WO2020001374A1 - 一种共价交联透明质酸气凝胶及其水凝胶以及制备方法 - Google Patents

一种共价交联透明质酸气凝胶及其水凝胶以及制备方法 Download PDF

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WO2020001374A1
WO2020001374A1 PCT/CN2019/092172 CN2019092172W WO2020001374A1 WO 2020001374 A1 WO2020001374 A1 WO 2020001374A1 CN 2019092172 W CN2019092172 W CN 2019092172W WO 2020001374 A1 WO2020001374 A1 WO 2020001374A1
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hyaluronic acid
hydrogel
cross
aerogel
covalently crosslinked
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PCT/CN2019/092172
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English (en)
French (fr)
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黄瑜
胡碧煌
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湖南玉津医疗科技有限公司
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Priority to US16/977,651 priority Critical patent/US20200399431A1/en
Publication of WO2020001374A1 publication Critical patent/WO2020001374A1/zh

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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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0023—Polysaccharides
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0031—Hydrogels or hydrocolloids
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0042—Materials resorbable by the body
    • 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
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/08—Polysaccharides
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0085—Porous materials, e.g. foams or sponges
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/009—Materials resorbable by the body
    • 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
    • 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
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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
    • 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
    • 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

Definitions

  • the invention belongs to the field of tissue engineering and medical materials, and particularly relates to a hyaluronic acid aerogel formed by covalent crosslinking, a hydrogel thereof, and a preparation method thereof.
  • Hydrogel is a cross-linked hydrophilic polymer three-dimensional network structure, and the product of the polymer network structure remains unchanged after removing water is an aerogel. Aerogels have properties not available in hydrogels, such as high porosity, high specific surface area, low density, high stability, and ease of transportation, storage, and use.
  • Hydrogels formed by physical interactions between polymer chains instead of cross-linking by covalent bonds are called physical hydrogels, which are characterized by poor physical properties and gradually dissolve with increasing solvent; chemical hydrogels are The network structure formed by covalent bond crosslinking is insoluble in any organic solvents and aqueous solutions.
  • the two types of hydrogels can be distinguished by dissolution experiments and rheological frequency scanning experiments.
  • Covalently crosslinked hydrogels can be highly swelled in water, have good biocompatibility and water permeability, and show good application prospects in the fields of tissue engineering and medical materials, such as the application of slow-release drug carriers and Tissue Engineering.
  • covalently crosslinked hydrogels have excellent biological and physical properties, and can also be used in cosmetic surgery, surgical sealants and adhesives, and used in wound dressings to promote wound healing. Therefore, the research and development of covalently crosslinked hydrogels has always attracted the attention of scholars at home and abroad.
  • Hyaluronic acid is a natural linear anionic macromolecular polysaccharide. It is a non-sulfated linear glycosaminoglycan composed of N-acetyl-D-glucosamine and D-glucuronic acid disaccharide units. Among them, monosaccharides are connected by ⁇ -1,3-glycosidic bonds, and disaccharide units are connected by ⁇ -1,4-glycosidic bonds.
  • Hyaluronic acid is widely present in human tissues and organs, and has the characteristics of biodegradability, non-immunity and non-cytotoxicity, but hyaluronic acid is easily degraded by hyaluronidase in the body.
  • the covalently cross-linked hyaluronic acid hydrogel formed by chemical crosslinking can prolong its residence time in the body and shows its superiority.
  • Q-Med AB produced The product was approved by the US FDA as a hyaluronic acid hyaluronic acid facial injection filler and officially entered the US market; in 2005, Became the first CFDA-approved modified hyaluronic acid gel in China. To date, the FDA has approved Other hyaluronic acid gels are on the market.
  • hyaluronic acid hydrogel dry glue
  • the preparation process of this method is more complicated, and the reaction temperature is higher, which easily leads to high hyaluronic acid. Molecular chain breaks.
  • WO8600079 also uses 1,4-butanediol diglycidyl ether as a cross-linking agent to prepare a hyaluronic acid hydrogel. This reaction is cross-linked at 50 ° C. The reaction temperature is relatively high and it is easy to cause the hyaluronic acid chain to break. And degradation.
  • CN101502677 is a method of mixing glycidyl ether and hyaluronic acid in a sodium hydroxide solution, and maintaining the temperature at 40 ° C to 80 ° C for 2 to 8 hours to prepare a water-insoluble hydrogel.
  • hyaluronic acid will be deteriorated due to hydrolysis in a strong alkali solution, while the cross-linking agent BDDE has thermal instability under alkaline conditions, and the high-temperature decomposition of BDDE affects the biocompatibility of the hydrogel huge.
  • the strongly alkaline products need to be further neutralized before they can be used as biocompatible materials.
  • Hydrogels prepared using divinyl sulfone (e.g. CN102813961A), carbodiimide (e.g. patent CN1893989A), and aldehydes (e.g. patent CN101062017) as crosslinkers show uneven cross-linking effects, poor stability, and The degree of cross-linking is uneven, and glutaraldehyde is more toxic.
  • the prepared hyaluronic acid nanoparticles are not highly biocompatible, and are prone to adverse reactions such as implant calcification.
  • the purpose of the present invention is to provide a new type of covalently crosslinked hyaluronic acid aerogel as a surgical dressing directly or covalently crosslinked hyaluronic acid hydrogel as a surgical sealant and tissue filling after adding physiological saline. Agents and drug carriers.
  • the cross-linking reaction is performed in a non-alkaline (18.2M megohm ultrapure water) and freeze-dried environment to obtain a cross-linked hyaluronic acid aerogel with excellent properties and its hydrogel.
  • the cross-linking agent is added to the hyaluronic acid solution, and after shaking and mixing well, it is poured into a petri dish and immediately freeze-dried to obtain a covalently cross-linked hyaluronic acid aerogel.
  • the molecular weight of the hyaluronic acid is 3.5 ⁇ 10 5 to 1.5 ⁇ 10 6 Da.
  • the hyaluronic acid concentration in the aqueous hyaluronic acid solution is 0.5% to 4% (w / v).
  • the cross-linking agent is 1,4-butanediol diglycidyl ether (BDDE), as shown in Formula 1.
  • BDDE 1,4-butanediol diglycidyl ether
  • the cross-linking agent is ethylene glycol diglycidyl ether (GDGE), as shown in Formula 2.
  • GDGE ethylene glycol diglycidyl ether
  • the concentration of the crosslinking agent in the hyaluronic acid solution is: 0.1% to 1% (v / v).
  • a cross-linking agent (BDDE, GDGE, or PEG500) containing a diglycidyl ether group and three polysaccharides such as chondroitin sulfate, alginic acid, and hyaluronic acid are selected for freeze-drying experiments. It was surprisingly found that the two polysaccharides, chondroitin sulfate and alginic acid, could not chemically crosslink with a cross-linking agent containing a diglycidyl ether group under lyophilization conditions to obtain a covalently crosslinked aerogel and its coagulation.
  • BDDE BDDE, GDGE, or PEG500
  • hyaluronic acid can be chemically cross-linked with a cross-linking agent containing a diglycidyl ether group under lyophilization conditions to obtain a covalently cross-linked hyaluronic acid aerogel, soaked in ultrapure water or In physiological saline, covalently crosslinked hyaluronic acid aerogels absorb water and swell to form covalently crosslinked hyaluronic acid hydrogels.
  • the present invention uses freeze-drying technology for the first time to promote the covalent cross-linking of a crosslinking agent containing diglycidyl ether and hyaluronic acid under neutral conditions, and The same reaction system did not form a covalently cross-linked hydrogel after standing for 72 hours at normal temperature (28 ° C).
  • the oxirane group in diglycidyl ether undergoes a ring-opening reaction with a hydroxyl group on hyaluronic acid under freeze-drying and neutral conditions, and forms covalently crosslinked hyaluronic acid gas coagulation by generating ether bonds gum.
  • Such aerogels can be directly used in clinical applications as new surgical dressings.
  • the covalently crosslinked hyaluronic acid aerogel is immersed in purified water, and the covalently crosslinked aerogel absorbs water and swells to obtain a covalently crosslinked hyaluronic acid hydrogel.
  • the crosslinking agent used in the method of the present invention is relatively safe (such crosslinking agents have been used in products of the same category approved by the US FDA for marketing, such as, ), The preparation process is simple and easy, and the production cost is low.
  • the invention prepares covalently cross-linked hyaluronic acid aerogel and its hydrogel, which can slow the degradation of hyaluronic acid in the body and prolong its residence time in the body.
  • the covalently cross-linked aerogel obtained by one step of freeze-drying can be directly used as a surgical dressing.
  • the hydrogel obtained can be used as a cell scaffold, Physiological activities such as cell proliferation and directional differentiation provide a place and suitable conditions; at the same time, it can create a moist and closed environment for the healing wound, allowing the wound to heal faster.
  • the covalently crosslinked hyaluronic acid hydrogel prepared by the present invention has good biocompatibility and stable structure, which is of great significance for the development of cell scaffolds and medical wound dressings with stable structure, excellent performance and good biocompatibility.
  • the crosslinked hyaluronic acid of the present invention does not need to add strong basic substances such as NaOH, it reacts under neutral conditions. After the reaction is completed, the step of removing the basic substances of NaOH is not required, which saves operation steps and reduces production costs . It also avoids the adverse reactions caused by the residue of NaOH and other substances to the subsequent implantation.
  • FIG. 1 shows the morphology of the covalently crosslinked hyaluronic acid hydrogel prepared in Examples 9, 10, 11, and 12.
  • FIG. 2 shows the morphology of the covalently crosslinked hyaluronic acid hydrogels prepared in Examples 45, 46, 47, and 48.
  • FIG. 3 is the degradation time of the covalently crosslinked hyaluronic acid hydrogel prepared in Examples 1 to 12 in a PBS buffer solution.
  • FIG. 4 is the degradation time of the covalently cross-linked hyaluronic acid hydrogel prepared in Examples 13 to 24 in a PBS buffer solution.
  • Figure 5 is a time scan of a "4.0% HA 35 + 0.1% BDDE" sample.
  • FIG. 6 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 9.
  • FIG. 7 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 9.
  • Figure 8 is a time scan of a "4.0% HA 35 + 0.2% BDDE" sample.
  • FIG. 9 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 10.
  • FIG. 10 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 10.
  • Figure 11 is a time scan of a "4.0% HA 35 + 0.4% BDDE" sample.
  • FIG. 12 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 11.
  • FIG. 13 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 11.
  • Figure 14 is a time scan of a "4.0% HA 35 + 1.0% BDDE" sample.
  • Example 15 is a frequency scan of a covalently cross-linked hyaluronic acid hydrogel prepared in Example 12.
  • FIG. 16 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 12.
  • Figure 17 is a time scan of a "4.0% HA 35 10.1% GDGE" sample.
  • FIG. 18 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 21.
  • FIG. 19 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 21.
  • FIG. 19 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 21.
  • Figure 20 is a time scan of a "4.0% HA 35 + 0.2% GDGE" sample.
  • FIG. 21 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 22.
  • FIG. 21 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 22.
  • FIG. 22 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 22.
  • Figure 23 is a time scan of a "4.0% HA 35 + 0.4% GDGE" sample.
  • FIG. 24 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 23.
  • Example 25 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 23.
  • Figure 26 is a time scan of a "4.0% HA 35 + 1.0% GDGE" sample.
  • FIG. 27 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 24.
  • FIG. 27 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 24.
  • FIG. 28 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 24.
  • FIG. 28 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 24.
  • Figure 29 is a time scan of a "4.0% HA 35 + 0.1% PEG500" sample.
  • FIG. 30 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 33.
  • FIG. 31 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 33.
  • FIG. 31 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 33.
  • Figure 32 is a time scan of a "4.0% HA 35 + 0.2% PEG500" sample.
  • FIG. 33 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 34.
  • FIG. 34 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 34.
  • Figure 35 is a time scan of a "4.0% HA 35 + 0.4% PEG500" sample.
  • FIG. 36 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 35.
  • FIG. 37 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 35.
  • FIG. 37 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 35.
  • Figure 38 is a time scan of a "4.0% HA 35 + 1.0% PEG500" sample.
  • FIG. 39 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 36.
  • FIG. 40 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 36.
  • Figure 41 is a time scan of a "1.5% HA 150 + 0.1% BDDE" sample.
  • FIG. 42 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 45.
  • FIG. 42 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 45.
  • Figure 43 is a frequency scan of a "1.5% HA 150 + 0.1% BDDE" sample.
  • FIG. 44 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 45.
  • FIG. 44 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 45.
  • Figure 45 is a time scan of a "1.5% HA 150 + 0.2% BDDE" sample.
  • FIG. 46 is a frequency scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 46.
  • Figure 47 is a frequency scan of a "1.5% HA 150 + 0.2% BDDE" sample.
  • FIG. 48 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 46.
  • FIG. 48 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 46.
  • Figure 49 is a time scan of a "1.5% HA 150 + 0.4% BDDE" sample.
  • FIG. 50 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 47.
  • Figure 51 is a frequency scan of a "1.5% HA 150 + 0.4% BDDE" sample.
  • FIG. 52 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 47.
  • FIG. 52 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 47.
  • Figure 53 is a time scan of a "1.5% HA 150 + 1.0% BDDE" sample.
  • FIG. 54 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 48.
  • Figure 55 is a frequency scan of a "1.5% HA 150 + 1.0% BDDE" sample.
  • FIG. 56 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 48.
  • FIG. 56 is a strain scan of the covalently cross-linked hyaluronic acid hydrogel prepared in Example 48.
  • Figure 57 is a time scan of a "1.5% HA 150 + 0.1% GDGE" sample.
  • FIG. 58 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 57.
  • FIG. 58 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 57.
  • Figure 59 is a frequency scan of a "1.5% HA 150 + 0.1% GDGE" sample.
  • FIG. 60 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 57.
  • FIG. 60 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 57.
  • Figure 61 is a time scan of a "1.5% HA 150 + 0.2% GDGE" sample.
  • FIG. 62 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 58.
  • FIG. 62 is a frequency scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 58.
  • Figure 63 is a frequency scan of a "1.5% HA 150 + 0.2% GDGE" sample.
  • FIG. 64 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 58.
  • FIG. 64 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 58.
  • Figure 65 is a time scan of a "1.5% HA 150 + 0.4% GDGE" sample.
  • FIG. 66 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 59.
  • FIG. 66 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 59.
  • Figure 67 is a frequency scan of a "1.5% HA 150 + 0.4% GDGE" sample.
  • FIG. 68 is a strain scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 59.
  • FIG. 68 is a strain scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 59.
  • Figure 69 is a time scan of a "1.5% HA 150 + 1.0% GDGE" sample.
  • FIG. 70 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 60.
  • FIG. 70 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 60.
  • Figure 71 is a frequency scan of a "1.5% HA 150 + 1.0% GDGE" sample.
  • FIG. 72 is a strain scan of a covalently cross-linked hyaluronic acid hydrogel prepared in Example 60.
  • FIG. 72 is a strain scan of a covalently cross-linked hyaluronic acid hydrogel prepared in Example 60.
  • Figure 73 is a time scan of a "1.5% HA 150 + 0.1% PEG500" sample.
  • FIG. 74 is a frequency scan of a covalently cross-linked hyaluronic acid hydrogel prepared in Example 69.
  • FIG. 74 is a frequency scan of a covalently cross-linked hyaluronic acid hydrogel prepared in Example 69.
  • Figure 75 is a frequency scan of a "1.5% HA 150 + 0.1% PEG500" sample.
  • FIG. 76 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 69.
  • FIG. 76 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 69.
  • Figure 77 is a time scan of a "1.5% HA 150 + 0.2% PEG500" sample.
  • FIG. 78 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 70.
  • FIG. 78 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 70.
  • Figure 79 is a frequency scan of a "1.5% HA 150 + 0.2% PEG500" sample.
  • FIG. 80 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 70.
  • FIG. 80 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 70.
  • Figure 81 is a time scan of a "1.5% HA 150 + 0.4% PEG500" sample.
  • FIG. 82 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 71.
  • FIG. 82 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 71.
  • Figure 83 is a frequency scan of a "1.5% HA 150 + 0.4% PEG500" sample.
  • FIG. 84 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 71.
  • FIG. 84 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 71.
  • Figure 85 is a time scan of a "1.5% HA 150 + 1.0% PEG500" sample.
  • FIG. 86 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 72.
  • FIG. 86 is a frequency scan of a covalently crosslinked hyaluronic acid hydrogel prepared in Example 72.
  • Figure 87 is a frequency scan of a "1.5% HA 150 + 1.0% PEG500" sample.
  • FIG. 88 is a strain scan of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 72.
  • Figure 89 is the degradation time of covalently cross-linked hyaluronic acid hydrogel in plasma.
  • Figure 90 shows the D-glucuronic acid standard solution and the regression equation.
  • FIG. 91 is the enzymatic degradation time of a covalently crosslinked hyaluronic acid hydrogel in vitro (hyaluronic acid molecular weight is 3.5 ⁇ 10 5 Da, hyaluronic acid concentration is 4% (w / v)).
  • FIG. 92 shows the enzymatic degradation time of a covalently crosslinked hyaluronic acid hydrogel in vitro (hyaluronic acid molecular weight is 1.5 ⁇ 10 6 Da, hyaluronic acid concentration is 1.5% (w / v)).
  • Figure 93 shows the in vitro enzymatic degradation rate of covalently cross-linked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 3.5 ⁇ 10 5 Da, hyaluronic acid concentration is 4% (w / v), cross-linking agent: BDDE ).
  • Figure 94 shows the in vitro enzymatic degradation rate of covalently cross-linked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 3.5 ⁇ 10 5 Da, hyaluronic acid concentration is 4% (w / v), cross-linking agent: GDGE ).
  • Figure 95 shows the in vitro enzymatic degradation rate of covalently crosslinked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 3.5 ⁇ 10 5 Da, hyaluronic acid concentration is 4% (w / v), crosslinking agent: PEG500 ).
  • Figure 96 is the in vitro enzymatic degradation rate of covalently crosslinked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 1.5 ⁇ 10 6 Da, hyaluronic acid concentration is 1.5% (w / v), cross-linking agent: BDDE ).
  • Figure 97 is the in vitro enzymatic degradation rate of covalently crosslinked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 1.5 ⁇ 10 6 Da, hyaluronic acid concentration is 1.5% (w / v), cross-linking agent: GDGE ).
  • Figure 98 shows the in vitro enzymatic degradation rate of covalently crosslinked hyaluronic acid hydrogel (hyaluronic acid molecular weight is 1.5 ⁇ 10 6 Da, hyaluronic acid concentration is 1.5% (w / v), crosslinking agent: PEG500 ).
  • Figure 101 shows a cell fluorescence chart of the "HA 35 -P500-4-3" sample cultured for 2 days
  • Fig. 102 is a cell fluorescence chart of the "HA 150 -P500-3-3" sample cultured for 2 days
  • Figure 103 shows the growth and culture of L929 cells on the "HA 35 -P500-4-3" sample for 2 days
  • Fig. 104 shows the growth and culture of L929 cells on the "HA 150 -P500-3-3" sample for 2 days.
  • hyaluronic acid hyaluronic acid molecular weight 3.5 ⁇ 10 5 Da (0.25g) and dissolve it in ultrapure water (25mL) to obtain 1% aqueous hyaluronic acid solution (w / v) (pH7.0). Shake with a shaker overnight at room temperature to completely dissolve and let it stand to defoam; add 1,4-butanediol diglycidyl ether (0.025mL, crosslinker concentration 0.1% (v / v)), mix well It was then poured into a petri dish with a diameter of 7.0 cm and immediately freeze-dried.
  • the freeze-drying step is to first put the petri dish in a freeze dryer to pre-freeze to -40 ° C, and then program the temperature under a certain degree of vacuum while keeping the sample frozen to obtain a crosslinked aerogel (72h in total);
  • the crosslinked hyaluronic acid aerogel is immersed in a buffer solution or purified water, and the covalently crosslinked hyaluronic acid aerogel swells with water absorption to obtain a covalently crosslinked hyaluronic acid hydrogel.
  • the chondroitin sulfate and alginic acid solution and the cross-linking agent were freeze-dried to obtain a solid substance, which was easily soluble in water, indicating that the structure could not be covalently cross-linked.
  • Example 2-72 The procedure of Example 2-72 is the same as that of Example 1, and is different from Example 1 in the molecular weight, concentration of hyaluronic acid, and the type and concentration of the crosslinking agent.
  • the reaction parameters of Examples 1 to 72 are shown in Table 1 below.
  • Example 2-72 According to the formula and reaction system of hyaluronic acid and cross-linking agent in Example 2-72 in Table 1, a. Freeze-drying for 72h and b. Standing experiment at room temperature for 72h. Crosslinked products.
  • the covalently crosslinked hyaluronic acid aerogel was immersed in 3 mL of PBS buffer solution, and the covalently crosslinked hyaluronic acid aerogel was swelled to obtain a covalently crosslinked hyaluronic acid hydrogel.
  • the degradation rate of this hydrogel was 1.0% HA> 2.0% HA> 4.0% HA .
  • hyaluronic acid with a molecular weight of 1.5 ⁇ 10 6 Da was chemically crosslinked with different crosslinking agents and different crosslinking doses under lyophilization conditions to obtain covalently crosslinked hyaluronic acid aerogels. Soaked in PBS, the covalently crosslinked hyaluronic acid aerogel swelled to obtain a covalently crosslinked hyaluronic acid hydrogel.
  • the degradation rate of this hydrogel was 0.5% HA > 1.0% HA > 1.5% HA. It can be seen from Figs.
  • covalently crosslinked hyaluronic acid aerogels Long; and HA with molecular weight 1.5 ⁇ 10 6 Da and 1.5% concentration reacted with different cross-linking agents and different cross-linking doses under freeze-drying conditions to obtain covalently cross-linked hyaluronic acid aerogels.
  • the covalently crosslinked hyaluronic acid hydrogel was immersed in a PBS buffer solution, and the covalently crosslinked hyaluronic acid hydrogel was swelled in the PBS buffer solution for the longest degradation time.
  • W s is the weight of the hydrogel
  • W d is the weight of the aerogel.
  • Liquid absorption mass of liquid absorbed per 1 g of covalently crosslinked hyaluronic acid aerogel (g)
  • the parameters are set to: scan mode: oscillation; rotor: PP25, gap 1mm; take point frequency: 1 / 20s; test temperature: 20 ° C.
  • Time sweep time (f) is 1Hz, strain (Strain) is 1%, and points are taken at constant time;
  • Frequency sweep Frequency (f) is 10 ⁇ 0.01Hz, Strain is 1%, and 19 points are taken in total, No time setting;
  • strain sweep frequency (f) is 1Hz, strain (Strain) is 0.1% to 100%,
  • 11 and 12 illustrate that the present invention promotes chemical cross-linking of a cross-linking agent with hyaluronic acid through a freeze-drying technique.
  • 14 and 15 illustrate that the present invention promotes chemical cross-linking of a cross-linking agent with hyaluronic acid by a freeze-drying technique.
  • the strain scan of FIG. 10 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 10 has excellent viscoelasticity.
  • the strain scan of FIG. 13 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 11 has excellent viscoelasticity.
  • the strain scan of FIG. 16 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 12 has excellent viscoelasticity.
  • the strain scan of FIG. 19 shows that at a fixed frequency and temperature, when the shear force gradually increases, the storage modulus of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 21 changes and starts to decrease; Under this strain, the network structure of the hydrogel has begun to break. When G 'is less than G ", it indicates that the network structure of the hydrogel is completely destroyed, indicating that the viscoelasticity of the hydrogel is average.
  • the strain scan of FIG. 22 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 22 It has excellent viscoelasticity.
  • the strain scan of FIG. 25 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 23 has excellent viscoelasticity.
  • Figure 29 and Figure 30, Figure 32 and Figure 33, Figure 35 and Figure 36, Figure 38 and Figure 39 illustrate that the present invention promotes chemical crosslinking of the cross-linking agent with hyaluronic acid by freeze-drying technology.
  • the strain scan of FIG. 31 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 33 has excellent viscoelasticity.
  • the strain scan of FIG. 34 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 34 has excellent viscoelasticity.
  • the strain scan of FIG. 37 shows that the covalently crosslinked hyaluronic acid hydrogel prepared in Example 35 has excellent viscoelasticity.
  • Example 40 illustrates that at a fixed frequency and temperature, when the shear force gradually increases, the storage modulus of the covalently crosslinked hyaluronic acid hydrogel prepared in Example 36 changes and starts to decrease; Under this strain, the network structure of the hydrogel has begun to break. When G 'is less than G ", it indicates that the network structure of the hydrogel is completely destroyed, indicating that the viscoelasticity of the hydrogel is average.
  • the sample of “HA150 + 0.4% BDDE” changed from a state of G ′ greater than G to a state of G ′ less than G ”, indicating that the“ 1.5% HA150 + 0.4% BDDE ”sample is a physically cross-linked hydrogel, which is because of HA itself. Contains hydroxyl and carboxyl groups. When HA is dissolved in purified water, hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • the sample of “HA150 + 1.0% BDDE” changed from a state of G ′ greater than G ”to a state of G ′ less than G”, indicating that the “1.5% HA150 + 1.0% BDDE” sample is a physically cross-linked hydrogel, which is because of HA itself. Contains hydroxyl and carboxyl groups. When HA is dissolved in purified water, hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • the HA150 + 0.1% PEG500 "sample changed from a state where G 'is greater than G" to a state where G' is less than G ", indicating that the" 1.5% HA150 + 0.1% PEG500 "sample is a physically cross-linked hydrogel.
  • HA itself Contains hydroxyl and carboxyl groups.
  • hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • HA150 + 0.2% PEG500 changed from a state where G 'is greater than G" to a state where G' is less than G ", indicating that the" 1.5% HA150 + 0.2% PEG500 "sample is a physically cross-linked hydrogel.
  • HA itself Contains hydroxyl and carboxyl groups.
  • hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • HA150 + 0.4% PEG500 changed from a state where G 'is greater than G" to a state where G' is less than G ", indicating that the" 1.5% HA150 + 0.4% PEG500 "sample is a physically cross-linked hydrogel.
  • HA itself Contains hydroxyl and carboxyl groups.
  • hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • HA150 + 1.0% PEG500 changed from a state where G 'is greater than G" to a state where G' is less than G ", indicating that the" 1.5% HA150 + 1.0% PEG500 "sample is a physically cross-linked hydrogel.
  • HA itself Contains hydroxyl and carboxyl groups.
  • hyaluronic acid itself has hydrogen bonds formed in the molecule, and can form intermolecular hydrogen bonds with water molecules. The hydrogen bonds formed between polymer chains and Van der Waals forces cause physics. Cross-linking to form a physical hydrogel with a network structure.
  • covalently crosslinked hyaluronic acid aerogels prepared in Examples 9 to 12, 21 to 24, 33 to 36, 45 to 48, 57 to 60, and 69 to 72 were respectively taken and cut into 0.5 cm ⁇ 0.5cm in size, and soaked in 3mL PBS buffer solution for 30min, covalently crosslinked hyaluronic acid aerogel was swelled by suction to obtain covalently crosslinked hyaluronic acid hydrogel, using filter paper and disposable straws Aspirate the petri dish and the PBS buffer solution attached to the hydrogel surface, weigh the hydrogel mass and record it as W 0 (g), then completely soak the hydrogel in the plasma and place it at 37 ° C for biochemical culture Let stand in the box. The mass was weighed every 8h, weighed and recorded as W t (g) until the hydrogel was completely degraded. The experiment was repeated three times.
  • the experimental results are shown in FIG. 89.
  • the stability of the covalently cross-linked hyaluronic acid hydrogel in the plasma was measured to determine its stability in the plasma.
  • the covalently crosslinked hyaluronic acid hydrogel prepared in Example 45 (sample name is HA 150 -B-3-1) was completely degraded at 152 h; the covalently crosslinked hyaluronic acid water prepared in Example 33
  • the gel (sample name HA 35- P500-4-1) and the covalently cross-linked hyaluronic acid hydrogel (sample name HA 35 -G-4-1) prepared in Example 21 were completely degraded at 144 h. This shows that the prepared covalently crosslinked hyaluronic acid hydrogel has good stability in plasma.
  • D-glucuronic acid standard curve Prepare 1.5 mg / mL D-glucuronic acid solution, and then take 0.5 mL of water and dilute to 50 mL to prepare D-glucuronic acid standard solution. Use this standard solution to dilute to different concentrations, the concentrations are 0.0015, 0.0045, 0.0075, 0.0105, 0.0150 mg / mL. Take 60uL of the test solution in a centrifuge tube and put it in an ice-water bath. Slowly add 300uL of 0.025mol / L borax sulfuric acid solution to each tube, and mix after adding.
  • a covalently cross-linked hyaluronic acid hydrogel and a lyophilized hyaluronic acid sample without a cross-linking agent were immersed in 3 mL of a hyaluronidase solution (10 U / mL), and an enzyme degradation experiment was performed at 37 ° C. Take the time when the sample is put into the hyaluronidase solution as the starting time, take 60uL of the supernatant as the test solution every 24h, and supplement with 60uL of fresh hyaluronidase solution (10U / mL); take 60uL of the test solution in Centrifuge the tube and place in an ice-water bath.
  • the covalently crosslinked hyaluronic acid hydrogel prepared by the present invention has excellent resistance to enzymatic degradation. This is because chemical modification and cross-linking of hyaluronic acid can improve hyaluronic acid. Hydrogels are resistant to enzymatic degradation, thereby extending their enzymatic degradation time in vitro.
  • the above experimental results show that the covalently crosslinked hyaluronic acid aerogel prepared by the present invention has good liquid absorption performance, and can quickly absorb water and swell to obtain a covalently crosslinked hyaluronic acid hydrogel.
  • the following can be obtained:
  • the covalently crosslinked hyaluronic acid hydrogel prepared by the present invention is chemically crosslinked under freeze-drying conditions, and the prepared hydrogel has good stability and resistance to enzyme degradation. It has good application prospects in the fields of tissue engineering and medical materials.
  • the 96-well plate was placed in a cell incubator with a volume fraction of 5% CO 2 at 37 ° C. and distributed culture was performed for 2 days.
  • CCK-8 Cell Counting Kit-8
  • CCK-8 reagent contains [2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4 -Benzene disulfonic acid) -2H-tetrazolium monosodium salt] (Water soluble tetrazolium (WST-8)), which is reduced by living cell mitochondrial dehydrogenase to a water-soluble yellow formazan dye (Li Hongyan, Et al., 2005). The OD value was then measured at 450 nm using a microplate reader. The larger the OD value, the greater the number of living cells. Based on this rule, the cell proliferation rate is analyzed (Ying Tingfei, 1999).
  • Relative cell proliferation rate absorbance value of the sample group / absorbance value of the control group ⁇ 100%.
  • the CCK-8 experimentally measured absorbance value was analyzed by one-way ANOVA with SPSS 17.0.
  • the sample group and blank group were analyzed by q test. When q ⁇ 0.05 indicates that there is a difference between the two samples, when q ⁇ 0.01 indicates that there is a significant difference between the two, but when q> 0.05, it indicates that there is no difference between the two samples.
  • Cell Viability Imaging Kit (Blue / Green) includes Live reagents and Dead reagent, Live reagent stains the nucleus of living cells, Dead reagent stains only nuclei with impaired cell integrity. The nucleus of living cells is blue, and the nucleus of dead cells is green.
  • cytotoxicity of the hydrogel was measured and the results were discussed and analyzed.
  • L929 cells that were normally cultured in the same period were used as blank controls, and L929 cells that were cultured in the same period with 0.64% phenol were used as positive controls to calculate the relative cell proliferation rate of the sample group.
  • Fig. 99 shows the aerogel obtained by cross-linking reaction of HA with molecular weight 1.5 ⁇ 10 6 Da, different types of cross-linking agents and different cross-linking agents under lyophilization conditions. Relative proliferation rate of cells after 2 days of culture in the box. From this result, it can be concluded that this hydrogel is not cytotoxic when used as a medical dressing, but needs to be replaced regularly.
  • the hydrogel prepared in this study creates a moist and closed environment for the wound surface and improves the speed of wound healing.
  • the hydrogel can be used as a temporary cell delivery carrier for various tissue regeneration.
  • the three-dimensional network structure of the hydrogel is beneficial to the diffusion of oxygen and nutrients, and creates a good environment for the cells to support the survival and proliferation of the cells.
  • Figure 100 is aerogel obtained by cross-linking reaction of HA with a molecular weight of 3.5 ⁇ 10 5 Da, different types of cross-linking agents and different cross-linking doses under lyophilization conditions.
  • the hydrogel obtained by soaking the PBS buffer solution was cultured. Relative proliferation rate of cells after 2 days of culture in the box. From this result, it can be concluded that when this hydrogel is used as a medical dressing, it has almost no cytotoxicity, but it needs to be replaced regularly. It provides a moist and closed environment for wound healing and improves the speed of wound healing.
  • the hydrogel can be used as a temporary cell delivery carrier for various tissue regeneration.
  • the three-dimensional network structure of the hydrogel is beneficial to the diffusion of oxygen and nutrients, and provides a good environment for cells to support their survival and proliferation.
  • Figures 101 and 102 are fluorescence images of the cells of the "HA35-P500-4-3" sample and the "HA150-P500-3-3" sample after being cultured for 2 days.
  • the hydrogel can be used as a cell delivery carrier, provide a good environment for the cells to support the survival and proliferation of the cells, and can also be used as a medical dressing to provide a moist and closed wound surface. Environment to increase the speed of wound healing.
  • the hydrogel prepared by the invention can be used as a cell delivery carrier to provide a good environment for cells to support the survival and proliferation of the cells; it can also be used as a medical dressing to provide a moist and closed environment for the wound surface and improve the speed of wound healing.

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Abstract

一种共价交联形成的透明质酸气凝胶及其水凝胶以及制备方法。该方法以透明质酸为原料配制成透明质酸水溶液，以1,4-丁二醇二缩水甘油醚、乙二醇二缩水甘油醚或聚乙二醇二缩水甘油醚为交联剂，利用冷冻干燥技术促使交联剂与透明质酸通过共价键发生化学交联，制得共价交联的透明质酸气凝胶，将其浸泡于纯化水中，共价交联的气凝胶吸水溶胀得到共价交联的透明质酸水凝胶。该共价交联的透明质酸气凝胶有良好的吸液性能，而且能快速吸水溶胀成共价交联的透明质酸水凝胶，而且共价交联的气凝胶吸水溶胀得到共价交联的透明质酸水凝胶具有良好的稳定性和抗酶降解性能。

Description

一种共价交联透明质酸气凝胶及其水凝胶以及制备方法 技术领域
本发明属于组织工程和医用材料领域，具体涉及一种共价交联形成的透明质酸气凝胶及其水凝胶以及制备方法。
背景技术
水凝胶是交联的亲水性的聚合物三维网状结构，而其除去水分后聚合物网络结构保持不变所得的产物为气凝胶。气凝胶具有水凝胶不具备的性质，如高孔隙率、高比表面积、低密度、较高的稳定性、便于运输、储存和使用。
通过高分子链之间物理相互作用、而非共价键交联形成的水凝胶称为物理水凝胶，其特点是物理性能较差而且随着溶剂增多逐渐溶解；而化学水凝胶是通过共价键交联形成的网络结构，不溶于任何有机溶剂和水溶液。可以通过溶解实验和流变学频率扫描实验区分这两类水凝胶。
共价交联的水凝胶能在水中高度溶胀，具有良好的生物相容性和水渗透性，在组织工程和医用材料领域展现出很好的应用前景，如应用于药物缓控释载体和组织工程。同时由于共价交联的水凝胶具有优良的生物学特性和物理性质，还可应用于整容外科、外科封闭剂与粘合剂以及用于创伤敷料促进创面愈合。因此，共价交联的水凝胶的研发一直备受国内外学者们的关注。
透明质酸(Hyaluronic acid，HA)是一种天然直链阴离子大分子多糖，为非硫酸化的线性糖胺聚糖，由N-乙酰-D-葡萄糖胺和D-葡萄糖醛酸双糖单位组成，其中单糖间由β-1，3-糖苷键相连，而双糖单位之间则由β-1，4-糖苷键相连。透明质酸广泛存在于人体组织和器官中，具有生物降解性、无免疫性和无细胞毒性等特性，但透明质酸易被体内的透明质酸酶降解。通过化学交联形成的共价交联的透明质酸水凝胶可以延长其体内的居留时间就显示出了它的优越性。2003年，Q-Med AB公司生产的
Figure PCTCN2019092172-appb-000001
产品获美国FDA批准作为透明质酸玻尿酸类面部注射填充剂正式进入美国市场；2005年，
Figure PCTCN2019092172-appb-000002
成为国内首个获CFDA批准的注射用修饰透明质酸凝胶。迄今为止，美国FDA已经批准了
Figure PCTCN2019092172-appb-000003
等透明质酸凝胶上市。
同时，也有国内外专利和文献公开了对化学交联的透明质酸水凝胶的研究结果。制备化学交联的透明质酸水凝胶最常用的交联剂有缩水甘油醚、二乙烯基砜、碳二亚胺、醛类、京尼平、聚乙二醇等。其中采用1，4-丁二醇二缩水甘油醚的，如USP5827937用1，4-丁二醇二缩水甘油醚作为交联剂与透明质酸发生交联反应，但该反应需要在40℃时活化4h，然后稀释至0.5～1％，再经过减压蒸馏过程才能得到透明质酸水凝胶(干胶)，此方法的制备工艺较为复杂，且反应温度较高，容易造成透明质酸高分子链断裂。WO8600079也是利用1，4-丁二醇二缩水甘油醚作为交联剂制备透明质酸水凝胶，该反应是在50℃条件下进行交联，反应温度较高，容易造成透明质酸链断裂和降解。CN101502677是将缩水甘油醚和透明质酸在氢氧化钠溶液中混合，于40℃～80℃保温2h～8h，制得水不溶性水凝胶。在反应过程中，透明质酸会因为强碱的溶液中水解作用而劣化，而交联剂BDDE在碱性条件下具有热不稳定性，高温分解的BDDE对于水凝胶的生物相容性影响巨大。而且，强碱性的产物需要进一步中和处理才能作为生物相容的材料使用。
采用二乙烯基砜(如专利CN102813961A)、碳二亚胺(如专利CN1893989A)、醛类(如专利CN101062017)作为交联剂的制备的水凝胶分别出现了交联效果不均一、稳定性差和交联程度不均匀、以及戊二醛毒性较大，制得的透明质酸纳米微粒的生物相容性不高，且容易出现植入物钙化等不良反应。
目前，尚未见共价交联的透明质酸气凝胶及其制备方法的报道。
虽然共价交联的水凝胶在组织工程和医用材料领域的应用取得了巨大的进展，但是，由于原材料上的缺陷、制备工艺复杂与性能上的不足限制了其在组织工程和医用材料领域的广泛应用。所以发展在温和条件下不使用有毒试剂且性能优异的共价交联的水凝胶仍然面临着挑战。
发明内容
本发明的目的在于提供一种新型的共价交联的透明质酸气凝胶直接作为外科敷料、或加入生理盐水后成为共价交联的透明质酸水凝胶作为外科封闭剂、组织填充剂和药物载体。在非碱性(18.2M兆欧超纯水中)、冷冻干燥的环境下进行交联反应，获得优异性能的交联透明质酸气凝胶及其水凝胶。
本发明所采取的技术方案是：
(1)用超纯水(18.2M兆欧)将透明质酸作为原料配制成透明质酸溶液，静置消泡。
(2)将交联剂加到透明质酸溶液中，充分振荡混合均匀后，将其倒入培养皿中立即进行冷冻干燥，制得共价交联的透明质酸气凝胶。
(3)将共价交联的透明质酸气凝胶浸泡于溶剂中，共价交联的气凝胶吸水溶胀得到共价交联的透明
质酸水凝胶。
优选的，透明质酸的分子量为3.5×10 5～1.5×10 6Da。
优选的，上述透明质酸水溶液中透明质酸的浓度为0.5％～4％(w/v)。
优选的，上述交联剂为1，4-丁二醇二缩水甘油醚(BDDE)，如式1所示。
Figure PCTCN2019092172-appb-000004
优选的，上述交联剂为乙二醇二缩水甘油醚(GDGE)，如式2所示。
Figure PCTCN2019092172-appb-000005
优选的，上述交联剂为聚乙二醇二缩水甘油醚(n＝10，分子量为500)(PEG500)，如式3所示。
Figure PCTCN2019092172-appb-000006
优选的，上述交联剂在透明质酸溶液的浓度为：0.1％～1％(v/v)。
本发明选用含有二缩水甘油醚基团的交联剂(BDDE、或GDGE、或PEG500)与硫酸软骨素、海藻酸和透明质酸三种多糖分别进行冷冻干燥实验。惊讶的发现，硫酸软骨素和海藻酸这两种多糖不能与含有二缩水甘油醚基团的交联剂在冻干条件下发生化学交联反应得到共价交联的气凝胶及其水凝胶，而透明质酸可以与含有二缩水甘油醚基团的交联剂在冻干条件下发生化学交联反应得到共价交联的透明质酸气凝胶，将其浸泡于超纯水或生理盐水中，共价交联的透明质酸气凝胶吸水溶胀成共价交联的透明质酸水凝胶。
本发明与现有的技术相比，具有以下优点与有益效果：本发明首次利用冷冻干燥技术促使含有二缩水甘油醚的交联剂与透明质酸在中性条件下进行共价交联，而相同的反应体系在常温下(28℃)静置72小时并不能形成共价交联的水凝胶。其中，二缩水甘油醚中的环氧乙烷基团在冷冻干燥和中性条件下与透明质酸上的羟基发生开环反应，通过生成醚键而形成共价交联的透明质酸气凝胶。这样的气凝胶可直接作为新型的外科敷料用于临床。将共价交联的透明质酸气凝胶浸泡于纯化水中，共价交联的气凝胶吸水溶胀得到共价交联的透明质酸水凝胶。本发明方法使用的交联剂相对安全(此类交联剂已用于美国FDA批准上市的同类别的产品中，如，
Figure PCTCN2019092172-appb-000007
)，制备工艺简单易行，生产成本较低。
本发明制备的是共价交联的透明质酸气凝胶及其水凝胶，可以减缓透明质酸在体内的降解，延长其在体内的滞留时间。经冷冻干燥一步得到的共价交联气凝胶可直接作为外科敷料应用，同时由于除去了水溶剂，更易于保存和在常温下更稳定；由此得到的水凝胶可作为细胞支架，为细胞的增殖和定向分化等生理活动提供场所和适宜的条件；同时还可以为正在痊愈的伤口创造一个较湿润密闭环境，使伤口以更快的速度痊愈。本发明制备的共价交联的透明质酸水凝胶生物相容性好、结构稳定，这对研制结构稳定、性能优异、生物相容性好的细胞支架和医用伤口敷料具有重要意义。
由于本发明的交联透明质酸在不需要加入NaOH等强碱性物质，在中性条件下反应，反应完成后不需要进行除去NaOH碱性物质的步骤，节省了操作步骤，降低了生产成本。也避免了NaOH等物质的残留给后续植入人体带来的不良反应。
附图说明
图1为实施例9、10、11、12制备的共价交联的透明质酸水凝胶的形态。
图2为实施例45、46、47、48制备的共价交联的透明质酸水凝胶的形态。
图3为实施例1～12制备的共价交联的透明质酸水凝胶在PBS缓冲溶液中的降解时间。
图4为实施例13～24制备的共价交联的透明质酸水凝胶在PBS缓冲溶液中的降解时间。
图5为“4.0％HA 35+0.1％BDDE”样品的时间扫描。
图6为实施例9制备的共价交联的透明质酸水凝胶的频率扫描。
图7为实施例9制备的共价交联的透明质酸水凝胶的应变扫描。
图8为“4.0％HA 35+0.2％BDDE”样品的时间扫描。
图9为实施例10制备的共价交联的透明质酸水凝胶的频率扫描。
图10为实施例10制备的共价交联的透明质酸水凝胶的应变扫描。
图11为“4.0％HA 35+0.4％BDDE”样品的时间扫描。
图12为实施例11制备的共价交联的透明质酸水凝胶的频率扫描。
图13为实施例11制备的共价交联的透明质酸水凝胶的应变扫描。
图14为“4.0％HA 35+1.0％BDDE”样品的时间扫描。
图15为实施例12制备的共价交联的透明质酸水凝胶的频率扫描。
图16为实施例12制备的共价交联的透明质酸水凝胶的应变扫描。
图17为“4.0％HA 3510.1％GDGE”样品的时间扫描。
图18为实施例21制备的共价交联的透明质酸水凝胶的频率扫描。
图19为实施例21制备的共价交联的透明质酸水凝胶的应变扫描。
图20为“4.0％HA 35+0.2％GDGE”样品的时间扫描。
图21为实施例22制备的共价交联的透明质酸水凝胶的频率扫描。
图22为实施例22制备的共价交联的透明质酸水凝胶的应变扫描。
图23为“4.0％HA 35+0.4％GDGE”样品的时间扫描。
图24为实施例23制备的共价交联的透明质酸水凝胶的频率扫描。
图25为实施例23制备的共价交联的透明质酸水凝胶的应变扫描。
图26为“4.0％HA 35+1.0％GDGE”样品的时间扫描。
图27为实施例24制备的共价交联的透明质酸水凝胶的频率扫描。
图28为实施例24制备的共价交联的透明质酸水凝胶的应变扫描。
图29为“4.0％HA 35+0.1％PEG500”样品的时间扫描。
图30为实施例33制备的共价交联的透明质酸水凝胶的频率扫描。
图31为实施例33制备的共价交联的透明质酸水凝胶的应变扫描。
图32为“4.0％HA 35+0.2％PEG500”样品的时间扫描。
图33为实施例34制备的共价交联的透明质酸水凝胶的频率扫描。
图34为实施例34制备的共价交联的透明质酸水凝胶的应变扫描。
图35为“4.0％HA 35+0.4％PEG500”样品的时间扫描。
图36为实施例35制备的共价交联的透明质酸水凝胶的频率扫描。
图37为实施例35制备的共价交联的透明质酸水凝胶的应变扫描。
图38为“4.0％HA 35+1.0％PEG500”样品的时间扫描。
图39为实施例36制备的共价交联的透明质酸水凝胶的频率扫描。
图40为实施例36制备的共价交联的透明质酸水凝胶的应变扫描。
图41为“1.5％HA 150+0.1％BDDE”样品的时间扫描。
图42为实施例45制备的共价交联的透明质酸水凝胶的频率扫描。
图43为“1.5％HA 150+0.1％BDDE”样品的频率扫描。
图44为实施例45制备的共价交联的透明质酸水凝胶的应变扫描。
图45为“1.5％HA 150+0.2％BDDE”样品的时间扫描。
图46为实施例46制备的共价交联的透明质酸水凝胶的频率扫描。
图47为“1.5％HA 150+0.2％BDDE”样品的频率扫描。
图48为实施例46制备的共价交联的透明质酸水凝胶的应变扫描。
图49为“1.5％HA 150+0.4％BDDE”样品的时间扫描。
图50为实施例47制备的共价交联的透明质酸水凝胶的频率扫描。
图51为“1.5％HA 150+0.4％BDDE”样品的频率扫描。
图52为实施例47制备的共价交联的透明质酸水凝胶的应变扫描。
图53为“1.5％HA 150+1.0％BDDE”样品的时间扫描。
图54为实施例48制备的共价交联的透明质酸水凝胶的频率扫描。
图55为“1.5％HA 150+1.0％BDDE”样品的频率扫描。
图56为实施例48制备的共价交联的透明质酸水凝胶的应变扫描。
图57为“1.5％HA 150+0.1％GDGE”样品的时间扫描。
图58为实施例57制备的共价交联的透明质酸水凝胶的频率扫描。
图59为“1.5％HA 150+0.1％GDGE”样品的频率扫描。
图60为实施例57制备的共价交联的透明质酸水凝胶的应变扫描。
图61为“1.5％HA 150+0.2％GDGE”样品的时间扫描。
图62为实施例58制备的共价交联的透明质酸水凝胶的频率扫描。
图63为“1.5％HA 150+0.2％GDGE”样品的频率扫描。
图64为为实施例58制备的共价交联的透明质酸水凝胶的应变扫描。
图65为“1.5％HA 150+0.4％GDGE”样品的时间扫描。
图66为实施例59制备的共价交联的透明质酸水凝胶的频率扫描。
图67为“1.5％HA 150+0.4％GDGE”样品的频率扫描。
图68为实施例59制备的共价交联的透明质酸水凝胶的应变扫描。
图69为“1.5％HA 150+1.0％GDGE”样品的时间扫描。
图70为实施例60制备的共价交联的透明质酸水凝胶的频率扫描。
图71为“1.5％HA 150+1.0％GDGE”样品的频率扫描。
图72为实施例60制备的共价交联的透明质酸水凝胶的应变扫描。
图73为“1.5％HA 150+0.1％PEG500”样品的时间扫描。
图74为实施例69制备的共价交联的透明质酸水凝胶的频率扫描。
图75为“1.5％HA 150+0.1％PEG500”样品的频率扫描。
图76为实施例69制备的共价交联的透明质酸水凝胶的应变扫描。
图77为“1.5％HA 150+0.2％PEG500”样品的时间扫描。
图78为实施例70制备的共价交联的透明质酸水凝胶的频率扫描。
图79为“1.5％HA 150+0.2％PEG500”样品的频率扫描。
图80为实施例70制备的共价交联的透明质酸水凝胶的应变扫描。
图81为“1.5％HA 150+0.4％PEG500”样品的时间扫描。
图82为实施例71制备的共价交联的透明质酸水凝胶的频率扫描。
图83为“1.5％HA 150+0.4％PEG500”样品的频率扫描。
图84为实施例71制备的共价交联的透明质酸水凝胶的应变扫描。
图85为“1.5％HA 150+1.0％PEG500”样品的时间扫描。
图86为实施例72制备的共价交联的透明质酸水凝胶的频率扫描。
图87为“1.5％HA 150+1.0％PEG500”样品的频率扫描。
图88为实施例72制备的共价交联的透明质酸水凝胶的应变扫描。
图89为共价交联的透明质酸水凝胶在血浆中的降解时间。
图90为D-葡萄糖醛酸标准溶液及回归方程。
图91为共价交联的透明质酸水凝胶在体外的酶降解时间(透明质酸分子量为3.5×10 5Da，透明质酸浓度为4％(w/v))。
图92为共价交联的透明质酸水凝胶在体外的酶降解时间(透明质酸分子量为1.5×10 6Da，透明质酸浓度为1.5％(w/v))。
图93为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为3.5×10 5Da，透明质酸浓度为4％(w/v)，交联剂：BDDE)。
图94为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为3.5×10 5Da，透明质酸浓度为4％(w/v)，交联剂：GDGE)。
图95为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为3.5×10 5Da，透明质酸浓度为4％(w/v)，交联剂：PEG500)。
图96为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为1.5×10 6Da，透明质酸浓度为1.5％(w/v)，交联剂：BDDE)。
图97为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为1.5×10 6Da，透明质酸浓度为1.5％(w/v)，交联剂：GDGE)。
图98为共价交联的透明质酸水凝胶在体外的酶降解率(透明质酸分子量为1.5×10 6Da，透明质酸浓度为1.5％(w/v)，交联剂：PEG500)。
图99为HA水凝胶在培养2天后的细胞相对增殖率(Mr(HA)＝1.5×10 6Da)
图100为HA水凝胶在培养2天后的细胞相对增殖率(Mr(HA)＝3.5×10 5Da)
图101为“HA 35-P500-4-3”样品的细胞荧光图培养2d
图102为“HA 150-P500-3-3”样品的细胞荧光图培养2d
图103为L929细胞在“HA 35-P500-4-3”样品上生长培养2d
图104为L929细胞在“HA 150-P500-3-3”样品上生长培养2d。
具体实施方式
为了使本发明的发明目的、技术方案以及有益效果更加清楚明显，以下对发明中的实施例进行详细的描述。
实施例1
称取透明质酸(透明质酸分子量为3.5×10 5Da)(0.25g)溶于超纯水中(25mL)，得到1％透明质酸水溶液(w/v)(pH7.0)，在室温下摇摆机振荡过夜，使其完全溶解，并静置消泡；加入1，4-丁二醇二缩水甘油醚(0.025mL，交联剂浓度为0.1％(v/v))，混匀后倒入直径为7.0cm的培养皿中，立即进行冷冻干燥。冷冻干燥步骤为先将培养皿放入冻干机中进行预冻到-40℃，然后在一定真空度下程序升温同时保持样品冷冻状态得到交联的气凝胶(共72h)；将共价交联的透明质酸气凝胶浸泡于缓冲液或纯化水中，共价交联的透明质酸气凝胶吸水溶胀得到共价交联的透明质酸水凝胶。
作为对照，在冷冻干燥的同时，相同的透明质酸和交联剂溶液在室温放置与冷冻干燥相同的时间，样品仍为流动的溶液。
使用实施例1相同的条件和步骤，硫酸软骨素和海藻酸溶液分别与交联剂经冷冻干燥得到固体物质，易溶于水，说明不能形共价交联的结构。
实施例2-72的步骤与实施例1相同，在透明质酸分子量、浓度以及交联剂的种类和浓度与实施例1不同。实施例1～72的反应参数，如下表1所示。
表1反应参数
Figure PCTCN2019092172-appb-000008
Figure PCTCN2019092172-appb-000009
根据表1中实施例2-72的透明质酸与交联剂的配方和反应系统进行a.冷冻干燥72h和b.室温静置72h的比较实验，结果显示，室温静置72h不能形成共价交联的产品。
实施例73透明质酸水凝胶在PBS缓冲溶液中的降解实验
通过研究共价交联的透明质酸水凝胶在PBS缓冲溶液中的稳定性，从而对制备的共价交联的透明质酸水凝胶进行初步筛选。
1.取实施例1～72所制备的共价交联的透明质酸气凝胶，分别将其切为0.5cm×0.5cm大小。
2.将共价交联的透明质酸气凝胶浸泡在3mL PBS缓冲溶液中，共价交联的透明质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶。
3.在室温下进行PBS缓冲溶液的降解实验，将共价交联的透明质酸水凝胶放置在PBS缓冲溶液中，每隔一天称重一次，直至水凝胶完全降解，并每隔一天更换一次PBS缓冲溶液。重复实验三次。
实验结果如图3～8所示。无论透明质酸的分子量是3.5×10 5Da还是1.5×10 6Da，当透明质酸的浓度增大时，水凝胶的抗降解能力越强。其中分子量为3.5×10 5Da的透明质酸分别与不同交联剂和不同交联剂量在冻干条件下发生化学交联反应，得到共价交联的透明质酸气凝胶，将其浸泡于PBS中，共价交联的透明 质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶，此水凝胶的降解速率：1.0％HA＞2.0％HA＞4.0％HA。另外分子量为1.5×10 6Da的透明质酸分别与不同的交联剂和不同交联剂量在冻干条件下发生化学交联反应，得到共价交联的透明质酸气凝胶，将其浸泡于PBS中，共价交联的透明质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶，此水凝胶的降解速率：0.5％HA＞1.0％HA＞1.5％HA。由图3～8可知，分子量为3.5×10 5Da、浓度为4％的HA与不同交联剂和不同交联剂量在冻干条件下发生化学交联反应，得到共价交联的透明质酸气凝胶，将其浸泡于PBS缓冲溶液中，共价交联的透明质酸气凝胶吸液溶胀得到的共价交联的透明质酸水凝胶在PBS缓冲溶液中的降解时间最长；而分子量为1.5×10 6Da、浓度为1.5％的HA与不同交联剂和不同交联剂量在冻干条件下发生化学交联反应，得到共价交联的透明质酸气凝胶，将其浸泡于PBS缓冲溶液中，共价交联的透明质酸气凝胶吸液溶胀得到的共价交联的透明质酸水凝胶在PBS缓冲溶液中的降解时间最长。
实施例74气凝胶的吸液生实验
检测分子量为1.5×10 6Da、浓度为1.5％的透明质酸与不同交联剂和不同交联剂量冻干得到的共价交联的透明质酸气凝胶的吸液性能，同时检测分子量为3.5×10 5Da、浓度为4.0％的透明质酸与不同交联剂和不同交联剂量冻干得到的共价交联的透明质酸气凝胶的吸液性能。
1.取实施例9～12、21～24、33～36、45～48、57～60、69～72所制备的共价交联的透明质酸气凝胶，分别将其切为0.5cm×0.5cm大小，在室温干燥状态下称取质量为W 0(g)。将待测样品放入培养皿中，称取质量并记录为W d(g)。
2.向盛放共价交联的透明质酸气凝胶的培养皿中加入过量的PBS缓冲溶液，共价交联的透明质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶。
3.用滤纸和一次性吸管吸净培养皿和附着在水凝胶表面的PBS缓冲溶液，称取质量并记录为W s(g)，并再向培养皿中加入过量的PBS缓冲溶液。每10min称取质量一次，并向培养皿中加入过量的PBS缓冲溶液，直到水凝胶的质量保持恒定不变为止。重复实验三次。
4.利用公式1计算各个气凝胶的吸液性：
吸液性＝(W s-W d)/W d  (公式1)
其中W s为水凝胶的重量；W d为气凝胶的重量。实验结果如下表2所示。
表2共价交联的气凝胶的吸液性
Figure PCTCN2019092172-appb-000010
Figure PCTCN2019092172-appb-000011
吸液性：每1g共价交联的透明质酸气凝胶吸收的液体质量(g)
实验结果表明，所有共价交联的透明质酸气凝胶均在10min内完全吸液溶胀成共价交联的透明质酸水凝胶。从表2数据可以看出，共价交联的透明质酸气凝胶的吸液质量是气凝胶本身的22～45倍，说明共价交联的透明质酸气凝胶具有良好的吸液能力。
实施例75透明质酸水凝胶的流变学实验
流变学研究为分析粘弹性材料结构与性能的有效工具。在对水凝胶的研究时，通过对水凝胶的形成时间、频率和应变扫描，得出其储存模量(G’，Storage modulus)与损失模量(G”，Loss modulus)变化曲线，来表征水凝胶的性质。
流变学测试中参数设置为，扫描模式：振荡；转子：PP25，gap 1mm；取点频率：1/20s；测试温度：20℃。
Time sweep(时间扫描)：频率(f)为1Hz，应变(Strain)为1％，恒定时间取点；
Frequency sweep(频率扫描)：频率(f)为10～0.01Hz，应变(Strain)为1％，共取19个点，No time Setting；
Strain sweep(应变扫描)：频率(f)为1Hz，应变(Strain)为0.1％～100％，
共取19个点，No time Setting。实验样品分组如表3所示。
表3实验样品参数
Figure PCTCN2019092172-appb-000012
Figure PCTCN2019092172-appb-000013
a.冷冻干燥后加水；b.室温静置72h。
通过图15的时间扫描可以知道：“4.0％HA 35+0.1％BDDE”样品的G’均小于G”，说明此样品不是水凝胶。图16的频率扫描可以发现G’均大于G”，可以证明实施例9制备的透明质酸水凝胶是化学交联的水凝胶。图15和图16说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图17的应变扫描说明在 固定频率和温度的情况下，当剪切力逐渐增大，实施例9制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
通过图8、11、14的时间扫描可以知道：“4.0％HA 35+0.2％BDDE”、“4.0％HA 35+0.4％BDDE”、“4.0％HA 35+1.0％BDDE”样品的G’均小于G”，说明此样品不是水凝胶。图9、12、15的频率扫描可以发现G’均大于G”，可以证明实施例10、11、12制备的透明质酸水凝胶是化学交联的水凝胶。图18和图19说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图11和图12说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图14和图15说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图10的应变扫描表明实施例10制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图13的应变扫描表明实施例11制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图16的应变扫描表明实施例12制备的共价交联的透明质酸水凝胶具有优异的粘弹性。
通过图17、20、23、26的时间扫描可以知道：“4.0％HA 35+0.1％GDGE”、“4.0％HA 35+0.2％GDGE”、“4.0％HA 35+0.4％GDGE”、“4.0％HA 35+1.0％GDGE”样品的G’均小于G”，说明此样品不是水凝胶。图18、21、24、27的频率扫描可以发现G’均大于G”，可以证明实施例21、22、23、24制备的透明质酸水凝胶是化学交联的水凝胶。图17和图18、图20和图21、图23和图24、图26和图27说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。
图19的应变扫描说明在固定频率和温度的情况下，当剪切力逐渐增大，实施例21制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。图22的应变扫描表明实施例22制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图25的应变扫描表明实施例23制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图38的应变扫描说明在固定频率和温度的情况下，当剪切力逐渐增大，实施例24制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
通过图29、32、35、38的时间扫描可以知道：“4.0％HA 35+0.1％PEG500”、“4.0％HA 35+0.2％PEG500”、“4.0％HA 35+0.4％PEG500”、“4.0％HA 35+1.0％PEG500”样品的G’均小于G”，说明此样品不是水凝胶。图30、33、36、39的频率扫描可以发现G’均大于G”，可以证明实施例33、34、35、36制备的透明质酸水凝胶是化学交联的水凝胶。图29和图30、图32和图33、图35和图36、图38和图39分别说明本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图31的应变扫描表明实施例33制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图34的应变扫描表明实施例34制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图37的应变扫描表明实施例35制备的共价交联的透明质酸水凝胶具有优异的粘弹性。图40的应变扫描说明在固定频率和温度的情况下，当剪切力逐渐增大，实施例36制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
根据表2中使用1.5×10 6Da、浓度为1.5％的透明质酸与不同交联剂和不同交联剂量的配方和反应系统也进行了a.冷冻干燥后加水和b.室温静置72h的比较实验和流变学研究，结果也证明冷冻干燥促进共价交联的产品形成而室温静置72h不能形成共价交联的产品。
观察图41的时间扫描可以知道：“1.5％HA150+0.1％BDDE”样品的G’均大于G”，说明此样品为水凝胶。通过图42的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例45制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图43的频率扫描可以知道随着频率的变小，“1.5％HA150+0.1％BDDE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.1％BDDE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图44的应变扫描可以看出当频率和温度不变的情况下，剪切力逐 渐增大，但水凝胶的储存模量(G’)依然基本稳定，这说明水凝胶的网络结构不会因为应变的增大而改变，说明此水凝胶网络有优异的粘弹性。
观察图45的时间扫描可以知道：“1.5％HA150+0.2％BDDE”样品的G’均大于G”，说明此样品为水凝胶。通过图46的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例46制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图47的频率扫描可以知道随着频率的变小，“1.5％HA150+0.2％BDDE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.2％BDDE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图48的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例46制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图49的时间扫描可以知道：“1.5％HA150+0.4％BDDE”样品的G’均大于G”，说明此样品为水凝胶。通过图50的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例47制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图51的频率扫描可以知道随着频率的变小，“1.5％HA150+0.4％BDDE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.4％BDDE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图52的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例47制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图53的时间扫描可以知道：“1.5％HA150+1.0％BDDE”样品的G’均大于G”，说明此样品为水凝胶。通过图54的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例48制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图55的频率扫描可以知道随着频率的变小，“1.5％HA150+1.0％BDDE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+1.0％BDDE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图56的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例48制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图57的时间扫描可以知道：“1.5％HA150+0.1％GDGE”样品的G’均大于G”，说明此样品为水凝胶。通过图58的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例57制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图59的频率扫描可以知道随着频率的变小，“1.5％HA150+0.1％GDGE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.1％GDGE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术 促使交联剂与透明质酸发生化学交联。图60的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例57制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图61的时间扫描可以知道：“1.5％HA150+0.2％GDGE”样品的G’均大于G”，说明此样品为水凝胶。通过图62的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例58制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图63的频率扫描可以知道随着频率的变小，“1.5％HA150+0.2％GDGE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.2％GDGE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图64的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例58制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图65的时间扫描可以知道：“1.5％HA150+0.4％GDGE”样品的G’均大于G”，说明此样品为水凝胶。通过图66的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例59制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图67的频率扫描可以知道随着频率的变小，“1.5％HA150+0.4％GDGE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.4％GDGE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图68的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例59制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图69的时间扫描可以知道：“1.5％HA150+1.0％GDGE”样品的G’均大于G”，说明此样品为水凝胶。通过图70的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例60制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图71的频率扫描可以知道随着频率的变小，“1.5％HA150+1.0％GDGE”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+1.0％GDGE”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图72的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例60制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图73的时间扫描可以知道：“1.5％HA150+0.1％PEG500”样品的G’均大于G”，说明此样品为水凝胶。通过图74的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例69制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图75的频率扫描可以知道随着频率的变小，“1.5％HA150+0.1％PEG500”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.1％PEG500”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联， 形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图76的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例69制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图77的时间扫描可以知道：“1.5％HA150+0.2％PEG500”样品的G’均大于G”，说明此样品为水凝胶。通过图78的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例70制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图79的频率扫描可以知道随着频率的变小，“1.5％HA150+0.2％PEG500”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.2％PEG500”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图80的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例70制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图81的时间扫描可以知道：“1.5％HA150+0.4％PEG500”样品的G’均大于G”，说明此样品为水凝胶。通过图82的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例71制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图83的频率扫描可以知道随着频率的变小，“1.5％HA150+0.4％PEG500”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+0.4％PEG500”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图84的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例71制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
观察图85的时间扫描可以知道：“1.5％HA150+1.0％PEG500”样品的G’均大于G”，说明此样品为水凝胶。通过图86的频率扫描可以发现无论频率如何变化，G’一直都大于G”，说明实施例72制备的共价交联的透明质酸水凝胶为化学交联的水凝胶；通过图87的频率扫描可以知道随着频率的变小，“1.5％HA150+1.0％PEG500”样品从G’大于G”的状态变成G’小于G”的状态，说明“1.5％HA150+1.0％PEG500”样品为物理交联的水凝胶，这是因为HA本身含有羟基和羧基，当HA溶于纯化水中时，透明质酸本身有分子内形成的氢键，又可以与水分子形成分子间氢键，高分子链之间形成的氢键和范德华力导致物理交联，形成具有网络结构的物理水凝胶。随着频率的增加，物理交联的水凝胶结构会被最终破坏，从而G’小于G”。同时也可以说明物理交联水凝胶不会因为静置72h而变成化学交联水凝胶，证明了本发明是通过冷冻干燥技术促使交联剂与透明质酸发生化学交联。图88的应变扫描可以看出在固定频率和温度的情况下，当剪切力逐渐增大，实施例72制备的共价交联的透明质酸水凝胶的储存模量发生改变，开始下降；说明在这个应变之下，水凝胶的网络结构已经开始破坏。当G’小于G”时，说明水凝胶的网络结构彻底破坏，表明此水凝胶的粘弹性一般。
实施例76透明质酸水凝胶在血浆中的降解实验
分别取实施例9～12、21～24、33～36、45～48、57～60、69～72所制备的共价交联的透明质酸气凝胶，分别将其切为0.5cm×0.5cm大小，并将其浸泡在3mLPBS缓冲溶液中静置30min，共价交联的透明质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶，用滤纸和一次性吸管吸净培养皿和附着在水凝胶表面 的PBS缓冲溶液，称取水凝胶质量并记录为W 0(g)，接着将水凝胶完全浸泡在血浆中，并将其放在37℃生化培养箱中静置。每隔8h称取质量一次，称取质量并记录为W t(g)，直至水凝胶完全降解。重复实验三次。
实验结果如图89所示，通过检测共价交联的透明质酸水凝胶在血浆中的降解性，从而判断分析其在血浆中的稳定性。其中实施例45制备的的共价交联的透明质酸水凝胶(样品名称为HA 150-B-3-1)在152h完全降解；实施例33制备的共价交联的透明质酸水凝胶(样品名称为HA 35-P500-4-1)和实施例21制备的共价交联的透明质酸水凝胶(样品名称为HA 35-G-4-1)均在144h完全降解；这说明制得的共价交联的透明质酸水凝胶在血浆中具有良好的稳定性。
实施例77透明质酸水凝胶的体外酶降解实验
1.D-葡萄糖醛酸标准曲线的制作：制备1.5mg/mL D-葡萄糖醛酸液，然后取0.5mL加水稀释至50mL制备D-葡萄糖醛酸标准溶液。用此标准溶液分别稀释至不同浓度，浓度分别为0.0015、0.0045、0.0075、0.0105、0.0150mg/mL。取60uL待测液于离心管中，并放入冰水浴中。分别向每管中缓慢加入0.025mol/L的硼砂硫酸溶液300uL，加毕后混匀。然后将离心管放入100℃水浴中加热15min，取出放入冰水浴中，待其冷却至室温。各管均加入12uL咔唑乙醇溶液，加毕后混匀，将离心管放入100℃水浴中加热15min，取出后冷却至室温。用酶标仪检测在530nm处的吸光度。根据测得的吸光度和已知的D-葡萄糖醛酸溶液的浓度作D-葡萄糖醛酸的标准曲线。
D-葡萄糖醛酸的标准曲线如图90所示。
2.分别取实施例9～12、21～24、33～36、45～48、57～60、69～72所制备的共价交联的透明质酸气凝胶和未加交联剂并冻干后的透明质酸样品作对照品，实验样品分组如表4所示。
然后分别将其切为0.5cm×0.5cm大小，并将其浸泡在3mLPBS缓冲溶液中静置30min；共价交联的透明质酸气凝胶吸液溶胀得到共价交联的透明质酸水凝胶，而未加交联剂并冻干后的透明质酸样品也吸液溶胀，用滤纸和一次性吸管吸净附着在共价交联的透明质酸水凝胶表面的PBS缓冲溶液和未加交联剂并冻干后的透明质酸样品表面的PBS缓冲溶液。将共价交联的透明质酸水凝胶和未加交联剂并冻干后的透明质酸样品浸泡在3mL的透明质酸酶溶液(10U/mL)中，37℃进行酶降解实验。以样品放入透明质酸酶溶液的时间为起始时间，每24h取60uL上清液作为待测液，并补充60uL新鲜的透明质酸酶溶液(10U/mL)；取60uL待测液于离心管中，并放入冰水浴中。分别向每管中缓慢加入0.025mol/L的硼砂硫酸溶液300uL，加毕后混匀。然后将离心管放入100℃水浴中加热15min，取出放入冰水浴中，待其冷却至室温。各管均加入12uL咔唑乙醇溶液，加毕后混匀，将离心管放入100℃水浴中加热15min，取出后冷却至室温。用酶标仪检测在530nm处的吸光度。依据D-葡萄糖醛酸标准曲线计算共价交联的透明质酸水凝胶的降解百分率。
表4实验样品及具体配方
Figure PCTCN2019092172-appb-000014
Figure PCTCN2019092172-appb-000015
实验结果如图91和图92所示。通过化学交联反应制得的水凝胶的抗酶降解性能远远好于非交联的透明质酸样品的抗酶解性能；而且当透明质酸的分子量和浓度相同时，采用不同交联剂和不同交联剂量所制备的共价交联的透明质酸水凝胶在体外的酶降解时间一样。
通过图93～图98可以发现，透明质酸酶会逐渐降解透明质酸水凝胶。相对于未交联的透明质酸样品，本发明制备的共价交联的透明质酸水凝胶具有优异的抗酶降解性能，这是因为透明质酸的化学修饰和交联可以提高透明质水凝胶的抗酶降解性能，从而延长其在体外的酶降解时间。
以上实验结果表明，本发明制备的共价交联的透明质酸气凝胶有良好的吸液性能，而且能快速吸水溶胀得到共价交联的透明质酸水凝胶。通过共价交联的透明质酸水凝胶在PBS缓冲溶液、血浆和透明质酸酶溶液中的降解实验以及共价交联的透明质酸水凝胶的流变学实验，可以得出下述结论：本发明制备的共价交联的透明质酸水凝胶是在冷冻干燥条件下发生化学交联反应的，而且制得的水凝胶具有良好的稳定性和抗酶降解性能。在组织工程和医用材料领域具有良好的应用前景。
实施例78透明质酸水凝胶的细胞毒性实验与细胞荧光实验
(1)细胞毒性实验
①实验样品分组
表5实验样品及具体配方
Figure PCTCN2019092172-appb-000016
Figure PCTCN2019092172-appb-000017
②水凝胶与细胞的共同培养
a.对L929小鼠成纤维细胞进行传代培养；
b.设置空白组：全培养基培养L929小鼠成纤维细胞，阳性对照组：0.64％苯酚培养L929小鼠成纤维细胞，样品组：不同的水凝胶分别与L929小鼠成纤维细胞在全培养基的条件下进行培养。
c.将一定质量编号为1-24号的各种气凝胶在PBS缓冲溶液浸泡24h，气凝胶吸水溶胀成水凝胶，接着用滤纸吸净水凝胶表面附着的PBS缓冲溶液，并将水凝胶放入96孔板中，用紫外灯照射96孔板30min。其中每种水凝胶需三个样本进行对照；
d.收集第5代L929小鼠成纤维细胞，并弃掉原有的培养基；用玻璃胶头滴管吸取一定的PBS缓冲溶液，用其冲洗细胞两至三次；
e.用0.25％胰蛋白酶溶液消化细胞，当60％以上的细胞从培养瓶中脱落时，用玻璃胶头滴管收集细胞悬浮液到15mL离心管中；
f.将离心管放入离心机中，在转速1000rpm下室温离心5min，然后弃上清；
g.加入全培养基，用玻璃胶头滴管吹打并制备细胞悬液。用血球计数板计数，然后将细胞浓度稀释至1×10 4/cm 2，再用玻璃胶头滴管吹打多次，使细胞平均分布其中；
h.往样品组和空白组中直接加入细胞悬液200uL，其中样品组是将细胞均匀接种到水凝胶上，而阳性对照组是用0.64％苯酚培养细胞；
i.将96孔板置于体积分数5％CO 2、37℃细胞培养箱内，分布培养2d。
③L929小鼠成纤维细胞的CCK-8实验
a.CCK-8实验原理
Cell Counting Kit-8(简称CCK-8)常用于检测分析细胞毒性、细胞活性和细胞增殖。CCK-8试剂盒使用简便，并且其的检测结果准确，因而被广泛使用。CCK-8检测法的原理为：CCK-8试剂中含有[2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2，4-二磺酸苯)-2H-四唑单钠盐](Water soluble tetrazolium，WST-8)，在电子载体的作用下被活细胞线粒体脱氢酶还原成水溶性的黄色甲鑚染料(李红艳，等，2005)。然后用酶标仪在450nm波长处测定OD值。OD值越大，活细胞的数量越多，以此规律对细胞增殖率进行分析(奚廷斐，1999)。
b.CCK-8实验操作
96孔板中的细胞在5％CO 2、37℃细胞培养箱中分别培养2d后，往每组相对应的96孔板中加入20uL CCk-8溶液，在加入CCK-8检测液的过程中不要在产生气泡，避免影响吸光度值；加入完毕后将96孔板继续放入培养箱中孵育1h，紧接着用酶标仪检测其在450nm处的吸光度，采用以下公式进行计算，并得出每组的细胞相对增殖率(Relative growth rate，RGR)。
细胞相对增殖率＝样品组吸光度值/对照组吸光度值×100％。
根据每组样品的细胞相对增殖率均值，对每组样品的细胞相对增殖率进行描述与分析，并按表6对材料毒性进行相应评价(卫生部药政管理局，1997)。
表6细胞毒性评价标准
Figure PCTCN2019092172-appb-000018
c.统计学分析
对CCK-8实验测得的吸光度值用SPSS 17.0做单因素方差分析。样本组与空白组采用q检验法进行数据分析。当q＜0.05表示两样本有差异，当q＜0.01时表示两者存在显著差异，但当q＞0.05时，表示两样本之间并无差异。
(2)细胞荧光实验
①细胞荧光原理
Figure PCTCN2019092172-appb-000019
Cell Viability Imaging Kit(Blue/Green)包括
Figure PCTCN2019092172-appb-000020
Live试剂和
Figure PCTCN2019092172-appb-000021
Dead试剂，
Figure PCTCN2019092172-appb-000022
Live试剂对活细胞的细胞核染色，
Figure PCTCN2019092172-appb-000023
Dead试剂仅对细胞完整性受损的细胞核染色。活细胞的细胞核显蓝色，死细胞的细胞核显绿色。
②细胞荧光实验步骤
96孔板中的细胞在培养箱中培养相应的时间后，将96孔板中原有的培养液全部吸出，并加入200ul细胞全培养液-荧光染料混合液(全培养液-荧光染料混合液的配制比例为每毫升细胞全培养液中分别加入2滴染料A和2滴染料B，并且在使用前需混合均匀)，避免有气泡产生影响吸光度值。加入完毕后将96孔板放在培养箱中孵育25min。紧接着用倒置显微镜观察每组样品中细胞的生长状态。
(1)透明质酸水凝胶的细胞毒性实验结果
检测水凝胶的细胞毒性，并对其结果进行讨论分析。同期正常培养的L929细胞作为空白对照，并选取同期用0.64％苯酚培养的L929细胞作为阳性对照，计算样品组的细胞相对增殖率。图99是分子量为1.5×10 6Da的HA、不同交联剂种类和不同交联剂在冻干条件下发生交联反应得到气凝胶，将其浸泡PBS缓冲溶液得到的水凝胶在培养箱中分别培养2d后的细胞相对增殖率。通过此结果可以得出结论：此水凝胶作为医用敷料使用时，无细胞毒性，但需要定期更换。本研究制备的水凝胶为创伤面创造一个较湿润密闭环境，提高了伤口愈合的速度。同时此水凝胶可以作为暂时性的细胞递送载体，用于各种组织的再生。而且水凝胶的三维网络状结构有利于氧气和营养物质的扩散，为细胞创造一个良好的环境支持细胞的存活与增殖。
图100是分子量为3.5×10 5Da的HA、不同交联剂种类和不同交联剂量在冻干条件下发生交联反应得到气凝胶，将其浸泡PBS缓冲溶液得到的水凝胶在培养箱中分别培养2d后的细胞相对增殖率。通过此结果可以得出结论：此水凝胶作为医用敷料使用时，几乎无细胞毒性，但需要定期更换，其为伤口痊愈提供一个较湿润密闭的环境条件，提高伤口愈合的速度。同时此水凝胶可以作为暂时性的细胞递送载体，用于各种组织的再生。而且水凝胶的三维网络状结构有利于氧气和营养物质的扩散，为细胞提供一个良好的环境支持细胞的存活与增殖。
表7细胞毒性实验结果
(细胞培养2d后)
Figure PCTCN2019092172-appb-000024
Figure PCTCN2019092172-appb-000025
(2)水凝胶的细胞荧光实验
图101、图102是“HA35-P500-4-3”样品和“HA150-P500-3-3”样品分别培养2d后的细胞荧光图。
从L929小鼠成纤维细胞与水凝胶共同培养的荧光图片(图101-图102)可以得知，随着培养时间的增长，L929细胞的生长密度逐渐增高。细胞荧光实验结果与CCK-8实验结果相似，由于水凝胶具有三维网络状结构，可以促进氧气和营养物质的扩散，有利于细胞的生存和增殖，这表明本发明制备的水凝胶有良好的生物相容性。从细胞荧光图和相对增殖率可以看出此水凝胶可以作为细胞递送载体，为细胞提供一个良好的环境支持细胞的存活与增殖；而且还可以作为医用敷料，为创伤面提供一个较湿润密闭环境，提高伤口愈合的速度。
观察图103-图104，可见凝胶的微观结构、细胞适当的接种密度以及培养液的营养物质充足均是影响细胞存活和增殖的重要因素。本发明制备的水凝胶可以作为细胞递送载体，为细胞提供一个良好的环境支持细胞的存活与增殖；而且还可以作为医用敷料，为创伤面提供一个较湿润密闭环境，提高伤口愈合的速度。

Claims (10)

  1. 一种共价交联的透明质酸气凝胶及其水凝胶制备方法，其特征在于：在透明质酸溶液中加入交联剂制得交联反应体系，立即将交联反应体系在冷冻干燥环境下进行交联反应，制得共价交联的透明质酸气凝胶，将所得透明质酸气凝胶吸水溶胀得到共价交联的透明质酸水凝胶。
  2. 权利要求1中所述的交联透明质酸气凝胶及其水凝胶制备方法，其特征在于：所述交联剂为二缩水甘油醚类交联剂。
  3. 权利要求1中所述的交联透明质酸气凝胶及其水凝胶制备方法，所述二缩水甘油醚类交联剂为1，4-丁二醇二缩水甘油醚、乙二醇二缩水甘油醚、或聚乙二醇二缩水甘油醚(n＝10，分子量为500)。
  4. 权利要求1中所述的交联透明质酸气凝胶及其水凝胶制备方法，所述交联剂浓度为0.1％～1％(v/v)。
  5. 权利要求1中所述的交联透明质酸气凝胶及其水凝胶制备方法，所述交联反应体系的pH为6-8，优选约为7的中性条件下反应。
  6. 权利要求1中所述的交联透明质酸气凝胶及其水凝胶制备方法，所述的透明质酸的分子量为3.5×10 5～1.5×10 6Da。
  7. 权利要求1中所述的共价交联透明质酸气凝胶及其水凝胶制备方法，所述的透明质酸溶液浓度为0.5％～4％(w/v)。
  8. 一种根据权利要求1-6所述方法制得的共价交联透明质酸气凝胶及其水凝胶。
  9. 权利要求7所述交联透明质酸气凝胶及其水凝胶在细胞工程、组织工程学、药物载体或整容外科方面的用途。
  10. 根据权利要求9所述的用途，其特征在于，作为外科封闭剂与粘合剂或用于创伤敷料促进创面愈合。
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