients of the amniotic membrane and the decline of its repair function after traditional treatment.

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Unless otherwise specified, the technical and scientific terms used herein are those commonly understood by those of ordinary skill in the art to which the present invention belongs. If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, all of them are conventional products that can be obtained commercially.

[0049] Sources:

[0050] The amniotic membrane was donated by the Obstetrics Department of Shandong Provincial Hospital.

[0051] Proanthocyanidins were purchased from Maclean, product number P815549.

[0052] Methylcobalamin injection was purchased from Eisai (China) Pharmaceutical Co., Ltd.

[0053] N-Hydroxysuccinimide (NHS) was purchased from Aladdin, product number H109330.

[0054] New Zealand white rabbits were purchased from Xilingjiao Breeding and Propagation Center in Jinan.

[0055] Example 1

[0056] Using proanthocyanidins and methylcobalamin as raw materials and NHS (N-hydroxysuccinimide) as a cross-linking agent, an amniotic membrane loaded with proanthocyanidins and methylcobalamin is prepared, and the steps are as follows:

[0057] S0. Pretreatment: Take fresh amniotic membrane, rinse with sterile saline, and place in antibiotic saline (2.5ug / mL amphotericin B, 1:1000 tobramycin, 1:2000 penicillin) for 20-60 minutes, for example, 40 minutes. Scrape off the sponge layer, part of the fibroblast layer and serous exudate under a microscope. Attach a sterile nitrocellulose membrane (NC membrane) to it, with the amniotic epithelial surface facing up, and trim it to a suitable size according to actual needs. In this embodiment, the amniotic membrane (sheet) is trimmed into a size of 2.5cm×2.5cm.

[0058] S1. Prepare a mixed solution of proanthocyanidins and methylcobalamin: dissolve methylcobalamin and proanthocyanidins in a solution at a mass ratio of (2-10):(30-100). The preferred mass ratio is (2-8):(50-100), and more preferably (5-8):

[0059] (50-80). In this example, 25 mg of proanthocyanidins, 2.5 mg of methylcobalamin, and 50 mg of NHS were dissolved in 5 mL of PBS buffer.

[0060] In some embodiments, the pH of the mixed solution is adjusted to 6.5-7.5, because the pH value of tears is usually between 6.5 and 7.5, and in this range, it can be similar to tears and reduce irritation to the eyes. In this embodiment, 0.3% boric acid is added to adjust the pH value to between 6.5-7.5.

[0061] In this embodiment, the dissolution method is to subject the mixed solution to ultrasonic treatment in a 37° C. water bath for 20 min at an energy of 20 Hz to completely dissolve it and form a uniform solution.

[0062] S2. Cross-linking the amniotic membrane: Place the amniotic membrane into the mixed solution prepared in step S1, ensuring that the entire surface is covered, and let it react at room temperature for 30 minutes to 3 hours, for example, 45 minutes, 1 hour, 1.5 hours, or 2 hours.

[0063] S3. Washing: Remove the amniotic membrane and wash it in PBS for 5 times, 20 minutes each time, to remove unreacted reagents and by-products.

[0064] In some embodiments, ultra-high performance liquid chromatography is used to measure the loading rates of proanthocyanidins and methylcobalamin, and the loading concentration of methylcobalamin is measured to be 1.5-2.3 ug / mL, and the loading concentration of proanthocyanidins is 3-4 ug / mL.

[0065] In this embodiment, the average loading concentration of methylcobalamin is 2.3 ug / mL; the average loading concentration of proanthocyanidins is 3.6 ug / mL.

[0066] Example 2

[0067] Load rate determination:

[0068] 1. Prepare mixed solution:

[0069] Mixed solution 1: The concentrations of methylcobalamin solution, proanthocyanidin solution, and NHS solution are (0.1 mg / mL, 3 mg / mL, 8 mg / mL), respectively.

[0070] Mixed solution 2: The concentrations of methylcobalamin solution, proanthocyanidin solution, and NHS solution are (0.5 mg / mL, 5 mg / mL, and 10 mg / mL), respectively.

[0071] Mixed solution 3: The concentrations of methylcobalamin solution, proanthocyanidin solution, and NHS solution are (0.8 mg / mL, 8 mg / mL, and 15 mg / mL), respectively.

[0072] Mixed solution 4: The concentrations of methylcobalamin solution, proanthocyanidin solution, and NHS solution are (1.0 mg / mL, 10 mg / mL, 20 mg / mL), respectively.

[0073] Ultrasonicate the mixture in a 37°C water bath for 20 minutes at an energy of 20 Hz to completely dissolve it and form a uniform solution. Place the amniotic membrane in the mixture to ensure that the entire surface is covered, and let it stand at room temperature for 2 hours to react. Place the loaded amniotic membrane in a centrifuge tube, grind it into a gel-like state, and centrifuge it at 13,000 rpm for 10 minutes.

[0074] The loading rate was determined by ultra-high performance liquid chromatography (UPLC) to obtain the loading concentrations of methylcobalamin and proanthocyanidins. Three replicates were set for each group of experiments, and the average value was taken. The results are as follows:

[0075] Group Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Methylcobalamin concentration (mg / mL) 0 0.1 0.5 0.8 1.0 Procyanidin concentration (mg / mL) 0 3 5 8 10 NHS concentration (mg / mL) 0 8 10 15 20 Methylcobalamin loading concentration (μg / mL) - 1.5 2.3 2.0 1.9 Proanthocyanidin loading concentration (μg / mL) - 3 3.6 4.0 4.2

[0076] The results showed that the loading rate began to increase with the increase of concentration. When methylcobalamin and proanthocyanidins reached an equilibrium concentration, as the concentration of proanthocyanidins increased, they would compete for the binding sites between methylcobalamin and the amniotic membrane, affecting the loading rate of methylcobalamin.

[0077] Example 3

[0078] Amniotic membranes with different loading rates were prepared and tested.

[0079] 1. Prepare solution:

[0080] Methylcobalamin solutions of different concentrations (0.1 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.8 mg / mL) were prepared.

[0081] Proanthocyanidin solutions of different concentrations (3 mg / mL, 5 mg / mL, 10 mg / mL) were prepared.

[0082] Preparation of NHS solutions of different concentrations (5 mg / mL, 10 mg / mL, 15 mg / mL)

[0083] 2. Mixed reaction:

[0084] Different concentrations of methylcobalamin and proanthocyanidins were mixed with NHS and reacted at 37°C for 3 hours to ensure sufficient mixing to obtain a complex.

[0085] 3. Detection of complex formation:

[0086] The absorbance change of each solution before and after the reaction was measured using a UV-Vis spectrophotometer to confirm the formation of the complex.

[0087] The molecular weight of the complexes was analyzed by gel electrophoresis or polyacrylamide gel difference analysis.

[0088] 4. Stability test:

[0089] The composite solution after the reaction was stored at different temperatures (4° C., 25° C., and 37° C.), and samples were taken regularly to measure the change in particle size.

[0090] Dynamic light scattering (DLS) technique was used to obtain the particle size change data of the composite.

[0091] 5. Cytotoxicity assay:

[0092] The human corneal epithelial cells were treated with amniotic membrane treated with the above complex, and an untreated group was set as a control.

[0093] The cell viability was determined by MTT method or cell counting method.

[0094] 6. Load rate determination:

[0095] The loading rate was determined using ultra performance liquid chromatography (UPLC).

[0096] The experimental results are shown in the following table:

[0097] Group Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Methylcobalamin concentration (mg / mL) 0 0.5 0.8 0.4 0.4 Procyanidin concentration (mg / mL) 0 5 3 10 5 NHS concentration (mg / mL) 0 10 10 10 15 Absorbance before reaction 0.05 0.05 0.05 0.05 0.05 Absorbance after reaction 0.05 0.45 0.32 0.4 0.38 Particle size (4℃ / 24h) - 145 nm 158nm 160nm 150nm Particle size (25℃ / 24h) - 170 nm 185nm 190nm 180nm Cell survival rate (%) 95 92 87 85 86 Methylcobalamin loading concentration (μg / mL) - 2.3 1.8 1.5 2 Proanthocyanidin loading concentration (μg / mL) - 3.6 3 4 3.8

[0098] Example 4

[0099] Cytotoxicity experiment: CCK-8 method was used to detect cell viability.

[0100] 1) Prepare complete culture medium: 5 ml serum, 500 ul double antibody, and DMEM / F-12 culture medium, mix and make up to 50 ml.

[0101] 2) Cell seeding: Human corneal epithelial cells were seeded into a 96-well plate. 100 μL of complete culture medium was added to each well, and the plates were cultured in an incubator at 37° C. and 5% CO 2 for 24 hours to allow the cells to adhere to the plate.

[0102] 3) Treating cells: In the experimental group, methylcobalamin, proanthocyanidins and NHS were dissolved in complete culture medium at different concentrations and added to each well of a 96-well plate (100 μL per well). The untreated group served as the control group. The 96-well plate was returned to the incubator and cultured for 24 hours.

[0103] 4) CCK-8 assay: Add 10 μL of CCK-8 reagent to each well and mix gently. Return the 96-well plate to the incubator and incubate for 2 hours.

[0104] 5) Calculate cell viability: Cell viability (%) = ((experimental) - (blank)) / ((control) - (blank)) × 100. Wherein, (experimental) is the OD value of the wells with cells, CCK-8 solution and methylcobalamin, proanthocyanidin, NHS mixed solution; (control) is the OD value of the wells with cells, CCK-8 solution but no drug solution; (blank) is the OD value of the wells with culture medium, CCK-8 solution but no cells.

[0105] The experimental results are shown in the following table:

[0106] Group Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Experimental Group 4 Methylcobalamin concentration (mg / mL) 0 0.1 0.5 0.8 1.0 Procyanidin concentration (mg / mL) 0 3 5 8 10 NHS concentration (mg / mL) 0 8 10 15 20 Cell survival rate (%) 100％ 100％ 100％ 94.3％ 89％

[0107] Comparative Example 1

[0108] After anesthetizing 12 New Zealand white rabbits, the corneal epithelium and basement membrane within 11mm of the rabbit cornea were scraped with an epithelial knife. Six rabbits were randomly selected and treated with the amniotic membrane loaded with proanthocyanidins and methylcobalamin prepared in Example 1 (the loading concentration of methylcobalamin was 2.3ug / mL; the loading concentration of proanthocyanidins was 3.6ug / mL) (amniotic membrane group), and the remaining 6 rabbits were not treated (control group). Three and five days after the operation, corneal slit lamp photography and sodium fluorescein staining were used to record the healing of the corneal epithelium, redness of the eyes, secretions and other abnormalities. The results are as follows: Figure 1 As shown in the figure, the conjunctiva of the control group was obviously congested after the corneal epithelium was scraped off, and the inflammation persisted. Seven days after surgery, the corneal epithelium was still nearly 3mm in the range of fluorescein sodium staining. In the amniotic membrane group, the conjunctiva was not obviously congested, the inflammatory reaction was mild, the corneal epithelium was nearly healed 5 days after surgery, and the corneal epithelium was completely healed 7 days after surgery, indicating that the amniotic membrane can reduce the inflammatory reaction of the ocular surface and promote the healing of the corneal epithelium.

[0109] One month after surgery, Image J software was used to calculate the healing rates of corneal epithelium in the two groups. Figure 2 As shown in the figure, the healing speed of corneal epithelium in the amniotic membrane group was significantly faster than that in the blank control group, and the difference was statistically significant. There was one corneal ulcer in the control group, and no infection occurred during the epithelial healing period in the amniotic membrane group. No obvious purulent secretions were found in the amniotic membrane group, and the amniotic membrane dissolution rate was 0% after 1 week of observation. No obvious mechanical damage was found in the conjunctival sac, and no bleeding, exudation, or granulation hyperplasia occurred, indicating that amniotic membrane can effectively promote corneal epithelial healing and has anti-inflammatory, antibacterial, and repair-promoting functions.

[0110] One month after surgery, the rabbits were killed, their eyeballs were removed, and corneal tissue was obtained and observed using a confocal laser microscope. Figure 3 As shown, the corneal nerve fibers in the control group did not regenerate, but the nerve fibers in the amniotic membrane group did regenerate, confirming that the amniotic membrane loaded with proanthocyanidins and methylcobalamin can promote the regeneration of corneal nerves.

[0111] Comparative Example 2

[0112] After anesthesia, a 6mm trephine drill and a 45-degree knife were used to make a lamellar implant bed with a diameter of 6mm and a depth of about 300μm along the natural lamellar layer of the corneal stroma in the center of the rabbit cornea. The 6mm corneal implant (corneal stroma piece) was sutured and fixed on the implant bed. Twelve rabbits whose corneal implant epithelium had not healed one week after surgery were selected for experimental observation.

[0113] Six New Zealand white rabbits were randomly selected and treated with the amniotic membrane loaded with proanthocyanidins and methylcobalamin prepared according to the method of Example 1 (the loading concentration of methylcobalamin was 2.0 ug / mL; the loading concentration of proanthocyanidins was 4.0 ug / mL) (amniotic membrane group), and the remaining six rabbits were not treated (control group). One week and two weeks after treatment, corneal slit lamp photography and sodium fluorescein staining were used to record the healing of the corneal epithelium and complications such as red eyes and neovascularization. The results are as follows Figure 4 As shown in the figure, a large number of new blood vessels grew in the control group, and there was 1 case of corneal dissolution and perforation. The healing speed of corneal epithelium in the amniotic membrane group was significantly faster than that in the blank control group, with fewer new blood vessels and higher corneal nerve repair than that in the control group. No obvious purulent secretions were observed, and no amniotic membrane dissolution was observed. In addition, no obvious mechanical damage was observed in the conjunctival sac of the amniotic membrane group, and no bleeding, exudation, or granulation hyperplasia occurred, indicating that amniotic membrane can effectively promote corneal epithelial healing and has anti-inflammatory, antibacterial, and repair-promoting functions.

[0114] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing amniotic membrane loaded with proanthocyanidins and methylcobalamin, characterized in that: include: The amniotic membrane is immersed in a solution containing proanthocyanidins, methylcobalamin and N-hydroxysuccinimide, and the amniotic membrane loaded with proanthocyanidins and methylcobalamin is obtained after the reaction.

2. The preparation method according to claim 1, characterized in that The concentration ratio of methylcobalamin to proanthocyanidins is 2-10:30-100.

3. The preparation method according to claim 1, characterized in that: The concentration of proanthocyanidins is 3-10 mg / mL; the concentration of methylcobalamin is 0.1-0.8 mg / mL; and the concentration of NHS is 8-20 mg / mL.

4. The preparation method according to claim 3, characterized in that: The concentration of proanthocyanidins is 5-8 mg / mL; the concentration of methylcobalamin is 0.4-0.8 mg / mL; and the concentration of NHS is 10-15 mg / mL.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The pH of the solution is 6.5-7.

5.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The reaction is carried out under the conditions of standing at room temperature for 30 minutes to 3 hours.

7. The amniotic membrane loaded with proanthocyanidins and methylcobalamin prepared according to the preparation method according to any one of claims 1 to 6.

8. The amniotic membrane according to claim 7, wherein The loading concentration of methylcobalamin was 1.5-2.3ug / mL, and the loading concentration of proanthocyanidins was 3-4ug / mL.

9. The amniotic membrane according to claim 8, wherein The loading concentration of methylcobalamin is 2.3ug / mL, and the loading concentration of proanthocyanidins is 3.6ug / mL; or the loading concentration of methylcobalamin is 2.0ug / mL, and the loading concentration of proanthocyanidins is 4.0ug / mL.

10. Use of the preparation method according to any one of claims 1 to 6 or the amniotic membrane loaded with proanthocyanidins and methylcobalamin according to any one of claims 7 to 9 in the preparation of a medical device for treating or alleviating corneal epithelial defects and promoting corneal epithelial cell repair.

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