Slow-release gel preparation for endometrial repair and preparation method thereof

Abstract

The invention relates to the technical field of medical gel, and particularly discloses a sustained-release gel preparation for endometrial repair and a preparation method of the sustained-release gel preparation. Keratinocyte factor sustained-release microspheres containing keratinocyte factor-glucan particles and lactic acid-glycolic acid copolymer shells are constructed around keratinocyte factors, and the keratinocyte factor sustained-release microspheres are loaded by hyaluronic acid hydrogel containing polylysine, collagen and silk fibroin to form the sustained-release gel preparation for endometrial repair. The sustained-release gel preparation has the effect of promoting proliferation of human endometrial cells, can repair endometrial injury, promote injury healing and prevent uterine cavity adhesion, and has important significance in treating endometrial injury and improving physical and psychological health of women.

Description

Technical Field

[0001] The present invention relates to the technical field of medical gels, and particularly to a sustained-release gel preparation for endometrial repair and a preparation method thereof. Background Art

[0002] The endometrium is a complex tissue, which can be divided into a temporary superficial functional layer composed of dense glandular tissue and loose connective matrix, and a permanent deep basal layer composed of matrix, glandular basal part, supporting vascular system and various immune cell populations. Under the regulation of hormones such as estrogen and progesterone secreted by the ovaries, the temporary superficial functional layer of the endometrium continuously undergoes the cycle of proliferation-secretion-shedding. There are more than 400 cycle periods on average during a female's reproductive period. The permanent deep basal layer of the endometrium is the place where stem cells grow, which can not only provide cells for renewing the functional layer after menstruation, but also is related to embryo implantation, the connection between the fetus and the mother, and the maintenance of pregnancy.

[0003] There are many factors that can cause endometrial injury, such as bacterial and viral infections, the use of certain drugs, insufficient blood supply to the body, frequent intrauterine operations (artificial abortion), etc. All of these may damage the basal layer of the endometrium, impair the endometrial regeneration function, cause fibrosis or scarring, and then lead to thinning of the endometrium, reduced endometrial receptivity, intrauterine adhesion, etc. The clinical symptoms include irregular menstruation, abnormal menstruation, secondary infertility, habitual abortion, etc. in women, which seriously affect the psychological and physical health of women.

[0004] At present, the methods for treating endometrial injury include surgical removal of adhesions, hormone therapy, and drug intervention, etc. However, there are still some problems that cannot be ignored: the high recurrence rate of the surgical method, the low efficiency of the hormone method, and the unsatisfactory effect of drug intervention. Therefore, developing a sustained-release gel preparation for endometrial repair, which can repair endometrial injury, promote injury healing, and prevent intrauterine adhesion, is of great significance for treating endometrial injury and improving women's physical and mental health. Summary of the Invention

[0005] The purpose of the present invention is to provide a sustained-release gel preparation for endometrial repair and a preparation method thereof, which can repair endometrial injury, promote injury healing, prevent intrauterine adhesion, and solve the problems of high recurrence rate of the surgical method, low efficiency of the hormone method, and unsatisfactory effect of drug intervention.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0008] Add keratinocyte growth factor sustained-release microspheres to the dopamine-grafted hyaluronic acid mixed solution, stir for 0.5 - 2 h, then adjust the pH of the solution to 8 - 9, add sodium periodate, and react in a water bath at 35 - 40 °C in the dark for 10 - 15 h to form a gel. Rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0009] As a limitation of the present invention, the mass ratio of the keratinocyte growth factor sustained-release microspheres to sodium periodate is (0.01 - 0.03):(0.4 - 0.5).

[0010] As a limitation of the present invention, the preparation method of the dopamine-grafted hyaluronic acid mixed solution is as follows:

[0011] Mix sodium hyaluronate and deionized water, stir evenly to obtain a sodium hyaluronate solution, adjust the pH of the sodium hyaluronate solution to 4 - 5, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, after fully dissolving, add dopamine hydrochloride, and continuously stir for 10 - 15 h to obtain a dopamine-grafted hyaluronic acid solution. Add polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 20 - 40 min, then use ultrapure water as the dialysis solution, and dialyze the solution with an 8 - 14 KDa dialysis membrane for 48 - 72 h for purification treatment. After the purification is completed, add collagen and silk fibroin, and stir evenly to obtain a dopamine-grafted hyaluronic acid mixed solution.

[0012] As a limitation of the present invention, by mass, the dopamine-grafted hyaluronic acid mixed solution includes 0.5 - 2 parts of sodium hyaluronate, 0.3 - 0.7 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.2 - 0.4 parts of N-hydroxysuccinimide, 0.2 - 0.4 parts of dopamine hydrochloride, 0.05 - 0.2 parts of polylysine, 0.05 - 0.2 parts of collagen, and 0.05 - 0.2 parts of silk fibroin.

[0013] As a limitation of the present invention, the preparation method of the keratinocyte growth factor sustained-release microspheres is as follows:

[0014] Dissolve the poly(lactic-co-glycolic acid) copolymer in dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix sodium chloride, polyvinyl alcohol, and deionized water, stir, and disperse them evenly to form a polyvinyl alcohol solution. Dissolve sodium chloride in deionized water to form a sodium chloride solution. Add keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, and stir magnetically for 60 - 120 s to form the primary emulsion. Subsequently, transfer the primary emulsion to the polyvinyl alcohol solution and stir at 1000 - 2000 rpm for 60 - 120 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 - 300 rpm for 2 - 4 h for curing. Collect the cured microspheres, wash them with deionized water, and freeze-dry them to obtain keratinocyte growth factor sustained-release microspheres.

[0015] As a limitation of the present invention, the mass ratio of the poly(lactic-co-glycolic acid) copolymer to the keratinocyte growth factor-dextran microparticles is (18 - 22):(0.5 - 0.7).

[0016] As a limitation of the present invention, the mass fraction of sodium chloride in the polyvinyl alcohol solution is 4 - 6%, and the mass fraction of polyvinyl alcohol is 0.5 - 2%; the mass fraction of sodium chloride in the sodium chloride solution is 5 - 15%.

[0017] As a limitation of the present invention, the preparation method of the keratinocyte growth factor-dextran microparticles is as follows:

[0018] Mix keratinocyte growth factor, dextran, and polyethylene glycol, and stir at 1000 - 2000 rpm for 0.5 - 2 min to make them evenly mixed. Then freeze at -80 °C for 10 - 15 h. After freezing is completed, take it out and vacuum freeze-dry. Wash the obtained dry powder with dichloromethane, centrifuge, remove PEG in the supernatant, and dry at room temperature for 20 - 30 h to obtain keratinocyte growth factor-dextran microparticles.

[0019] As a limitation of the present invention, the mass ratio of keratinocyte growth factor, dextran, and polyethylene glycol is (0.05 - 0.2):(0.4 - 0.6):(4 - 6).

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] Keratinocyte growth factor is an alkaline protein growth factor secreted by tissue cells, which can stimulate the proliferation of epithelial cells, thereby achieving the function of promoting wound healing, and has good therapeutic and repair effects on skin, corneal, or other tissue injuries. However, due to the complex internal environment of the human body and the influence of tissue absorption or enzymatic hydrolysis, the half-life of keratinocyte growth factor in the body is very short. Therefore, using dextran to protect keratinocyte growth factor to form keratinocyte growth factor-dextran microparticles can extend the effective action time of keratinocyte growth factor and also avoid protein inactivation during the processing process.

[0022] Poly(lactic-co-glycolic acid) has good biodegradability, biocompatibility, non-toxicity and non-immunogenicity. Encapsulating keratinocyte growth factor-dextran microparticles with poly(lactic-co-glycolic acid) to form keratinocyte growth factor sustained-release microspheres and constructing an in vivo sustained-release system for keratinocyte growth factor can prolong the effective time of keratinocyte growth factor in vivo and avoid the increase in local hormone or drug levels caused by direct administration, which may lead to irritation and discomfort in the human body.

[0023] Hyaluronic acid is a glycosaminoglycan commonly present in the extracellular matrix. Dopamine is grafted onto hyaluronic acid, and the phenolic hydroxyl groups on dopamine are oxidized with sodium periodate, a strong oxidant, and crosslinked to form a dopamine-hyaluronic acid hydrogel with good biodegradability, biocompatibility, non-toxicity and non-immunogenicity. On this basis, adding the cationic polypeptide polylysine improves the bioadhesion of the hydrogel, providing a basis for the hydrogel to adhere to the endometrial wound surface and achieve precise release of keratinocyte growth factor. Adding collagen and silk fibroin can improve the strength, toughness, elasticity and environmental stability of the hydrogel, which is beneficial to the loading of the sustained-release microspheres and the transport of substances, thus facilitating the provision of a channel for the transport of nutrients to cells. Specific embodiments

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] Keratinocyte growth factor (KGF-2, biological activity > 2 × 10 6 IU / mg), polyethylene glycol (PEG400), poly(lactic-co-glycolic acid) (lactic acid / glycolic acid ratio: 50 / 50, Mw = 10000), polyvinyl alcohol (PVA1788, degree of alcoholysis: 87 - 89%), polylysine (PDL, Mw = 150000), collagen (high-purity type I collagen, Mw = 300000), silk fibroin (Mw = 100000).

[0026] Example 1: A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0027] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0028] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make them evenly mixed, then place them in a -80 °C refrigerator and freeze for 12 h. After freezing, take them out, vacuum freeze-dry, wash the obtained dry powder with dichloromethane, centrifuge, remove PEG in the supernatant, and dry at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0029] Step 2: Prepare keratinocyte growth factor sustained-release microspheres

[0030] Dissolve 20 g of poly(lactic-co-glycolic acid) copolymer in 80 g of dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix 5 g of sodium chloride, 1 g of polyvinyl alcohol and 94 g of deionized water, stir to disperse evenly to form a polyvinyl alcohol solution. Dissolve 200 g of sodium chloride in 1800 g of deionized water to form a sodium chloride solution. Add 0.6 g of keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, stir and disperse magnetically for 60 s to form a primary emulsion. Then transfer the primary emulsion to the polyvinyl alcohol solution and stir at 2000 rpm for 60 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 rpm for 4 h to solidify. Collect the solidified microspheres, wash with deionized water, and freeze-dry to obtain keratinocyte growth factor sustained-release microspheres.

[0031] Step 3: Prepare a sustained-release gel preparation for endometrial repair

[0032] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After fully dissolving, add 0.3 g of dopamine hydrochloride and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification. After purification, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.02 g of keratinocyte growth factor sustained-release microspheres, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel. Wash with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0033] Example 2: A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0034] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0035] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make them evenly mixed, then place them in a -80 °C refrigerator and freeze for 12 h. After freezing, take them out, vacuum freeze-dry. Wash the obtained dry powder with dichloromethane, centrifuge, remove PEG in the supernatant, and dry at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0036] Step 2: Prepare keratinocyte growth factor sustained-release microspheres

[0037] Dissolve 20 g of poly(lactic-co-glycolic acid) copolymer in 80 g of dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix 5 g of sodium chloride, 1 g of polyvinyl alcohol and 94 g of deionized water, stir to disperse evenly to form a polyvinyl alcohol solution. Dissolve 200 g of sodium chloride in 1800 g of deionized water to form a sodium chloride solution. Add 0.6 g of keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, stir and disperse magnetically for 60 s to form a primary emulsion. Then transfer the primary emulsion to the polyvinyl alcohol solution and stir at 2000 rpm for 60 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 rpm for 4 h to solidify. Collect the solidified microspheres, wash with deionized water, and freeze-dry to obtain keratinocyte growth factor sustained-release microspheres.

[0038] Step 3: Prepare a sustained-release gel preparation for endometrial repair

[0039] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After fully dissolving, add 0.3 g of dopamine hydrochloride and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification treatment. After purification, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.017 g of keratinocyte growth factor sustained-release microspheres, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel. Wash with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0040] Example 3: A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0041] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0042] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make the mixture uniform, then place it in a -80 °C refrigerator and freeze for 12 h. After freezing, take it out and perform vacuum freeze-drying. Wash the obtained dry powder with dichloromethane, centrifuge to remove PEG in the supernatant, and dry at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0043] Step 2: Prepare keratinocyte growth factor sustained-release microspheres

[0044] Dissolve 20 g of poly(lactic-co-glycolic acid) copolymer in 80 g of dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix 5 g of sodium chloride, 1 g of polyvinyl alcohol and 94 g of deionized water, stir to disperse them evenly to form a polyvinyl alcohol solution. Dissolve 200 g of sodium chloride in 1800 g of deionized water to form a sodium chloride solution. Add 0.6 g of keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, stir magnetically for 60 s to disperse and form a primary emulsion. Then transfer the primary emulsion to the polyvinyl alcohol solution and stir at 2000 rpm for 60 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 rpm for 4 h to solidify. Collect the solidified microspheres, wash them with deionized water, and perform freeze-drying to obtain keratinocyte growth factor sustained-release microspheres.

[0045] Step 3: Prepare a sustained-release gel preparation for endometrial repair

[0046] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After complete dissolution, add 0.3 g of dopamine hydrochloride and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification. After purification, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.015 g of keratinocyte growth factor sustained-release microspheres, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel. Rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0047] Example 4: A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0048] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0049] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran, and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make the mixture homogeneous, then place it in a -80 °C refrigerator and freeze for 12 h. After freezing is completed, take it out, perform vacuum freeze-drying. Wash the obtained dry powder with dichloromethane, centrifuge, remove the PEG in the supernatant, and dry at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0050] Step 2: Prepare keratinocyte growth factor sustained-release microspheres

[0051] Dissolve 20 g of poly(lactic-co-glycolic acid) copolymer in 80 g of dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix 5 g of sodium chloride, 1 g of polyvinyl alcohol, and 94 g of deionized water, stir to disperse evenly to form a polyvinyl alcohol solution. Dissolve 200 g of sodium chloride in 1800 g of deionized water to form a sodium chloride solution. Add 0.6 g of keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, stir magnetically for 60 s to disperse and form a primary emulsion. Then transfer the primary emulsion to the polyvinyl alcohol solution and stir at 2000 rpm for 60 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 rpm for 4 h to solidify. Collect the solidified microspheres, wash with deionized water, and perform freeze-drying to obtain keratinocyte growth factor sustained-release microspheres.

[0052] Step 3: Prepare a sustained-release gel preparation for endometrial repair

[0053] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After complete dissolution, add 0.3 g of dopamine hydrochloride and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution and dialyze the solution through a 14KDa dialysis membrane for 48 h for purification treatment. After purification is completed, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.023 g of keratinocyte growth factor sustained-release microspheres, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel. Wash with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0054] Example 5: A preparation method of a sustained-release gel preparation for endometrial repair, specifically:

[0055] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0056] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make the mixture uniform, then place it in a -80 °C refrigerator and freeze for 12 h. After freezing, take it out, perform vacuum freeze-drying. The obtained dry powder is washed with dichloromethane, centrifuged to remove PEG in the supernatant, and dried at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0057] Step 2: Prepare sustained-release microspheres of keratinocyte growth factor

[0058] Dissolve 20 g of poly(lactic-co-glycolic acid) copolymer in 80 g of dichloromethane to prepare a poly(lactic-co-glycolic acid) copolymer solution. Mix 5 g of sodium chloride, 1 g of polyvinyl alcohol and 94 g of deionized water, stir to disperse evenly to form a polyvinyl alcohol solution. Dissolve 200 g of sodium chloride in 1800 g of deionized water to form a sodium chloride solution. Add 0.6 g of keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) copolymer solution, stir and disperse magnetically for 60 s to form a primary emulsion. Then transfer the primary emulsion to the polyvinyl alcohol solution and stir at 2000 rpm for 60 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution and stir at 100 rpm for 4 h to solidify. Collect the solidified microspheres, wash with deionized water, and perform freeze-drying to obtain sustained-release microspheres of keratinocyte growth factor.

[0059] Step 3: Prepare a sustained-release gel preparation for endometrial repair

[0060] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After fully dissolving, add 0.3 g of dopamine hydrochloride and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification. After purification, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.025 g of sustained-release microspheres of keratinocyte growth factor, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel. Rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0061] Based on Example 1 below, control experiments are carried out, specifically Comparative Example 1 and Comparative Example 2, as described below:

[0062] Comparative Example 1: This comparative example relates to a preparation method of a sustained-release gel preparation for endometrial repair. The difference from Example 1 is that keratinocyte growth factor-dextran microparticles are added to the hydrogel, specifically as follows:

[0063] Step 1: Prepare keratinocyte growth factor-dextran microparticles

[0064] Mix 0.1 g of keratinocyte growth factor, 0.5 g of dextran, and 5 g of polyethylene glycol, stir at 1500 rpm for 1 min to make them evenly mixed, then place them in a -80 °C refrigerator and freeze for 12 h. After freezing, take them out, perform vacuum freeze-drying. The obtained dry powder is washed with dichloromethane, centrifuged, the PEG in the supernatant is removed, and dried at room temperature for 24 h to obtain keratinocyte growth factor-dextran microparticles.

[0065] Step 2: Prepare a sustained-release gel preparation for endometrial repair

[0066] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution. Adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After fully dissolving, add 0.3 g of dopamine hydrochloride, and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution, and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification treatment. After purification, add 0.1 g of collagen, 0.15 g of silk fibroin, and 0.02 g of keratinocyte growth factor-dextran microparticles, continue to stir for 1 h, then adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel, and rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0067] Comparative Example 2: This comparative example relates to a preparation method of a sustained-release gel preparation for endometrial repair. The difference from Example 1 is that keratinocyte growth factor sustained-release microspheres are not added to the hydrogel, specifically as follows:

[0068] Step 1: Prepare a sustained-release gel preparation for endometrial repair

[0069] Mix 1 g of sodium hyaluronate and 100 g of deionized water, stir evenly to obtain a sodium hyaluronate solution, adjust the pH of the sodium hyaluronate solution to 5, add 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.3 g of N-hydroxysuccinimide. After fully dissolving, add 0.3 g of dopamine hydrochloride, and continuously stir for 12 h to obtain a dopamine-grafted hyaluronic acid solution. Add 0.09 g of polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 30 min, then use ultrapure water as the dialysis solution, and dialyze the solution with a 14KDa dialysis membrane for 48 h for purification treatment. After purification, add 0.1 g of collagen and 0.15 g of silk fibroin, adjust the pH of the solution to 9, add 0.45 g of sodium periodate, and react in a 37 °C water bath in the dark for 12 h to form a gel, and rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

[0070] Detection experiment:

[0071] Prepare samples of sustained-release gel preparations for endometrial repair according to the preparation methods in Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2, respectively.

[0072] In vitro release test: Put 1.5 g of the sustained-release gel preparation sample into 15 mL of simulated synthetic uterine fluid (pH = 7.2), store it in a 37 °C water bath for 30 days, collect the simulated synthetic uterine fluid every two days and add fresh simulated synthetic uterine fluid to keep the total volume unchanged. The collected simulated synthetic uterine fluid is used to measure the concentration of the released keratinocyte growth factor with a KGF-ELISA kit (PK678, Beyotime Biotechnology), and calculate the in vitro cumulative release rate of the sustained-release gel preparation sample.

[0073] Promoting cell proliferation test: Select the CCK-8 method to test the promoting proliferation effect of the sustained-release gel preparation for endometrial repair on human endometrial cells. First, place each sustained-release gel preparation sample in a 96-well plate to form a gel. After disinfection treatment, plant 4×10 4 human endometrial cells (1×10 6 cells / T25 flask, Qisai Biotechnology) on the gel in the well plate. The control group is not treated with gel. After culturing for 48 h, test. Mix the CCK-8 solution (No. C0038, Beyotime Biotechnology) and the complete medium according to a mass ratio of 1:10 to prepare a cytotoxicity detection solution. Before the test, aspirate the medium, wash 3 times with PBS buffer, and then add 500 μL of the prepared cytotoxicity detection solution, and continue to culture for 1.5 h. Finally, aspirate 100 μL of the test solution into a new 96-well plate, and measure the OD value at 450 nm with a multifunctional microplate reader. Calculate the cell proliferation rate of each group with the cell proliferation rate of the control group as 100%.

[0074] Repair effect test: 80 healthy female rats (8 weeks old, weighing 240 - 260 g) were selected as test subjects. They were randomly divided into 8 groups. After anesthesia, the abdominal cavity was incised to expose the uterus. The endometrium was scraped off with a surgical blade until the endometrium was pale to the naked eye. Subsequently, after rinsing with physiological saline, in the first 7 groups, a sustained-release gel preparation sample for endometrial repair was implanted on the endometrial wound surface of the rats. The last group was only rinsed with physiological saline without implanting the gel as a control. After implantation and suturing, all the postoperative rats were given antibiotic treatment for three consecutive days, and at the same time, appropriate light, sufficient feed, and pure water were provided. They were raised for 30 days. After 30 days, the rats were euthanized, the uterus was removed, and the average thickness of the regenerated endometrium of each group of rats was measured.

[0075]

[0076] Conclusion: It can be seen from the test data that the in vitro cumulative release amount of keratinocyte factor of the gel preparation prepared in Comparative Example 1 in the first 5 days is much higher than that of the Examples. And at 30 days, the in vitro cumulative release amounts of the keratinocyte factor of the sustained-release gel reagents prepared in the Examples and the Comparative Example are nearly equal. However, the cell proliferation promoting rate of the Comparative Example is greater than that of the Examples. This is due to the large amount of keratinocyte factor released in a short time in the Comparative Example. From the results of comparing the endometrial thickness, the sustained-release gel preparation for endometrial repair prepared by the preparation method of Example 1 can be slowly released, has a promoting proliferation effect on human endometrial cells, and can be used for the repair of the endometrium.

[0077] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims in the present invention.

Claims

1. A preparation method of a sustained-release gel preparation for endometrial repair, characterized in that: Specifically: Add keratinocyte growth factor sustained-release microspheres to the dopamine-grafted hyaluronic acid mixed solution, stir for 0.5 - 2 h, then adjust the pH of the solution to 8 - 9, add sodium periodate, and react in a water bath at 35 - 40 °C in the dark for 10 - 15 h to form a gel. Rinse with deionized water to obtain a sustained-release gel preparation for endometrial repair.

2. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 1, characterized in that: The mass ratio of keratinocyte growth factor sustained-release microspheres to sodium periodate is (0.01 - 0.03):(0.4 - 0.5).

3. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 1, wherein: The preparation method of the dopamine-grafted hyaluronic acid mixed solution is as follows: Mix sodium hyaluronate and deionized water, stir evenly to obtain a sodium hyaluronate solution, adjust the pH of the sodium hyaluronate solution to 4 - 5, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, fully dissolve, then add dopamine hydrochloride, and continuously stir for 10 - 15 h to obtain a dopamine-grafted hyaluronic acid solution. Add polylysine to the dopamine-grafted hyaluronic acid solution, continuously stir for 20 - 40 min, then use ultrapure water as the dialysis solution, and dialyze the solution with an 8 - 14 kDa dialysis membrane for 48 - 72 h for purification treatment. After purification, add collagen and silk fibroin, and stir evenly to obtain a dopamine-grafted hyaluronic acid mixed solution.

4. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 3, characterized in that: By mass, the dopamine-grafted hyaluronic acid mixed solution includes 0.5 - 2 parts of sodium hyaluronate, 0.3 - 0.7 parts of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 0.2 - 0.4 parts of N-hydroxysuccinimide, 0.2 - 0.4 parts of dopamine hydrochloride, 0.05 - 0.2 parts of polylysine, 0.05 - 0.2 parts of collagen, and 0.05 - 0.2 parts of silk fibroin.

5. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 1, characterized in that: The preparation method of the keratinocyte growth factor sustained-release microspheres is as follows: Dissolve poly(lactic-co-glycolic acid) in dichloromethane to prepare a poly(lactic-co-glycolic acid) solution. Mix sodium chloride, polyvinyl alcohol, and deionized water, stir, and disperse evenly to form a polyvinyl alcohol solution. Dissolve sodium chloride in deionized water to form a sodium chloride solution. Add keratinocyte growth factor-dextran microparticles to the poly(lactic-co-glycolic acid) solution, stir magnetically for 60 - 120 s to form a primary emulsion, then transfer the primary emulsion to the polyvinyl alcohol solution, stir at 1000 - 2000 rpm for 60 - 120 s to form an emulsion. Finally, transfer the emulsion to the sodium chloride solution, stir at 100 - 300 rpm for 2 - 4 h for curing, collect the cured microspheres, wash with deionized water, and freeze-dry to obtain keratinocyte growth factor sustained-release microspheres.

6. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 5, characterized in that: The mass ratio of poly(lactic-co-glycolic acid) to keratinocyte growth factor-dextran microparticles is (18 - 22):(0.5 - 0.7).

7. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 5, characterized in that: The mass fraction of sodium chloride in the polyvinyl alcohol solution is 4 - 6%, and the mass fraction of polyvinyl alcohol is 0.5 - 2%; the mass fraction of sodium chloride in the sodium chloride solution is 5 - 15%.

8. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 5, characterized in that: The preparation method of the keratinocyte growth factor-dextran microparticles is: Mix keratinocyte growth factor, dextran and polyethylene glycol, and stir at 1000 - 2000 rpm for 0.5 - 2 min to make them evenly mixed. Then freeze at -80 °C for 10 - 15 h. After the freezing is completed, take it out and conduct vacuum freeze-drying. Wash the obtained dry powder with dichloromethane, centrifuge, remove PEG in the supernatant, and dry at room temperature for 20 - 30 h to obtain keratinocyte growth factor-dextran microparticles.

9. The preparation method of a sustained-release gel preparation for endometrial repair according to claim 8, characterized in that: The mass ratio of keratinocyte growth factor, dextran and polyethylene glycol is (0.05 - 0.2):(0.4 - 0.6):(4 - 6).

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