CN113278170B - Chemically crosslinked hyaluronic acid hydrogel and preparation method and application thereof - Google Patents
Chemically crosslinked hyaluronic acid hydrogel and preparation method and application thereof Download PDFInfo
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- CN113278170B CN113278170B CN202110662302.5A CN202110662302A CN113278170B CN 113278170 B CN113278170 B CN 113278170B CN 202110662302 A CN202110662302 A CN 202110662302A CN 113278170 B CN113278170 B CN 113278170B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
Abstract
The invention discloses a chemically crosslinked hyaluronic acid hydrogel and a preparation method and application thereof. The hyaluronic acid hydrogel is obtained by crosslinking hyaluronic acid or a salt thereof in a lithium bromide solution through a chemical crosslinking agent. According to the invention, the lithium bromide crosslinking is adopted to replace the traditional alkaline reaction condition, so that the dosage of the crosslinking agent can be reduced, the crosslinking reaction speed is accelerated, the crosslinking efficiency is improved, and the gel conversion efficiency is improved.
Description
Technical Field
The invention relates to a chemically crosslinked hyaluronic acid hydrogel and a preparation method and application thereof, belonging to the technical field of materials.
Background
Due to a series of problems of defects and deformities of human tissues and organs caused by congenital or acquired reasons and the demand of the medical and American industry, researchers and medical instrument enterprises are always searching for a safe, convenient and reliable filler for reshaping and promoting tissue repair, and since the 20 th century and the 40 th century, due to the further development of material science, many implant materials are used as tissue fillers for human tissue filling, and the use frequency of the mode of treating patients by means of tissue filling is higher and higher in the last thirty years, and the number of operations related to soft tissue filling in the United states is increased from 160 million cases in 2011 to 240 million cases in 2015. Tissue augmentation is the filling of tissue by volume expansion, and dermal fillers consisting of suspensions of various high viscosity liquids or polymer particles have been used for recent decades to fill wrinkles and acne under scars. The tissue fillers currently on the market are mainly collagen and hyaluronic acid.
Hyaluronic acid is a high-molecular long-chain mucopolysaccharide, widely distributed in various parts of the human body, has good hydrophilicity, viscoelasticity, lubricity and biocompatibility, and has been widely applied in the medical field. Natural hyaluronic acid has poor stability, is sensitive to hyaluronidase and free radicals, and has short in vivo retention time, and a common method is to crosslink the natural hyaluronic acid by using a crosslinking agent (divinyl sulfone (DVS) or butanediol diglycidyl ether (BDDE)), and regulate the in vivo retention time by the crosslinking degree.
At present, the crosslinking process is performed in an alkaline environment, preferably at pH 11-13, in the reported literature. The method has low crosslinking efficiency and low reaction recovery rate (gel conversion rate), and inevitably increases the injection frequency of the product and has high product price. Therefore, the key point for solving the material requirements and cost reduction in the field of the current tissue filling agent is to find an efficient crosslinked hydrogel and a preparation method thereof.
In order to solve the problems, the invention provides a high-efficiency cross-linked hyaluronic acid, a preparation method and application thereof. The basic idea is that hyaluronic acid is a long-chain polysaccharide with a higher degree of freedom in lithium bromide solution, exposing more reactive groups. The reaction of chemical crosslinking reaction in lithium bromide solution can raise the reaction homogeneity, crosslinking efficiency and product recovering rate obviously.
Disclosure of Invention
In order to solve the above problems, the present invention provides a chemically crosslinked hyaluronic acid hydrogel and a method for preparing the same. The hyaluronic acid is a long-chain polysaccharide, so that the hyaluronic acid has higher degree of freedom in a lithium bromide solution, more reaction groups are exposed, and the reaction crosslinking efficiency and the product recovery rate are improved.
The first purpose of the invention is to provide a chemically cross-linked hyaluronic acid hydrogel, which is obtained by cross-linking hyaluronic acid or a salt thereof in a lithium bromide solution through a chemical cross-linking agent.
Further, the chemical crosslinking agent is one or more than two of diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane and sodium trimetaphosphate.
Further, the molecular weight of the hyaluronic acid is 400k-3000 kDa.
Further, the molecular weight of the hyaluronic acid is preferably 500k-2500kDa, and more preferably 1000k-2200 kDa.
The second object of the present invention is to provide a method for preparing the chemically crosslinked hyaluronic acid hydrogel, comprising the steps of:
s1, dissolving hyaluronic acid or a salt thereof in a lithium bromide solution to obtain a hyaluronic acid lithium bromide solution, and adding a chemical cross-linking agent into the hyaluronic acid lithium bromide solution, or adding the chemical cross-linking agent into the lithium bromide solution in advance;
and S2, carrying out a crosslinking reaction to obtain the chemically crosslinked hyaluronic acid hydrogel.
Further, in the step of S2, a step of removing lithium bromide, unreacted chemical crosslinking agent and unreacted hyaluronic acid after the reaction is further included.
Further, the mass fraction of the hyaluronic acid or the salt thereof is 1 mg/mL-300 mg/mL.
Further, the mass fraction of the hyaluronic acid or a salt thereof is preferably 10 mg/mL-250 mg/mL, and more preferably 20 mg/mL-200 mg/mL.
Further, the mass-to-volume ratio of the hyaluronic acid or the salt thereof to the chemical crosslinking agent is 1 g: 0.2-1000 μ L.
Further, the mass-to-volume ratio of the hyaluronic acid or the salt thereof to the chemical crosslinking agent is preferably 1 g: 2 to 800. mu.L, more preferably 1 g: 5-500 μ L.
Further, the concentration of the lithium bromide is 0.1-10M.
Further, the concentration of lithium bromide is preferably 1 to 10M, and more preferably 2 to 9.8M.
Further, the crosslinking reaction is carried out for 3 minutes to 72 hours at the reaction temperature of minus 80 ℃ to 80 ℃.
The third purpose of the invention is to provide the application of the chemically cross-linked hyaluronic acid hydrogel, wherein the application comprises the application in the filling or repairing material of soft tissues, hard tissues or cavities.
The fourth purpose of the invention is to provide a hydrogel composition, which comprises the chemically crosslinked hyaluronic acid hydrogel.
Further, the chemically cross-linked hyaluronic acid hydrogel also comprises one or more combinations of a humectant, a bioactive agent, a cosmetic active agent, a cell attachment agent, a cell, an extracellular matrix and a medicament.
Wherein the humectant comprises: glycerol, polyglutamic acid, and the like.
Wherein the bioactive agent comprises: cell growth factors, peptides, peptidomimetics, antibodies, nucleic acids, polysaccharides, and the like.
Wherein the cells comprise: stem cells, adult cells, osteoblasts, chondroblasts, adipoblasts, and the like.
Wherein the cosmetic active agent comprises: anti-aging agent, anti-free radical agent, antioxidant, hydrating agent, whitening agent, coloring agent, sunscreen agent, muscle relaxant, etc.
The invention has the beneficial effects that:
according to the invention, the lithium bromide crosslinking is adopted to replace the traditional alkaline reaction condition, so that the dosage of the crosslinking agent can be reduced, the crosslinking reaction speed is accelerated, and the product recovery rate (gel conversion rate) is improved.
Description of the drawings:
FIG. 1 shows the reaction conditions and reaction efficiency of hyaluronic acid in alkaline system (0.25M NaOH) and lithium bromide system. A: the reaction condition of hyaluronic acid in a sodium hydroxide and lithium bromide system; b: the reaction efficiency of hyaluronic acid in sodium hydroxide and lithium bromide systems.
FIG. 2 shows the modification degree of chemically cross-linked hyaluronic acid prepared by different reaction systems.
FIG. 3 shows the in vitro degradation of chemically cross-linked hyaluronic acid prepared by different reaction systems.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
Example 1: preparation of cross-linked hyaluronic acid without reaction system
In order to verify the applicable range of the present invention and the reaction conditions thereof, the inventors conducted tests on the molecular weight of hyaluronic acid or a salt thereof, the reaction mass fraction, the kind of the crosslinking agent, the amount of the crosslinking agent added, the reaction temperature and the reaction time.
Molecular weight of hyaluronic acid or salt thereof: 400kDa, 500kDa, 700kDa, 900kDa, 1000kDa, 1300kDa, 1400kDa, 1500kDa, 1600kDa, 1800kDa, 2000kDa, 2100kDa, 2200kDa, 2400kDa, 2500kDa, 2700kDa, 2800kDa, 3000 kDa.
Reaction mass fraction: 1mg/mL, 2mg/mL, 5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL, 30mg/mL, 50mg/mL, 75mg/mL, 90mg/mL, 100mg/mL, 130mg/mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, 210mg/mL, 230mg/mL, 250mg/mL, 290mg/mL, 300mg/mL
Types of crosslinking agents: diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane and sodium trimetaphosphate.
Addition amount of the crosslinking agent: 1 g: 0.2. mu.L, 1 g: 0.40. mu.L, 1 g: 0.7. mu.L, 1 g: 1 μ L, 1 g: 4 μ L, 1 g: 7 μ L, 1 g: 8 μ L, 1 g: 10 μ L, 1 g: 30 μ L, 1 g: 60 μ L, 1 g: 80 μ L, 1 g: 100. mu.L, 1 g: 300. mu.L, 1 g: 600 μ L, 1 g: 900 μ L, 1 g: 1000 μ L
Reaction temperature: -80, -20, 4, 25, 37, 45, 60, 80
Reaction time: 3 minutes, 5 minutes, 10 minutes, 30 minutes, 45 minutes, 60 minutes, 1 hour, 3 hours, 5 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours.
The control group only had the reaction system as 0.25M sodium hydroxide solution.
Experiments show that the sample group of the invention is superior to the control group in the modification degree test. The invention adopts a lithium bromide reaction system to obviously improve the crosslinking efficiency.
The inventor lists dissolving 0.05g, 0.1g, 0.5g and 1g hyaluronic acid (molecular weight 1400KDa) in 10mL 0.25M NaOH or 9.3M lithium bromide respectively, adding 500 μ L1, 4-butanediol diglycidyl ether (BDDE) into the reaction system rapidly, stirring uniformly, placing in an oven at 60 ℃ for 3 hours, observing whether block gel is formed in the reaction system, collecting the block gel, washing with water, freeze-drying and weighing, and calculating the crosslinking efficiency.
Efficiency of crosslinking W2/W1*100％
Wherein, W2For crosslinking hyaluronic acid, W1Is hyaluronic acid added to the reaction system before crosslinking. As can be seen from FIG. 1, when the reaction concentration of hyaluronic acid is 0.5mg/mL, neither reaction system is gelled; when the reaction concentration is 1mg/mL, the lithium bromide system forms gel, the recovery rate is about 5 percent, and the sodium hydroxide system does not form gel; the reaction concentrations of 5mg/mL and 10mg/mL both formed gels, but the recovery rate of the sodium hydroxide system was less than 20%, while the recovery rate of the lithium bromide system exceeded 60%. It can be seen that the reaction efficiency of BDDE and hyaluronic acid is much higher in the lithium bromide system than in the sodium hydroxide system.
The research finds that: the ideal hyaluronic acid hydrogel can be obtained by using diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane and sodium trimetaphosphate as cross-linking agents.
The molecular weight of hyaluronic acid is 400k-3000kDa, and the ideal hyaluronic acid hydrogel can be obtained. Within the range of 500k-2500kDa, the hyaluronic acid hydrogel has more excellent performance. The hyaluronic acid hydrogel performance is further excellent in the range of 1000k-2200 kDa.
The hyaluronic acid or the salt thereof can obtain more ideal hyaluronic acid hydrogel within the range of 1 mg/mL-300 mg/mL. Within the range of 10 mg/mL-250 mg/mL, the hyaluronic acid hydrogel has more excellent performance. The hyaluronic acid hydrogel has a further excellent performance within a range of 20mg/mL to 200 mg/mL.
When the mass-volume ratio of the hyaluronic acid or the salt thereof to the chemical crosslinking agent is 1 g: within the range of 0.2-1000 mu L, the more ideal hyaluronic acid hydrogel can be obtained. In the presence of 1 g: within the range of 2-800 mu L, the hyaluronic acid hydrogel has more excellent performance. In the presence of 1 g: the hyaluronic acid hydrogel has further excellent performance within the range of 5-500 mu L.
The ideal hyaluronic acid hydrogel can be obtained within the lithium bromide concentration range of 0.1-10M. Within the range of 1-10M, the hyaluronic acid hydrogel has more excellent performance. Within the range of 2-9.8M, the hyaluronic acid hydrogel has further excellent performance.
The ideal hyaluronic acid hydrogel can be obtained within the reaction temperature range of-80 to 80 ℃.
Within the reaction time range of 3 minutes to 72 hours, the more ideal hyaluronic acid hydrogel can be obtained.
Example 2: hyaluronic acid modification degree test
In the 0.5g and 1g groups of example 1, samples at 9 positions on the obtained crosslinked hyaluronic acid gel were measured for modification degrees of 9 samples of about 1g each, and RSD values were calculated. Modification degree test conditions: weighing 0.25g of the purified sample of the first embodiment in a 5mL volumetric flask, diluting the volume to 5mL with the enzymolysis solution, standing the solution at 42 ℃ for 2h, inactivating the solution, and standing the inactivated solution for later use. The modification degree was measured by using high performance liquid chromatography, column: superdex 75, sample size: 20 μ L, flow rate: 0.5mL/min, column temperature: 35 ℃, mobile phase: 20mmol/L, wavelength: 232 nm. In other cases where the experimental conditions were otherwise identical, a higher degree of modification indicates a higher crosslinking efficiency. The closer the degree of modification at the 9 sampling points, the higher the uniformity of crosslinking. As can be seen from the results of FIG. 2, the modification degrees of 9 samples on the crosslinked hyaluronic acid gel obtained by the method of the present invention are almost completely consistent, while the modification degree difference in the comparative example is larger, which indicates that the method of the present invention effectively ensures the crosslinking uniformity. The sample obtained by the reaction under the alkaline condition can be obtained by comparison, and the modification degree of the sample obtained by the method is far higher than that of a comparison group, which shows that the crosslinking efficiency is obviously improved by adopting a microwave reaction method.
Example 3: in vitro degradation test
Taking a proper amount of 0.5g and 1g of the group in the first embodiment, placing a chemically cross-linked hyaluronic acid gel (containing about HA 8mg) in a penicillin bottle, adding 4mL of self-made hyaluronic acid gel enzyme (with the enzyme activity of 60U/mL), and uniformly mixing by vortex. Shaking in a water bath at 42 deg.C, sampling 50 μ L every 10min and diluting appropriately, measuring absorbance at 232nm until absorbance no longer changes, considering degradation is complete, and recording the time taken for complete degradation. The test results are shown in FIG. 3. The time for degrading the crosslinked hyaluronic acid gel for injection in vitro prepared by the embodiment is relatively long, and the time for degrading the crosslinked hyaluronic acid gel for injection in vitro prepared by the comparative example is relatively short, so that the crosslinked hyaluronic acid gel for injection prepared by the method provided by the invention has excellent enzymolysis resistance, and the crosslinking efficiency of the crosslinked hyaluronic acid gel for injection is high.
Example 4: injectable gel composition preparation, biosafety evaluation and animal subcutaneous filling experiment
Dissolving 10g of high molecular weight hyaluronic acid (molecular weight 1400KDa) in 100mL of 9.3M lithium bromide, rapidly adding 5mL of 1, 4-butanediol diglycidyl ether (BDDE) into the reaction system, stirring well, placing in an oven at 60 ℃ for 3 hours, cutting the gel into about 1cm3Adding phosphate buffer solution into the particles to swell the gel block, dialyzing, removing unreacted cross-linking agent and lithium bromide, controlling the concentration of hyaluronic acid to be 1.5 percent, controlling the pH value of the gel to be 6.0-8.0, and sieving the gel with a 120-mesh stainless steel sieve to obtain the cross-linked sodium hyaluronate gel. The reaction system was compared with 0.25M sodium hydroxide.
Adding small molecular weight hyaluronic acid (molecular weight 80KDa)1.0g into glycerol water (0.5% physiological saline, phosphate, amino acid, etc.) 100mL, stirring to dissolve, and filtering with 5 μm filter core to obtain non-crosslinked sodium hyaluronate gel with pH of 6.0-8.0. Mixing the sodium hyaluronate gel and the non-crosslinked sodium hyaluronate gel in a mass ratio of 1:10, uniformly mixing, and performing heat treatment at 100 ℃ for 60 minutes to obtain an injectable hyaluronate gel system. And the following tests were performed:
1. the extrusion force is detected according to the test method in appendix B in the cross-linked sodium hyaluronate gel for the YY/T0962-2014 plastic surgery. The extrusion force of the hyaluronic acid composition prepared by the invention is 5-30N.
2. The osmolarity was tested according to the onset of freezing point depression in the fourth 0632 osmolarity determination of the pharmacopoeia of the people's republic of China (2015 edition). The particle size of the injectable gel system is 250-350 mOsmol.
3. The particle diameter was measured according to the color development in the light color she development in the particle size and particle size distribution measurement method of 0982 in the fourth pharmacopoeia of the people's republic of China (2015 edition). The particle size of the injectable gel system is 10-300 μm.
4. The rotational viscosity was measured by a rotational viscometer method according to the third part of the viscosity measuring method of 0633 in the fourth pharmacopoeia of the people's republic of China (2015 edition). The injectable gel system has a kinematic viscosity of 10-80mm2/s。
5. The pH was measured according to the pH measurement method of the fourth 0631 pharmacopoeia of the people's republic of China (2015 edition). The injectable gel system has a pH of 6.0-8.0.
6. Heavy metals were tested according to the second method of the 0821 metal detection method of the fourth pharmacopoeia of the people's republic of China (2015). The heavy metal residue of the injectable gel system is less than or equal to 10 microgram/g.
7. Hemolysis was carried out according to the method prescribed in GB/T16886.4-2003. The injectable gel system is less than or equal to 5 percent.
8. Cytotoxicity test was carried out according to the method prescribed in GB/T16886.5-2008. The injectable gel system is less than or equal to grade I.
9. Skin sensitization test was carried out according to the method prescribed in GB/T16886.10-2005. The injectable gel system is less than or equal to grade I.
10. The yamaman toxicity test was carried out according to the method prescribed in GB/T16886.11-2011. The injectable gel system is free of yamamotoxicity.
11. Experiments with subcutaneous implants. About 250g of 30 healthy rats are selected, the sample group is injected into the left side of the back, and the control group is injected into the right side, wherein the injection amount is 1 mL. The effect of the tested punts for one week, two weeks, one month, three months and six months is tracked and observed. The experimental result shows that the effective number of the samples in the three-month group is 30, and the effective number of the samples in the control group is 28; an effective number of 28 samples in the six-month group and 15 samples in the control group. The safety of the crosslinked hyaluronic acid gel for injection prepared by the method can meet the requirement of injection beauty filling operation with the traditional crosslinking method, and long-term effectiveness results show that the maintenance time of the crosslinked hyaluronic acid gel for injection prepared by the method in vivo is far longer than that of a product prepared by the traditional crosslinking method, and the crosslinked hyaluronic acid gel for injection is an ideal tissue filler.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (3)
1. A preparation method of a chemically crosslinked hyaluronic acid hydrogel is characterized by comprising the following steps:
s1, dissolving hyaluronic acid or a salt thereof in a lithium bromide solution to obtain a hyaluronic acid lithium bromide solution, and adding a chemical cross-linking agent into the hyaluronic acid lithium bromide solution, or adding the chemical cross-linking agent into the lithium bromide solution in advance;
s2, carrying out a crosslinking reaction to obtain the chemically crosslinked hyaluronic acid hydrogel;
the chemical cross-linking agent is one or more than two of diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane and sodium trimetaphosphate;
the mass fraction of the hyaluronic acid or the salt thereof is 1 mg/mL-300 mg/mL;
the mass-volume ratio of the hyaluronic acid or the salt thereof to the chemical crosslinking agent is 1 g: 0.2-1000 μ L;
the concentration of the lithium bromide is 0.1-10M.
2. The method for preparing a chemically crosslinked hyaluronic acid hydrogel according to claim 1, wherein said crosslinking reaction is carried out at a reaction temperature of-80 to 80 ℃ for 3 minutes to 72 hours.
3. The method of claim 1, wherein the molecular weight of the hyaluronic acid is 400 kDa-3000 kDa.
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| CN101538377A (en) * | 2008-03-20 | 2009-09-23 | 上海昊海生物科技有限公司 | Cross-linked hyaluronic acid gel and preparation method thereof |
| KR20100121169A (en) * | 2009-05-08 | 2010-11-17 | 한림대학교 산학협력단 | Artificial eardrum using silk protein and method for fabricating the same |
| WO2014039607A1 (en) * | 2012-09-06 | 2014-03-13 | Allergan, Inc. | Hyaluronic acid/collagen- based dermal filler compositions and methods for making same |
| CN106589424A (en) * | 2016-12-12 | 2017-04-26 | 华熙福瑞达生物医药有限公司 | Crosslinked hyaluronic acid gel for injection and preparation method thereof |
| CN107936272A (en) * | 2017-11-27 | 2018-04-20 | 华熙福瑞达生物医药有限公司 | A kind of preparation method of 3D cross-linked-hyaluronic acid salt gels for radiotherapy protection and products thereof |
| CN111643730A (en) * | 2020-07-14 | 2020-09-11 | 华熙生物科技股份有限公司 | Preparation method and application of crosslinked hyaluronic acid gel for injection |
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| WO2022262012A1 (en) | 2022-12-22 |
| CN113278170A (en) | 2021-08-20 |
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