WO2022116385A1 - 不饱和羟丁基壳聚糖和温敏水凝胶、其制备方法及应用 - Google Patents

不饱和羟丁基壳聚糖和温敏水凝胶、其制备方法及应用 Download PDF

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WO2022116385A1
WO2022116385A1 PCT/CN2021/073923 CN2021073923W WO2022116385A1 WO 2022116385 A1 WO2022116385 A1 WO 2022116385A1 CN 2021073923 W CN2021073923 W CN 2021073923W WO 2022116385 A1 WO2022116385 A1 WO 2022116385A1
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unsaturated
hydroxybutyl chitosan
group
chitosan
monomer
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French (fr)
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李睿智
武亚军
王志伟
李东风
陈雄伟
王世炜
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爱美客技术发展股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • 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
    • 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
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/042—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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/145—Hydrogels or hydrocolloids
    • 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
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/28—Treatment by wave energy or particle radiation
    • 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
    • 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 present disclosure relates to the technical field of biomedical materials, and relates to unsaturated hydroxybutyl chitosan and temperature-sensitive hydrogel, a preparation method and application thereof.
  • Chitosan also known as deacetylated chitin, is obtained by deacetylation of chitin, which is widely present in nature.
  • the chemical name is polyglucosamine (1-4)-2-amino-B-D glucose.
  • the excellent properties of this natural polymer such as biofunctionality and compatibility, blood compatibility, and microbial degradability, have attracted wide attention from all walks of life.
  • chitosan is insoluble in water, it can only be dissolved in acid. This limits the scope of application of chitosan.
  • HBCS hydroxybutyl chitosan
  • the present disclosure provides an unsaturated hydroxybutyl chitosan (UHBCS), its preparation method, its application in the preparation of a thermosensitive gel, its method in the preparation of a thermosensitive gel, a thermosensitive gel prepared therefrom, and a thermosensitive gel prepared therefrom.
  • UHBCS unsaturated hydroxybutyl chitosan
  • the application of the prepared temperature-sensitive gel in the preparation of soft tissue filling materials, soft tissue repair materials, body surface wound dressings, soft tissue, tendon and nerve tissue post-operative anti-adhesion materials can improve the mechanical strength of the gel and can be applied to biological soft tissue materials and in the preparation of unsaturated hydroxybutyl chitosan, the reaction conditions are simple and easy to operate; in the method for preparing temperature-sensitive gel of unsaturated hydroxybutyl chitosan, the reaction conditions are simple and fast;
  • the thermosensitive gel has higher mechanical strength and higher elastic modulus.
  • the present disclosure provides an unsaturated hydroxybutyl chitosan, the unsaturated hydroxybutyl chitosan is a hydroxybutyl chitosan molecule containing unsaturated groups in side chains, and the unsaturated groups include unsaturated Saturated acid anhydride group and/or unsaturated acid chloride group, the graft ratio of the unsaturated group is 10% to 100%; for example, it can be, but not limited to, 10%, 20%, 40%, 60%, 80% % or 100%.
  • the above-mentioned unsaturated group refers to a group containing a ⁇ bond or a ring
  • the hydroxybutyl chitosan grafted with the unsaturated group can pass the addition between the unsaturated bonds under the action of the cross-linking agent.
  • the reaction forms a gel with a network structure.
  • the hydroxybutyl chitosan molecule contains two types of side chains: hydroxybutyl and amino groups, and the present disclosure selects unsaturated acid anhydride groups and/or unsaturated acid chloride groups.
  • the graft ratio refers to the ratio of the amount of monomer or polymer branches incorporated into the graft copolymer in the copolymerization reaction to the total amount of the monomer to be grafted or the polymer branch to be grafted initially input. In the present disclosure, it refers to the ratio between the number of molecules of hydroxybutyl chitosan monomers to which unsaturated acyl groups have been grafted to the total number of hydroxybutyl chitosan molecules.
  • the graft ratio of the unsaturated group is 25-98%.
  • the graft ratio of the unsaturated group is 38-98%.
  • the graft ratio of the unsaturated group is 25%.
  • the graft ratio of the unsaturated group is 96%.
  • the unsaturated hydroxybutyl chitosan includes one or a combination of two or more of the following monomers (A) to (C):
  • Monomer (A) is Monomer (B) is Monomer (C) is
  • R1 includes unsaturated acid anhydride group and/or unsaturated acid chloride group
  • R2 includes unsaturated acid anhydride group and/or unsaturated acid chloride group
  • R3 includes unsaturated acid anhydride group and/or unsaturated acid chloride group.
  • the unsaturated hydroxybutyl chitosan contains monomer (A) and monomer (C) in a quantity ratio of 1:5-9.
  • the unsaturated hydroxybutyl chitosan contains the three monomers (A) to (C), and the number of monomers (A): the number of monomers (B): the number of monomers (C) ) is 1:0.01 to 4:5 to 9.
  • the unsaturated acid anhydride group includes one or more combinations of methacrylic anhydride, acrylic anhydride, maleic anhydride or itaconic anhydride;
  • the unsaturated acid chloride group includes acryloyl chloride and/or Methacryloyl chloride.
  • the unsaturated acid anhydride group is an itaconic acid group, and the quantity ratio of the three monomers is 1:1:8.
  • the unsaturated acid anhydride group is methacrylic anhydride, and the quantity ratio of the three monomers is 1:1.5:7.5.
  • the unsaturated acid anhydride group is acryloyl chloride, and the quantity ratio of the three monomers is 1:1.5:7.5.
  • the present disclosure provides a method for preparing the above-mentioned unsaturated hydroxybutyl chitosan, the preparation method comprising: mixing an aqueous solution of hydroxybutyl chitosan with an organic solution of an unsaturated acylating agent, and then performing an acylation reaction to obtain an unsaturated Hydroxybutyl chitosan.
  • the mass fraction of hydroxybutyl chitosan in the hydroxybutyl chitosan aqueous solution is 0.1% to 10%, including but not limited to 0.1%, 1%, 5%, and 10%.
  • the mass fraction of the organic solution of the unsaturated acylating reagent is 1% to 50%, including but not limited to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% %, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.
  • the acylation reaction is carried out at a natural pH, and the pH is adjusted to 7.0 after the reaction is completed.
  • the temperature of the acylation reaction is 20-60°C, including but not limited to 20°C, 30°C, 40°C, 50°C, and 60°C.
  • the temperature of the acylation reaction is 40 to 50°C, including but not limited to 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C , 50°C.
  • the time of the acylation reaction is 2-24h, including but not limited to 2h, 4h, 6h, 8h, 10h, 12h, 15h, 18h, 24h.
  • the time of the acylation reaction is 15-20h, including but not limited to 15h, 16h, 17h, 18h, 19h, and 20h.
  • the acylation reaction is carried out under the action of a catalyst.
  • the catalyst includes a weakly basic catalyst.
  • the weakly basic catalyst includes one or more combinations of sodium acetate, sodium phosphate, triethylamine or sodium carbonate.
  • the added amount of the catalyst is 1-50% of the added amount of hydroxybutyl chitosan, including but not limited to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.
  • the acylation reaction is carried out under a protective atmosphere of inert gas.
  • the inert gas includes nitrogen.
  • the steps of filtration and drying are included in sequence.
  • the drying method includes freeze drying.
  • the solvent used in the organic solution of the unsaturated acylating reagent is a water-soluble inactive hydrogen reagent
  • the inactive hydrogen reagent is an organic organic solution that does not contain groups such as hydroxyl, primary amine or secondary amine. solvent.
  • the solvent includes tetrahydrofuran, liquid polyethylene glycol, N,N-dimethylformamide, dimethylsulfoxide or N-methylpyrrolidone.
  • the filtering method includes ultrafiltration, and the duration of ultrafiltration is 2 to 24 hours, including but not limited to 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours.
  • the equipment used in the ultrafiltration step includes membrane packages or hollow fibers.
  • the molecular weight cut-off of the membrane package or the hollow fiber is 1000-30,000 Da
  • the molecular weight cut-off is based on the molecular weight of the prepared unsaturated hydroxybutyl chitosan
  • the molecular weight of the unsaturated hydroxybutyl chitosan is The molecular weight can be controlled by adjusting various parameters of the acylation reaction.
  • the present disclosure provides the application of the above-mentioned unsaturated hydroxybutyl chitosan in the preparation of thermosensitive hydrogel.
  • the present disclosure provides a preparation method of a thermosensitive hydrogel.
  • the preparation method comprises using the above-mentioned unsaturated hydroxybutyl chitosan, under the action of a crosslinking initiator, to prepare a thermosensitive hydrogel through a free radical crosslinking reaction .
  • the crosslinking initiator includes a photocrosslinking initiator or a thermal crosslinking initiator.
  • the cross-linking initiator is a photo-cross-linking initiator
  • the preparation method of the temperature-sensitive hydrogel comprises dissolving unsaturated hydroxybutyl chitosan and the photo-cross-linking initiator in water, and then heating the temperature above 35°C.
  • ultraviolet light or blue light with a light intensity of 0.05-50mW ⁇ cm -2 is used for 1-20min irradiation, and the initiator releases free radicals under the light condition, and initiates the free radical cross-linking reaction of unsaturated hydroxybutyl chitosan.
  • a photocrosslinked thermosensitive hydrogel is obtained; the mass fraction of the unsaturated hydroxybutyl chitosan is 0.5% to 20%, including but not limited to 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% %, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20% ;
  • the mass fraction of the photocrosslinking initiator is 0.01% to 1%, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% , 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%,
  • the unsaturated hydroxybutyl chitosan is reacted with a photocrosslinking initiator at 35°C to 100°C.
  • the photocrosslinking initiator includes I2959.
  • the wavelength of the ultraviolet light is 200-400 nm, including but not limited to 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280nm, 285nm, 290nm, 295nm, 300nm.
  • the blue light wavelength is 400-480 nm, including but not limited to 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm .
  • the cross-linking initiator is a thermal cross-linking agent
  • the preparation method of the heat-sensitive hydrogel comprises dissolving unsaturated hydroxybutyl chitosan and a thermal cross-linking initiator in water, and the unsaturated
  • the mass fraction of hydroxybutyl chitosan is 0.5% to 10%, including but not limited to 0.5% to 20%, including but not limited to 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%
  • the mass fraction of the thermal crosslinking initiator is 0.01% ⁇ 5%, including but not limited to 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3 %, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%
  • the thermal crosslinking initiator includes a water-soluble peroxygen initiator or a water-soluble azo initiator, and the peroxygen initiator or the water-soluble azo initiator can release free radicals under heating conditions, causing unsaturated Free radical crosslinking reaction of hydroxybutyl chitosan.
  • the water-soluble peroxygen initiator includes one or a combination of two or more of peroxybenzoic acid, potassium persulfate or peroxyacetic acid.
  • the water-soluble azo initiator includes azobisisobutyramidine hydrochloride and/or azobisisobutylimidazoline hydrochloride.
  • thermosensitive hydrogel prepared by the above preparation method.
  • the present disclosure provides applications of the above hydrogels in the preparation of soft tissue replacement materials, soft tissue repair materials, body surface wound dressings, and post-operative anti-adhesion materials for soft tissue, tendon and nerve tissue.
  • thermosensitive hydrogel comprising or consisting of the mutually cross-linked unsaturated hydroxybutyl chitosan of the present disclosure.
  • the present disclosure provides a biomedical material made of the temperature-sensitive hydrogel of the present disclosure.
  • the biomedical material is selected from soft tissue repair materials, body surface wound dressings, or post-operative anti-adhesion materials for soft tissue, tendon and nerve tissue.
  • the present disclosure provides biomedical materials for repairing soft tissue, treating body surface trauma, or preventing adhesion of soft tissue, tendon and nerve tissue after surgery.
  • the present disclosure provides a method of wound management in a subject comprising: administering the biomedical material to the subject in need thereof.
  • the biomedical material is administered to the subject's wound site.
  • trauma can be the destruction of human tissue or organs due to mechanical factors.
  • Any external factors added to the human body also include structural or functional damage caused by high temperature, cold, electric current, radiation, acid, alkali, poisonous gas, poisonous insects, mosquito bites, etc., including but not limited to: cuts, stab wounds, contusions ,sprain.
  • the subject suffers from soft tissue injury, trauma to the body surface, or has undergone surgery.
  • the surgery is a surgery related to soft tissue, tendon and nerve tissue.
  • Fig. 1 is UHBCS-96IA in embodiment 6 through 1H NMR detection result
  • Fig. 2 is UHBCS-38IA in embodiment 7 through 1H NMR detection result
  • Fig. 3 is the FTIR spectrogram obtained by infrared detection of UHBCS-38IA and HBCS, CS in Example 7;
  • Fig. 4 is the schematic diagram of utilizing UHBCS-96IA to prepare thermosensitive gel in embodiment 12;
  • Figure 5 is a schematic diagram of the in vitro degradation results of UHBCS-96IA gel film in Experimental Example 1;
  • Fig. 6 is the mechanical strength comparison diagram of UHBCS-96IA gel film and UHBCS-38IA gel film in Experimental Example 2;
  • Figure 7 is the in vitro cytotoxicity test results of UHBCS-96IA gel membrane and UHBCS-38IA gel membrane in Experimental Example 3;
  • Fig. 8 is the HE staining result in Experimental Example 4.
  • FIG. 9 shows the results of MASSON staining in Experimental Example 4.
  • the unsaturated hydroxybutyl chitosan (UHBCS) provided by the embodiments of the present disclosure is a hydroxybutyl chitosan molecule with an unsaturated group in the side chain, and the unsaturated group is used to realize the chain-like hydroxybutyl chitosan It is cross-linked into a network gel to improve its mechanical strength so that it can be applied to the repair and replacement of biological soft tissue materials.
  • the side chain hydroxyl groups of unsaturated acid anhydride and/or unsaturated acid chloride and hydroxybutyl chitosan are selected through simple and easy-to-operate reaction conditions. And/or the amino group undergoes acylation reaction, which can realize the grafting of unsaturated groups to the main chain of hydroxybutyl chitosan under natural pH conditions.
  • the unsaturated side chain of the above-mentioned unsaturated hydroxybutyl chitosan can be used under the action of various cross-linking reagents. Simple reaction conditions can achieve rapid cross-linking to form temperature-sensitive gels with a network structure.
  • thermosensitive gel prepared from unsaturated hydroxybutyl chitosan provided in the embodiments of the present disclosure
  • the thermosensitive gel utilizes the above-mentioned unsaturated hydroxybutyl chitosan.
  • the characteristics of thermosensitive gels are temperature-sensitive reversibility. By controlling the temperature change, the sol and gel states of the hydrogel can be adjusted freely, and the changes can be repeated many times.
  • the chemically cross-linked gel is irreversible, and a sol cannot be formed by physical means after gelation.
  • thermosensitive gel has higher mechanical strength and higher elastic modulus, and has a longer service time when used to prepare biological soft tissue materials.
  • thermosensitive gel prepared from unsaturated hydroxybutyl chitosan provided by the embodiments of the present disclosure in the preparation of soft tissue filling materials, soft tissue repair materials, body surface wound dressings, and anti-adhesion materials for soft tissue, tendon and nerve tissue after surgery , because the above-mentioned thermosensitive gel has higher elastic modulus and longer service time, it is more suitable for the above-mentioned materials.
  • the beneficial effects of the embodiments of the present disclosure include, for example:
  • the side chain of the unsaturated hydroxybutyl chitosan provided by the present disclosure contains unsaturated acid anhydride groups and/or unsaturated acid chloride groups, and the graft ratio is 10% to 100%.
  • the unsaturated hydroxybutyl chitosan has The unsaturated acid anhydride and/or the unsaturated acid chloride group is a grafting group, which can still maintain good water solubility when the grafting ratio reaches 100%.
  • the use of the unsaturated group can realize the cross-linking of the chain hydroxybutyl chitosan to obtain a network gel, and the network gel has higher mechanical strength when applied to the repair and replacement of biological soft tissue materials.
  • the unsaturated acid anhydride group and/or unsaturated acid chloride group selected in the present disclosure through a simple acylation reaction, replace the hydrogen in the hydroxyl group and/or the hydrogen in the amino group in the molecular side chain of hydroxybutyl chitosan. It is an unsaturated acyl group, the reaction is simple, and the grafting rate is easy to control.
  • the present disclosure selects acid anhydrides and/or acid chlorides containing double bonds as unsaturated groups, which can still ensure the hydrophilicity of the grafted groups after grafting, such as itaconic acid groups, and still retain carboxyl groups after grafting, especially when the grafting site is the amino group at the 2-position of chitosan, the side chain after grafting is a hydrophilic amide group, which can also ensure the hydrophilicity of the grafting group.
  • acryloyl chloride and amino groups are grafted to form acrylamide. group. Therefore, the method in the present disclosure can ensure good water solubility when the graft ratio of hydroxybutyl chitosan molecules reaches 100%.
  • the preparation method of unsaturated hydroxybutyl chitosan provided by the present disclosure successfully grafts unsaturated acid anhydride and/or unsaturated acid chloride on the side chain of hydroxybutyl chitosan through acylation reaction, and the reaction is in Under natural pH conditions, the reaction conditions are simple and easy to operate.
  • the catalyst can form a buffer system in the solution to avoid wide fluctuations in the pH value of the solution.
  • the addition of the catalyst can improve the efficiency of the acylation reaction and reduce the side reaction.
  • freeze-drying method uses the principle of ice crystal sublimation, in a high vacuum environment, the moisture of the frozen food material is directly removed from the ice solid without melting the ice It is sublimated to steam, which avoids the damage to the molecular structure of unsaturated hydroxybutyl chitosan during the liquefaction process of solid phase components, and can better maintain the spatial structure of the prepared unsaturated hydroxybutyl chitosan molecule; in optional
  • the solvent used in the organic solution of the unsaturated acylating reagent is an inactive hydrogen reagent soluble in water, which can ensure that the active group of the unsaturated acylating reagent is not destroyed by the solvent.
  • thermosensitive gel provided by the present disclosure realizes the rapid cross-linking of the unsaturated side chain of the above-mentioned unsaturated hydroxybutyl chitosan under the action of various cross-linking reagents and simple reaction conditions. Simple, easy to operate, suitable for mass production.
  • thermosensitive gel prepared by using the above-mentioned unsaturated hydroxybutyl chitosan, which is formed by using the unsaturated hydroxybutyl chitosan provided by the present disclosure under the action of a cross-linking reagent through simple operation steps. It has a mesh-like gel, the temperature-sensitive gel has good thermal stability, chemical stability and biocompatibility, high Young's modulus, non-toxic, more stable when used to prepare biological soft tissue materials, and has a longer service time .
  • This embodiment provides an unsaturated hydroxybutyl chitosan, the side chain branching rate of the unsaturated hydroxybutyl chitosan chitosan is 96%, and contains the following three monomers:
  • This embodiment provides an unsaturated hydroxybutyl chitosan, the graft ratio in the side chain of the unsaturated hydroxybutyl chitosan is 38%, and the grafted unsaturated group is an itaconic acid group,
  • the main chain and quantity of monomers contained are the same as those in Example 1.
  • Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Solubility 343 235 87 106 332 55 2
  • the itaconic acid group of Examples 1 and 2 still retains the carboxyl group after grafting, especially when the grafting site is the amino group at the 2-position of chitosan, the side chain after grafting is a hydrophilic amide group , the hydrophilicity of the grafted group can also be ensured, for example, the acryloyl chloride in Example 3 is grafted with an amino group to form an acrylamide group. Therefore, the methods in the embodiments of the present disclosure can ensure that when the graft ratio of the hydroxybutyl chitosan molecule reaches 100%, it has good water solubility, and its water solubility is higher than that of the comparison group.
  • This example provides a method for preparing the unsaturated hydroxybutyl chitosan provided in Example 1.
  • 20 g of itaconic anhydride was dissolved in 200 mL of tetrahydrofuran, and dropped into the aqueous solution of hydroxybutyl chitosan with continuous rapid stirring.
  • the proton at the chemical shift of 2.0 ppm (peak d) in Figure 1 is the proton on the carbon attached to the hydroxybutyl group and the 6-O or 2-N of chitosan, which proves the existence of the side chain hydroxybutyl group and the main chain is Hydroxybutyl chitosan.
  • the chemical shifts of 6.29ppm (peak a) and 5.81ppm (peak b) have obvious proton peaks of the alkene group. Due to the different chemical environments of the two protons on the alkene, the proton near the carboxyl group is affected by the carboxyl group.
  • peak c is the characteristic peak of the non-double-bonded methylene proton on itaconic acid, because the itaconic acid group is in the outermost layer of the molecular chain, resulting in a well-extended proton
  • the NMR signal is stronger than the signal in the inner layer of the molecular chain.
  • the detection of 1H NMR also proved that itaconic acid was grafted onto the main chain of hydroxybutyl chitosan, and UHBCS-IA was successfully synthesized.
  • the graft ratio of itaconic acid group can be determined by proton nuclear magnetic resonance spectroscopy. Since UHBCS as an intermediate has already determined the grafting rate of hydroxybutyl group by elemental analysis, by comparing the proton peak of methyl group in hydroxybutyl group (peak f in Fig. 1) with the double bond methylene group on itaconic acid The graft ratio of the IA group in UHBCS-IA can be calculated from the peak area of the proton peak (peak a or b in Figure 1). The UHBCS-IA obtained in this example was calculated by the peak area ratio in 1H NMR.
  • UHBCS-96IA For every 100 chitosan monosaccharide structural units, 96 structural units were substituted by itaconic acid. Therefore, the itaconic acid grafted The rate is 96%, therefore, the low-substituted UHBCS-IA prepared in this example is denoted as "UHBCS-96IA".
  • This example provides the method for the unsaturated hydroxybutyl chitosan provided in Example 2.
  • 5 g of itaconic anhydride was dissolved in 200 mL of tetrahydrofuran (THF), and dropped into the aqueous solution of hydroxybutyl chitosan under continuous rapid stirring. After the dropwise addition, the temperature was raised to 40 ° C.
  • UHBCS-IA was obtained by freeze-drying for 36 hours.
  • the UHBCS-IA obtained in this example was detected by 1H NMR.
  • the results are shown in Figure 2.
  • the peak positions of UHBCS-IA obtained in this example are basically the same as those in Figure 1, a, b
  • the peak area with c is lower than in Figure 1.
  • the UHBCS-IA obtained in this example was calculated by the peak area ratio in 1H NMR.
  • a C O peak appeared at 1670-1744 cm -1 in the UHBCS-38IA spectrum, and a weak absorption peak also appeared at a similar position in the infrared spectra of CS and HBCS, which was due to chitosan (degree of deacetylation 90% ), there are acetamino groups that have not been removed.
  • the strong absorption peak of amide at 1670cm -1 proves that itaconic anhydride has been successfully grafted to the main chain of HBCS, and it also shows that itaconic anhydride is mainly grafted on the secondary amine at the 2-position of hydroxybutyl chitosan.
  • the C O peak of carboxyl group or ester group appeared at 1744cm -1 . Therefore, the successful synthesis of UHBCS-38IA can be demonstrated by infrared spectroscopy.
  • This example provides the method for the unsaturated hydroxybutyl chitosan provided in Example 3.
  • the method is as follows: Weigh 5 g of hydroxybutyl chitosan and dissolve it in 100 mL of deionized water at 10°C to obtain hydroxybutyl chitosan. To the aqueous solution of polysaccharide, add 1 g of anhydrous sodium acrylate as a catalyst, and stir for 30 min to obtain a uniform solution. Under the protection of dry high-purity nitrogen, 5 g of acryloyl chloride was dissolved in 200 mL of tetrahydrofuran (THF), and dropped into the aqueous solution of hydroxybutyl chitosan under continuous rapid stirring.
  • THF tetrahydrofuran
  • This example provides the method for the unsaturated hydroxybutyl chitosan provided in Example 4.
  • the method is as follows: Weigh 5 g of hydroxybutyl chitosan and dissolve it in 100 mL of deionized water at 10°C to obtain hydroxybutyl chitosan. To the aqueous solution of polysaccharide, add 1 g of anhydrous sodium methacrylate as a catalyst, and stir for 30 min to obtain a uniform solution. Under the protection of dry high-purity nitrogen, 5 g of methacrylic anhydride was dissolved in 200 mL of tetrahydrofuran (THF), and dropped into the aqueous solution of hydroxybutyl chitosan under continuous rapid stirring.
  • THF tetrahydrofuran
  • This example provides a method for preparing the unsaturated hydroxybutyl chitosan provided in Example 5.
  • the method is as follows: Weigh 5 g of hydroxybutyl chitosan and dissolve it in 100 mL of deionized water at 10°C to obtain hydroxybutyl chitosan. To the aqueous solution of chitosan, add 1 g of anhydrous sodium acetate as a catalyst, and stir for 30 min to obtain a uniform solution. Under the protection of dry high-purity nitrogen, 30 g of itaconic anhydride was dissolved in 100 mL of tetrahydrofuran, and dropped into the aqueous solution of hydroxybutyl chitosan with continuous rapid stirring.
  • This embodiment provides a preparation method of hydroxybutyl chitosan containing two unsaturated groups.
  • the method is as follows: Weigh 5 g of hydroxybutyl chitosan and dissolve it in 100 mL of deionized water at 10°C to obtain hydroxybutyl chitosan. To the aqueous solution of butyl chitosan, add 1 g of anhydrous sodium methacrylate as a catalyst, and stir for 30 min to obtain a uniform solution. Under the protection of dry high-purity nitrogen, 5 g of methacrylic anhydride was dissolved in tetrahydrofuran (THF), and dropped into the aqueous solution of hydroxybutyl chitosan with continuous rapid stirring.
  • THF tetrahydrofuran
  • This example provides a method for preparing a temperature-sensitive gel by light curing using the UHBCS-96IA provided in Example 6. Dissolve 1.0 g UHBCS-96IA in 10 mL of deionized water, use 0.1% photoinitiator I2959 to initiate the chemical cross-linking of UHBCS-96IA, and heat up to 37 °C to cure the gel, and then use ultraviolet light with an intensity of 5 mW. ⁇ cm -2 , the irradiation time is 5 minutes.
  • the temperature-sensitive phase transition and chemical cross-linking schematic diagram of the gel is shown in Figure 4.
  • the UHBCS-96IA aqueous solution undergoes a temperature-sensitive phase transition at 37 °C, from a transparent sol to a white gel, and continues to be irradiated by ultraviolet light.
  • the double bond undergoes a free radical cross-linking reaction to form a cross-linked gel.
  • This example provides a method for preparing a temperature-sensitive gel by thermal curing using the UHBCS-96IA provided in Example 6.
  • 1.0 g of UHBCS-96IA was dissolved in 10 mL of deionized water, and the chemical crosslinking of UHBCS-96IA was initiated with a mass fraction of 1% thermal initiator AIBA.
  • the crosslinking temperature was 60 °C and the reaction time was 30 minutes.
  • a part of the obtained chemically cross-linked gel was taken out and placed in a vacuum drying oven, and dried to constant weight at 50°C to obtain UHBCS-96IA dry gel film.
  • This example provides a method for preparing a temperature-sensitive gel by light curing using the UHBCS-38IA provided in Example 7.
  • the aqueous solution of UHBCS-38IA undergoes a temperature-sensitive phase transition at 37 °C, from a transparent sol to a white gel, and continues to be irradiated by ultraviolet light, the double bond of the side chain undergoes a free radical cross-linking reaction to form a cross-linked gel.
  • This example provides a method for preparing a temperature-sensitive gel by thermal curing using the UHBCS-38IA provided in Example 7.
  • 1.0 g of UHBCS-38IA was dissolved in 10 mL of deionized water, and the chemical crosslinking of UHBCS-38IA was initiated with a mass fraction of 1% thermal initiator AIBA.
  • the crosslinking temperature was 60 °C and the reaction time was 30 minutes.
  • a part of the obtained chemically cross-linked gel was taken out and placed in a vacuum drying oven, and dried to constant weight at 50° C. to obtain UHBCS-38IA dry gel film.
  • the in vitro degradation performance was tested on the UHBCS-96IA hydrogel obtained in Example 12. Since the degradation test takes a long time, under the same molecular weight of the selected HBCS, the more unsaturated groups branched on the side chain, the greater the degree of cross-linking of the hydrogel formed by cross-linking, and the greater the degree of cross-linking. That is, the longer the degradation time is, so in the degradation test, the UHBCS-96IA hydrogel with the greatest degree of substitution was selected as the marking hydrogel.
  • lysozyme mainly decomposes chitosan by destroying the ⁇ -1,4 glycosidic bond between some residual N-acetylglucosamine in UHBCS-96IA. Since lysozyme directly destroys the main chain of chitosan, it will be found that the original intact gel film will be split into fragments during the degradation process, and then these fragments will continue to be degraded under the action of lysozyme. It can be seen from the figure that 14 hours after the addition of lysozyme, the enzyme activity decreased, so the residual UHBCS-96IA gel membrane degraded slowly in the time interval of 14-24 hours.
  • UHBCS-96IA gel membrane was basically degraded completely.
  • the degradation time of UHBCS-96IA was within 36 hours.
  • Curve B in the figure shows that in the initial stage of degradation, the mass loss rate of the gel film is slow. This is because at the initial stage of degradation, the dense cross-linked network structure in the gel film prevents lysozyme or ROS from destroying the internal structure of the gel film, and can only be advanced layer by layer from the outside. Once the internal structure of the gel film is destroyed, the network structure of the gel film will rapidly disintegrate, and the mass loss begins to accelerate until the final degradation is complete.
  • ROS will preferentially attack targets with relatively small steric hindrance, so in the process of ROS degradation, only occasional gel film fragments such as lysozyme degradation occur. In most cases, the volume of the gel film gradually decreases and eventually disappears completely. .
  • the mechanical strength test of the UHBCS-96IA hydrogel and UHBCS-38IA hydrogel obtained in Example 13 and Example 15 is carried out, and the method is as follows: -96IA dry gel film and UHBCS-38IA dry gel film were cut into standard splines, with a width of 2 mm and a thickness of 0.1 mm. The mechanical properties of the gel film were tested at room temperature by a universal testing machine. The ultimate strength (MPa), elongation at break (%) and Young's modulus (MPa) of the gel film were mainly studied. The tensile rate of the universal testing machine was 200 mm ⁇ min. -1 . The test results are shown in Figure 6, where a is the strain curve of the UHBCS-96IA xerogel film, and b is the strain curve of the UHBCS-38IA xerogel film.
  • the UHBCS-96IA gel film undergoes a process of strain softening and strain hardening during the stretching process. Strain softening is caused by the dynamic recrystallization of linear polymer chains present in the gel film, whereas strain hardening is caused by a rigid gel cross-linked network. At the same time, the elongation at break of UHBCS-96IA gel film was lower than that of UHBCS-38IA gel film, while the breaking strength and Young's modulus were higher than those of UHBCS-38IA gel film. These phenomena all indicate that the UHBCS-96IA gel film is not a typical elastomer, but more like an interpenetrating network structure.
  • polyitaconic acid (PIA) is formed during the cross-linking process, molecular chains of chitosan and poly-itaconic acid exist simultaneously in the gel structure, rather than the simple cross-linking of two itaconic acid functional groups.
  • the method of allowing UHBCS-96IA hydrogel sol to undergo temperature-sensitive gelation first and then chemically cross-linking provides conditions for the formation of polyitaconic acid molecular chains. After temperature-sensitive gelation, the molecular chain of UHBCS-96IA is tightly entangled, which makes the IA functional groups in the molecular chain group together.
  • UHBCS-38IA gel film is a typical elastomer. This also demonstrates that the Young's modulus of UHBCS-96IA gel film is much higher than that of UHBCS-38IA gel film.
  • L-929 cells were seeded in cell culture medium with 1% penicillin-streptomycin solution and 10% fetal bovine serum solution. L-929 cells were incubated for 3 days at 37°C in a humidified cell culture incubator containing 5% carbon dioxide. Then, the UHBCS-IA hydrogel was transferred into a 96-well plate, cured by UV light and sterilized. Again, the L-929 cell culture medium was added to the wells where the UHBCS-IA gel was placed, and 1 mL of trypsin solution containing 0.1% EDTA was added, and the number of L-929 cells in each well was 1 ⁇ 10 5 . And continue to place in the cell incubator to promote cell growth.
  • the cytotoxicity of UHBCS-IA crosslinked hydrogels was determined by MTT method. After culturing for 24, 48 and 72 hours, 100 ⁇ L of MTT aqueous solution (concentration of 5 mg ⁇ mL -1 ) was added to each well and placed in an incubator for 4 hours. The MTT solution was then removed, 150 ⁇ L of dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance of the solution was measured at a wavelength of 490 nm using a microplate reader to determine the cytotoxicity of the gel to L-929. Cell viability was calculated according to the following formula:
  • the test animals were divided into two groups, UHBCS-96IA test group and model control group. 5 rats in each group.
  • the experimental design was as follows: in the laminectomy, only the right lamina of the rat was excised, and the left side was not treated.
  • UHBCS-96IA test group the laminae were removed from the right side to stop bleeding and then UHBCS-96IA sol (sol pH 7.2) was administered. After the sol came into contact with the tissues in the body, the body temperature of the animal triggered the gelation of the sol, and the sol was sutured after the gel was solidified. Wound.
  • the lamina was removed from the right side to stop bleeding, and then the carboxymethyl chitosan solution was given and the wound was sutured.
  • the mice were sacrificed 8 weeks after the model was administered for histopathological observation.
  • the formal experimental procedure is as follows: anesthetized by intraperitoneal injection of 4.5% sodium pentobarbital (1ml/kg), the rat is placed in a prone position, the lower 2/3 of the mouse hair is removed from the back, and the position of the spine is determined by touch, and an incision is made just above the spine. The length is 6-7cm. After the cortex is incised, the muscle layers are incised on both sides of the spine to remove the excess muscles covering the spine. The 4th and 5th lamina of the lumbar vertebrae were exposed, the right nerve root was injured, the ligamentum flavum and epidural fat were removed, and the clean dura mater was exposed as much as possible.
  • the UHBCS-96IA test group was given UHBCS-96IA sol to suture the wound after coagulation, and the model control group was given carboxymethyl chitosan solution after hemostasis and the wound was sutured.
  • the spinal cord tissue and nerve root tissue in Figure 9 are red, and the scar tissue is blue-green. It can be seen from Fig. 9 that at 8 weeks after laminectomy, the nerve roots in the model control group have been wrapped by fibrous tissue, and the occurrence of scars and adhesions is very obvious. In the UHBCS-96IA gel administration group, almost no blue-green adhesions and scar tissue were found around the nerve heels. It can be seen that the anti-adhesion effect of UHBCS-96IA gel is very obvious.
  • the embodiments of the present disclosure provide an unsaturated hydroxybutyl chitosan (UHBCS), its preparation method, its application in the preparation of a thermosensitive gel, its method in the preparation of a thermosensitive gel, a thermosensitive gel prepared therefrom, and its preparation
  • UHBCS unsaturated hydroxybutyl chitosan
  • the prepared hydroxybutyl chitosan can improve the mechanical strength of its gel and can be applied to the repair and replacement of biological soft tissue materials; and in the preparation of unsaturated hydroxybutyl chitosan, the preparation method is simple and easy operate.
  • the reaction conditions of unsaturated hydroxybutyl chitosan are simple and fast, so that the prepared thermosensitive gel has higher mechanical strength and higher elastic modulus.

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Abstract

本公开涉及生物医用材料技术领域，涉及不饱和羟丁基壳聚糖和温敏水凝胶、其制备方法及应用。本公开的提供了一种不饱和羟丁基壳聚糖及其制备方法，该不饱和羟丁基壳聚糖侧链含有不饱和基团，利用该不饱和基团实现链状的羟丁基壳聚糖的交联成网状凝胶，得到一种机械强度更高，具有更高的弹性模量的温敏凝胶，该温敏凝胶用于制备生物软组织材料时，服役时间更长，能够用于制备软组织填充材料，软组织修复材料，体表创伤敷料，软组织、肌腱与神经组织手术后防粘连材料。

Description

不饱和羟丁基壳聚糖和温敏水凝胶、其制备方法及应用
相关申请的交叉引用
本申请要求于2020年12月01日提交中国专利局的申请号为2020113885193、名称为“不饱和羟丁基壳聚糖和温敏水凝胶、二者的制备方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物医用材料技术领域，涉及不饱和羟丁基壳聚糖和温敏水凝胶、其制备方法及应用。
背景技术
壳聚糖(Chitosan)又称脱乙酰甲壳素，是由自然界广泛存在的甲壳素(chitin)经过脱乙酰作用得到的，化学名称为聚葡萄糖胺(1-4)-2-氨基-B-D葡萄糖。这种天然高分子的生物官能性和相容性、血液相容性、微生物降解性等优良性能被各行各业广泛关注，但由于壳聚糖难溶于水，只能溶解于酸中，也就限制了壳聚糖的应用范围。
近年来报道的壳聚糖新型改性产物：羟丁基壳聚糖(HBCS)，由于其独特的温度敏感型与良好的生物学活性，已然成为了研究的热点。但是由于HBCS在体内环境下受到生物酶与体内活性氧簇自由基的作用易发生降解。而且温敏性HBCS凝胶的机械强度较差，弹性模量较低，当将HBCS凝胶作用生物材料对一些活动度较大的部位进行修复时，易造成HBCS凝胶的破裂。
发明内容
鉴于上述问题，本公开提供一种不饱和羟丁基壳聚糖(UHBCS)、其制备方法、其在制备温敏凝胶中的应用、其在制备温敏凝胶的方法、由其制备的温敏凝胶以及由其制备的温敏凝胶在制备软组织填充材料、软组织修复材料、体表创伤敷料、软组织、肌腱与神经组织手术后防粘连材料中的应用，能够提高其凝胶的机械强度从而能够应用于生物软组织材料的修复和替换；并且在制备不饱和羟丁基壳聚糖中，反应条件简单、易操作；不饱和羟丁基壳聚糖在制备温敏凝胶的方法中，反应条件简单、快速；使得制备得到的温敏凝胶机械强度更高，具有更高的弹性模量。
本公开的一种实施方式可以这样实现：
本公开提供了一种不饱和羟丁基壳聚糖，所述不饱和羟丁基壳聚糖为侧链含有不饱和基团的羟丁基壳聚糖分子，所述不饱和基团包括不饱和酸酐基团和/或不饱和酰氯基团，所述不饱和基团的接枝率为10％～100％；例如可以为，但不限于10％、20％、40％、60％、80％或100％。
上述不饱和基团指的是含有π键或环的基团，接枝不饱和基团的羟丁基壳聚糖之间能够在交联 剂的作用下，通过不饱和键之间的加成反应形成具有网状结构的凝胶。羟丁基壳聚糖分子含有羟丁基及氨基两类侧链，本公开选用了不饱和酸酐基团和/或不饱和酰氯基团。接枝率是指共聚合反应中，单体或聚合物支链接到接枝共聚物中的量与初始投入的待接枝的单体或待接枝的聚合物支链的总量之比。本公开中指已接枝不饱和酰基的羟丁基壳聚糖单体的分子数与总的羟丁基壳聚糖分子数之间的比值。
可选地，所述不饱和基团的接枝率为25～98％。
可选地，所述不饱和基团的接枝率为38～98％。
可选地，所述不饱和基团的接枝率为25％。
可选地，所述不饱和基团的接枝率为96％。
可选地，所述不饱和羟丁基壳聚糖包括如下单体(A)～(C)中的一种或两种以上组合：
单体(A)为
Figure PCTCN2021073923-appb-000001
单体(B)为
Figure PCTCN2021073923-appb-000002
单体(C)为
Figure PCTCN2021073923-appb-000003
其中，R1包括不饱和酸酐基团和/或不饱和酰氯基团；R2包括不饱和酸酐基团和/或不饱和酰氯基团；R3包括不饱和酸酐基团和/或不饱和酰氯基团。
可选地，所述不饱和羟丁基壳聚糖含有数量比为1:5～9的单体(A)和单体(C)。
可选地，所述不饱和羟丁基壳聚糖含有所述(A)～(C)三种单体，且单体(A)的数量：单体(B)的数量：单体(C)的数量为1：0.01～4：5～9。
可选地，所述不饱和酸酐基团包括甲基丙烯酸酐、丙烯酸酐、马来酸酐或衣康酸酐中的一种或两种以上组合；所述不饱和酰氯基团包括丙烯酰氯和/或甲基丙烯酰氯。
可选地，所述不饱和酸酐基团为衣康酸基团，且三种单体的数量比为1:1:8。
可选地，所述不饱和酸酐基团为甲基丙烯酸酐，且三种单体的数量比为1：1.5：7.5。
可选地，所述不饱和酸酐基团为丙烯酰氯，且三种单体的数量比为1：1.5：7.5。
本公开提供了上述不饱和羟丁基壳聚糖的制备方法，所述制备方法包括，将羟丁基壳聚糖水溶液与不饱和酰化试剂的有机溶液混合后经过酰化反应制得不饱和羟丁基壳聚糖。
可选地，所述羟丁基壳聚糖水溶液中羟丁基壳聚糖的质量分数为0.1％～10％，包括但不限于0.1％、1％、5％、10％。
可选地，所述不饱和酰化试剂的有机溶液的质量分数为1％～50％，包括但不限于1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％。
可选地，所述酰化反应在自然pH下进行，反应完成后调节pH至7.0。
可选地，所述酰化反应的温度为20～60℃，包括但不限于20℃、30℃、40℃、50℃、60℃。
可选地，所述酰化反应的温度为40～50℃，包括但不限于40℃、41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃。
可选地，所述酰化反应的时间为2～24h，包括但不限于2h、4h、6h、8h、10h、12h、15h、18h、24h。
可选地，所述酰化反应的时间为15～20h，包括但不限于15h、16h、17h、18h、19h、20h。
可选地，所述酰化反应在催化剂作用下进行。
可选地，所述催化剂包括弱碱性催化剂。
可选地，所述弱碱性催化剂包括醋酸钠、磷酸钠、三乙胺或碳酸钠中的一种或两种以上组合。
可选地，所述催化剂的加入量为羟丁基壳聚糖加入量的1～50％，包括但不限于1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％。
可选地，所述酰化反应在惰性气体保护气氛下进行。
可选地，所述惰性气体包括氮气。
可选地，所述酰化反应完成并调节pH至7.0之后还依次包括过滤和干燥的步骤。
可选地，所述干燥的方法包括冷冻干燥法。
可选地，所述不饱和酰化试剂的有机溶液所使用的溶剂为溶于水的非活性氢试剂，所述的非活性氢试剂为不含羟基，伯胺或仲胺等基团的有机溶剂。
可选地，所述溶剂包括四氢呋喃、液体聚乙二醇、N,N-二甲基甲酰胺、二甲基亚砜或N-甲基吡咯烷酮。
可选地，所述过滤的方法包括超滤，超滤时长为2～24h，包括但不限于2h、4h、6h、12h、24h。
可选地，所述超滤步骤所用的器材包括膜包或中空纤维。
可选地，所述膜包或中空纤维的截留分子量为1000～30000Da，所述截留分子量以制备的不饱和羟丁基壳聚糖的分子量为标准，所述不饱和羟丁基壳聚糖的分子量能够通过调整酰化反应的各项参数来控制。
本公开提供了上述不饱和羟丁基壳聚糖在制备温敏水凝胶中的应用。
本公开提供了一种温敏水凝胶的制备方法，所述制备方法包括利用上述的不饱和羟丁基壳聚糖，在交联引发剂的作用下，通过自由基交联反应制得温敏水凝胶。
可选地，所述交联引发剂包括光交联引发剂或热交联引发剂。
可选地，所述交联引发剂为光交联引发剂，所述温敏水凝胶的制备方法包括将不饱和羟丁基壳聚糖与光交联引发剂溶于水，然后在35℃以上条件下，采用光强度为0.05～50mW·cm -2的紫外光或蓝光照射1～20min，引发剂在光照条件下释放自由基，引发不饱和羟丁基壳聚糖的自由基交联反应，得到光交联温敏水凝胶；所述不饱和羟丁基壳聚糖质量分数为0.5％～20％，包括但不限于0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、10％、10.5％、11％、11.5％、12％、12.5％、13％、13.5％、14％、14.5％、15％、15.5％、16％、16.5％、17％、17.5％、18％、18.5％、19％、19.5％、20％；所述光交联引发剂质量分数为0.01％～1％，包括但不限于0.01％、0.02％、0.03％、0.04％、0.05％、0.06％、0.07％、0.08％、0.09％、0.1％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％、0.45％、0.5％、0.55％、0.6％、0.65％、0.7％、0.75％、0.8％、0.85％、0.9％、0.95％、1％；所述的光交联引发剂与不饱和羟丁基壳聚糖的质量比为1:1～200，包括但不限于1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50、1:55、1:60、1:65、1:70、1:75、1:80、1:85、1:90、1:95、1:100、1:110、1:120、1:130、1:140、1:150、1:160、1:170、1:180、1:190、1:200。
可选地，所述不饱和羟丁基壳聚糖与光交联引发剂在35℃～100℃条件下反应。
可选地，所述光交联引发剂包括I2959。
可选地，所述紫外光波长为200～400nm，包括但不限于200nm、205nm、210nm、215nm、220nm、225nm、230nm、235nm、240nm、245nm、250nm、255nm、260nm、265nm、270nm、275nm、280nm、285nm、290nm、295nm、300nm。
可选地，所述蓝光波长为400～480nm，包括但不限于400nm、405nm、410nm、415nm、420nm、425nm、430nm、435nm、440nm、445nm、450nm、455nm、460nm、465nm、470nm、475nm、480nm。
可选地，所述交联引发剂为热交联剂，所述热敏水凝胶的制备方法包括将不饱和羟丁基壳聚糖与热交联引发剂溶于水，所述不饱和羟丁基壳聚糖质量分数为0.5％～10％，包括但不限于0.5％～20％，包括但不限于0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、10％；所述热交联引发剂的质量分数为0.01％～5％，包括但不限于0.01％、0.02％、0.03％、0.04％、0.05％、0.06％、0.07％、0.08％、0.09％、0.1％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％、0.45％、0.5％、0.55％、0.6％、0.65％、0.7％、0.75％、0.8％、0.85％、0.9％、0.95％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％；然后在35℃条件下使不饱和羟丁基壳聚糖发生胶化后，再升温至60～80℃，持续反应10～120min，得到热交联温敏水凝胶；所述的热交联引发剂与不饱和羟丁基壳聚糖的质量比为1:2～200，包括但不限于1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50、1:55、1:60、1:65、1:70、1:75、 1:80、1:85、1:90、1:95、1:100、1:110、1:120、1:130、1:140、1:150、1:160、1:170、1:180、1:190、1:200。
进一步可选地，所述热交联引发剂包括水溶性过氧引发剂或水溶性偶氮引发剂，过氧引发剂或水溶性偶氮引发剂能够在加热条件下释放自由基，引发不饱和羟丁基壳聚糖的自由基交联反应。
进一步可选地，所述水溶性过氧引发剂包括过氧苯甲酸，过硫酸钾或过氧乙酸中的一种或两种以上组合。
进一步可选地，所述水溶性偶氮引发剂包括偶氮二异丁脒盐酸盐和/或偶氮二异丁咪唑啉盐酸盐。
本公开提供了一种上述制备方法制备得到的温敏水凝胶。
本公开提供了上述水凝胶在制备软组织替换材料，软组织修复材料，体表创伤敷料，软组织、肌腱与神经组织手术后防粘连材料中的应用。
本公开提供了一种温敏水凝胶，包括或由相互交联的本公开的不饱和羟丁基壳聚糖组成。
本公开提供了一种生物医用材料，所述生物医用材料由本公开的温敏水凝胶制成。
可选地，所述生物医用材料选自软组织修复材料，体表创伤敷料，或软组织、肌腱与神经组织手术后防粘连材料。
本公开提供了生物医用材料，用于修复软组织，治疗体表创伤，或软组织、肌腱与神经组织手术后防粘连的用途。
本公开提供了一种对受试者中创伤处理的方法，包括：向所述有此需要的受试者施用所述的生物医用材料。
可选地，向所述受试者创伤部位施用所述的生物医用材料。
例如，创伤可以为由于机械因素引起人体组织或器官的破坏。加于人体的任何外来因素还包括高温、寒冷、电流、放射线、酸、碱、毒气、毒虫、蚊咬等所造成的结构或功能方面的破坏，包括但不限于：割伤、刺伤、挫伤、扭伤。
可选地，所述受试者患有软组织损伤，体表创伤，或经历了手术。
可选地，所述手术是与软组织、肌腱与神经组织相关的手术。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为实施例6中UHBCS-96IA经1H NMR检测结果；
图2为实施例7中UHBCS-38IA经1H NMR检测结果；
图3为实施例7中UHBCS-38IA与HBCS、CS经红外检测得到的FTIR谱图；
图4为实施例12中利用UHBCS-96IA制备温敏凝胶的示意图；
图5为实验例1中UHBCS-96IA凝胶膜体外降解结果示意图；
图6为实验例2中UHBCS-96IA凝胶膜与UHBCS-38IA凝胶膜机械强度对比图；
图7为实验例3中UHBCS-96IA凝胶膜与UHBCS-38IA凝胶膜体外细胞毒性检测结果；
图8为实验例4中HE染色结果；
图9为实验例4中MASSON染色结果。
实施方式
本公开的实施例提供的不饱和羟丁基壳聚糖(UHBCS)为侧链含有不饱和基团的羟丁基壳聚糖分子，利用不饱和基团实现链状的羟丁基壳聚糖的交联成网状凝胶，以提高其机械强度从而能够应用于生物软组织材料的修复和替换。
本公开的实施例提供的不饱和羟丁基壳聚糖的制备方法中，通过简单、易操作的反应条件，选择不饱和酸酐和/或不饱和酰氯与羟丁基壳聚糖的侧链羟基和/或氨基发生酰化反应，能够在自然pH条件下，实现将不饱和基团接枝与羟丁基壳聚糖的主链上。本公开在将上述不饱和基团接枝于羟丁基壳聚糖时，只需要发生简单的酰化反应，反应简单，反应过程中无需对pH进行调节，即节约工序、提高了效率，又避免了pH调节过程中酸碱中和反应的放热对不饱和基团造成的破坏。
本公开的实施例提供的不饱和羟丁基壳聚糖在制备温敏凝胶的方法中，在上述不饱和羟丁基壳聚糖的不饱和侧链能够在多种交联试剂的作用下，利用简单的反应条件即可实现快速的交联，形成具有网状结构的温敏凝胶。
本公开的实施例提供的由不饱和羟丁基壳聚糖制备的温敏凝胶中，该温敏凝胶即利用上述不饱和羟丁基壳聚糖。温敏凝胶的特性为温度敏感可逆性，通过控制温度的变化可以自由调整水凝胶的溶胶与凝胶状态，并可以多次反复变化。而化学交联的凝胶则不可逆，凝胶化后不可通过物理手段形成溶胶。通过上述制备方法得到的，在温敏凝胶化之后，不饱和的基团紧密缠绕给化学交联提供了环境，使得上述不饱和羟丁基壳聚糖能够通过不饱和侧链实现网状交联，得到的温敏凝胶机械强度更高，具有更高的弹性模量，用于制备生物软组织材料时，服役时间更长。
本公开的实施例提供的由不饱和羟丁基壳聚糖制备的温敏凝胶在制备软组织填充材料，软组织修复材料，体表创伤敷料，软组织、肌腱与神经组织手术后防粘连材料中的应用中，由于上述温敏凝胶具有更高的弹性模量和更长的服役时间，因此，更适合用于上述材料中。
与现有的技术相比，本公开实施例的有益效果包括，例如：
本公开提供的不饱和羟丁基壳聚糖侧链中含有不饱和酸酐基团和/或不饱和酰氯基团，接枝率为10％～100％，该不饱和羟丁基壳聚糖以不饱和酸酐和/或不饱和酰氯基团为接枝基团，当接枝率达到100％时仍能保持良好的水溶性。且利用该不饱和基团能够实现链状的羟丁基壳聚糖的交联，得到网状凝胶，该网状凝胶应用于生物软组织材料的修复和替换时具有更高的机械强度。
同时，本公开选用的不饱和酸酐基团和/或不饱和酰氯基团，通过简单的酰化反应，将羟丁基壳聚糖分子侧链中羟基中的氢和/或氨基中的氢取代为不饱和酰基，反应简单，且接枝率容易控制。
在应用不饱和羟丁基壳聚糖进行交联制备水凝胶时，接枝率越高其交联度越高，形成的凝胶网络越致密，所得水凝胶的机械性能也就越好，但是，通常羟丁基壳聚糖的接枝率对其水溶性有着重 要的影响，由于本公开中使用的不饱和基团为疏水基团，所以随着接枝率的增加，羟丁基壳聚糖的侧链疏水基团增多，疏水性的增加会造成水溶性降低，不利于水溶液条件下的交联反应。本公开选择含有双键的酸酐和/或酰氯作为不饱和基团在接枝后仍然可以保证接枝基团的亲水性，例如衣康酸基团，接枝后仍保留羧基，尤其是当接枝位点为壳聚糖2位的氨基时，接枝后侧链为亲水的酰胺基团，亦可保证接枝基团的亲水性，例如丙烯酰氯与氨基接枝后形成丙烯酰胺基团。因此，本公开中的方法可以保证羟丁基壳聚糖分子的接枝率达到100％时都具有良好的水溶性。
同时，本公开提供的不饱和羟丁基壳聚糖的制备方法成功地将不饱和酸酐和/或不饱和酰氯通过酰化反应接枝与羟丁基壳聚糖的侧链上，该反应在自然pH条件下进行，反应条件简单，易操作。同时，在可选地酰化反应在催化剂作用下进行中，催化剂能够在溶液中形成缓冲体系，避免溶液pH值的大范围波动，同时，催化剂的加入可提高酰化反应效率，减少副反应的发生；在可选的干燥的方法为冷冻干燥法中：冷冻干燥法是利用冰晶升华的原理，在高度真空的环境下，将已冻结了的食品物料的水分不经过冰的融化直接从冰固体升华为蒸汽，避免了固相组分液化过程对不饱和羟丁基壳聚糖分子结构的破坏，能够更好地保持制备得到的不饱和羟丁基壳聚糖分子的空间结构；在可选地不饱和酰化试剂的有机溶液所使用的溶剂为溶于水的非活性氢试剂可保证不饱和酰化试剂的活性基团不被溶剂所破坏。
本公开提供的温敏凝胶的制备方法，实现了上述不饱和羟丁基壳聚糖的不饱和侧链在多种交联试剂的作用下及简单的反应条件下的快速交联，该方法反应条件简单，易操作，适于批量生产。
本公开还提供了采用上述不饱和羟丁基壳聚糖制备的温敏凝胶，利用本公开提供的不饱和羟丁基壳聚糖通过简单的操作步骤，使其在交联试剂的作用下形成的具有网状的凝胶，该温敏凝胶的热稳定性、化学稳定性及生物相容性好，杨氏模量高，无毒，用于制备生物软组织材料时，更加稳定，且服役时间更长。
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖壳聚糖的侧链接枝率为96％，共包含下三种单体：
单体(A)
Figure PCTCN2021073923-appb-000004
单体(B)
Figure PCTCN2021073923-appb-000005
和单体(C)
Figure PCTCN2021073923-appb-000006
其中R 1、R 2和R 3均为衣康酸基团，三种单体的数量比为n A：n B：n C＝1：1：8。
实施例2
本实施例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖的侧链中接枝率为38％，接枝的不饱和基团为衣康酸基团，包含的单体主链及数量与实施例1相同。
实施例3
本实施例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖的侧链中包含接枝率为40％的不饱和基团丙烯酰氯，n A：n B：n C＝1：1.5：7.5。
实施例4
本实施例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖的侧链中包含接枝率为25％的不饱和基团甲基丙烯酸酐，n A：n B：n C＝1：1.5：7.5。
实施例5
本实施例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖与实施例1的区别在于，n A：n B：n C＝为1:1.5:7.5。
对比例1
本对比例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖的侧链中包含接枝率为25％的甲基丙烯酸缩水甘油酯，包含的单体主链与实施例1相同，区别在于，R 1、R 2和R 3均为甲基丙烯酸缩水甘油酯，n A：n B：n C＝1：1.5：7.5。
对比例2本对比例提供了一种不饱和羟丁基壳聚糖，该不饱和羟丁基壳聚糖的侧链中包含接枝 率为25％的丙烯，不饱和试剂选用溴丙烯，包含的单体主链与实施例1相同，区别在于，R基为丙烯，n A：n B：n C＝1：1.5：7.5。
采用溶解度检测法，测定上述实施例上述实施例1～5以及对比例1和2提供的不饱和羟丁基壳聚糖水溶性，溶解度单位为g/L，结果如表1所示：
表1实施例1～5及对比例1和2所得不饱和羟丁基壳聚糖水溶性
  实施例1 实施例2 实施例3 实施例4 实施例5 对比例1 对比例2
溶解度 343 235 87 106 332 55 2
由表1可以看出，在本公开的实施例选择含有双键的酸酐和/或酰氯作为不饱和基团相较于对比例1-2的其他不饱和基团，本公开实施例中的基团在接枝后仍然可以保证接枝基团的亲水性。例如，实施例1和2的衣康酸基团，接枝后仍保留羧基，尤其是当接枝位点为壳聚糖2位的氨基时，接枝后侧链为亲水的酰胺基团，亦可保证接枝基团的亲水性，例如实施例3的丙烯酰氯与氨基接枝后形成丙烯酰胺基团。因此，本公开实施例中的方法可以保证羟丁基壳聚糖分子的接枝率达到100％时都具有良好的水溶性，其水溶性均高于对比组。
实施例6：制备实施例1的壳聚糖
本实施例提供了制备实施例1中提供的不饱和羟丁基壳聚糖的方法。称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水醋酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将20g的衣康酸酐溶解于200mL四氢呋喃中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中。滴加完毕之后，升温至40℃并持续反应16小时。反应结束后，采用截流分子量为10kDa的中空纤维柱(型号：UFP-10-C-6A，制造商：GE Healthcare(通用医疗公司))超滤24小时，以去除体系中多余的四氢呋喃、衣康酸和盐分。冷冻干燥36小时得到UHBCS-IA。将本实施例中得到的UHBCS-IA经1H NMR检测，结果如图1所示。图1中化学位移2.0ppm(d峰)处的质子为羟丁基与壳聚糖6-O或2-N相连碳上的质子，证明了侧链羟丁基的存在，证明了主链为羟丁基壳聚糖。在图1中化学位移为6.29ppm(a峰)与5.81ppm(b峰)处出现了明显的烯烃基团的质子峰，由于烯烃上的两个质子的化学环境不同，靠近羧基的质子受到羧基的拉电子效应而使化学位移转向低场，为6.29ppm；而远离羧基的质子则处在相对低场，为5.81ppm。同样，处在0.86ppm(f峰)处的为羟丁基上甲基的质子峰，1.45ppm(e峰)处为羟丁基上与甲基相连亚甲基的质子峰。在3.00-4.00ppm之间出现的宽峰为壳聚糖的糖环上的或羟丁基上与氧相连亚甲基或次甲基的质子峰。出现在3.34ppm(c峰)处的峰为衣康酸上的非双键亚甲基质子的特征峰，这是因为衣康酸基团处在分子链的最外层，得到良好伸展的质子的核磁共振信号要强于处在分子链内层的信号。通过核磁共振氢谱的检测同样证明了衣康酸接枝到羟丁基壳聚糖的主链之上，UHBCS-IA成功合成。
通过核磁共振氢谱可以确定衣康酸基团的接枝率，即取代度。由于作为中间体的UHBCS已经由元素分析法确定了羟丁基的接枝率，因此通过比较羟丁基中甲基的质子峰(图1中f峰)与衣康酸上的双键亚甲基质子峰(图1中a或b峰)的峰面积即可计算得到UHBCS-IA中IA基团的接枝 率。本实施例中得到的UHBCS-IA经1H NMR中的峰面积比计算，每100个壳聚糖单糖结构单元上，有96个结构单元被衣康酸取代，因此衣康酰化的接枝率为96％，因此，本实施例制备得到的低取代UHBCS-IA记为“UHBCS-96IA”。
实施例7：制备实施例2的壳聚糖
本实施例提供了实施例2中提供的不饱和羟丁基壳聚糖的方法。称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水醋酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将5g的衣康酸酐溶解于200mL四氢呋喃(THF)中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中，滴加完毕之后，升温至40℃并持续反应16小时。反应结束后，采用截流分子量为10kDa的中空纤维柱超滤24小时，以去除体系中多余的四氢呋喃，衣康酸和盐分。冷冻干燥36小时得到UHBCS-IA，将本实施例中得到的UHBCS-IA经1H NMR检测，结果如图2所示，本实施例获得的UHBCS-IA峰位置与图1基本一致，a，b与c的峰面积低于图1。本实施例中得到的UHBCS-IA经1H NMR中的峰面积比计算，每100个壳聚糖单糖结构单元上，有38个结构单元被衣康酸取代，因此衣康酰化的接枝率为38％，因此，本实施例制备得到的低取代UHBCS-IA记为“UHBCS-38IA”。
将本实施例得到的UHBCS-38IA与HBCS、CS进行红外检测，得到FTIR谱图如图3所示，图中A为UHBCS-38IA的红外图谱，B为HBCS的红外图谱，C为CS的红外图谱。从UHBCS-38IA、CS与HBCS的红外光谱，发现相比较CS的图谱中，2925-2966cm -1处出现了较为明显的C-H振动峰，这是由接枝到CS主链上羟丁基基团引起的。UHBCS-38IA图谱中在1670-1744cm -1处的出现了C＝O峰，在CS与HBCS的红外光谱中在相似位置也出现了弱吸收峰，这是由于壳聚糖(脱乙酰度90％)中存在尚未脱掉的乙酰氨基。但1670cm -1处出现的酰胺强吸收峰证明了衣康酸酐已经成功接枝到了HBCS的主链之上，也说明衣康酸酐主要接枝在羟丁基壳聚糖2位的仲胺上。同时，在1744cm -1处出现的羧基或酯基的C＝O峰。因此，通过红外光谱可以证明UHBCS-38IA的成功合成。
制备实施例8：制备实施例3的壳聚糖
本实施例提供了实施例3中提供的不饱和羟丁基壳聚糖的方法，方法如下：称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水丙烯酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将5g的丙烯酰氯溶解于200mL四氢呋喃(THF)中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中，滴加完毕之后，升温至40℃并持续反应18小时。反应结束后，用氢氧化钠中和体系pH至7.0左右，采用截流分子量为10kDa的中空纤维柱超滤24小时，以去除体系中多余的四氢呋喃，丙烯酸和盐分。冷冻干燥36小时得到UHBCS-AC。
制备实施例9：制备实施例4的壳聚糖
本实施例提供了实施例4中提供的不饱和羟丁基壳聚糖的方法，方法如下：称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水甲基丙烯酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将5g的甲基丙烯酸酐溶解 于200mL四氢呋喃(THF)中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中，滴加完毕之后，升温至40℃并持续反应18小时。反应结束后，用氢氧化钠中和体系pH至7.0左右，采用截流分子量为10kDa的中空纤维柱超滤24小时，以去除体系中多余的四氢呋喃，甲基丙烯酸和盐分。冷冻干燥36小时得到HBCS-MA。
制备实施例10：制备实施例5的壳聚糖
本实施例提供了制备实施例5中提供的不饱和羟丁基壳聚糖的方法，方法如下：称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水醋酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将30g的衣康酸酐溶解于100mL四氢呋喃中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中，滴加完毕之后，升温至50℃并持续反应16小时。反应结束后，采用截流分子量为10kDa的中空纤维柱超滤24小时，以去除体系中多余的四氢呋喃，衣康酸和盐分。冷冻干燥36小时得到UHBCS-IA。
制备实施例11
本实施例提供了一种含有两种不饱和基团的羟丁基壳聚糖的制备方法，方法如下：称取5g羟丁基壳聚糖在10℃条件溶解于100mL去离子水中，得到羟丁基壳聚糖的水溶液，加入1g无水甲基丙烯酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将5g的甲基丙烯酸酐溶解于四氢呋喃(THF)中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中。滴加完毕之后，继续当前状态，加入1g无水醋酸钠作为催化剂，搅拌30min得到均匀的溶液。在干燥的高纯氮保护下，将5g的衣康酸酐溶解于200mL四氢呋喃(THF)中，持续快速搅拌下滴入到羟丁基壳聚糖的水溶液中。滴加完毕之后，升温至40℃并持续反应18小时。反应结束后，用氢氧化钠中和体系pH至7.0左右，采用截流分子量为10kDa的中空纤维柱超滤24小时，以去除体系中多余的四氢呋喃，甲基丙烯酸和盐分，冷冻干燥36小时得到HBCS-MA-IA。经1H NMR检测，本实施例得到的不饱和羟丁基壳聚糖中甲基丙烯酸酐的接枝率为25％，衣康酸基团的接枝率为38％，且n A：n B：n C＝1：1.5：7.5。经溶解性检测，本实施例得到的不饱和羟丁基壳聚糖的溶解度为154g/L。
制备实施例12：制备温敏凝胶
本实施例提供了采用实施例6提供的UHBCS-96IA通过光固化制备温敏凝胶的方法。将1.0g UHBCS-96IA溶解于10mL去离子水中，采用质量分数为0.1％的光引发剂I2959引发UHBCS-96IA的化学交联，升温至37℃待凝胶固化之后，采用紫外光照射强度为5mW·cm -2，照射时间为5分钟。凝胶的温敏相转变与化学交联示意图如图4所示，UHBCS-96IA水溶液在37℃时发生温敏相转变，由透明溶胶转变为白色凝胶，继续经过紫外光照射，侧链的双键发生自由基交联反应而形成交联凝胶。
制备实施例13：制备温敏凝胶
本实施例提供了采用实施例6提供的UHBCS-96IA通过热固化制备温敏凝胶的方法。将1.0g UHBCS-96IA溶解于10mL去离子水中，采用质量分数为1％的热引发剂AIBA引发UHBCS-96IA的化学交联，交联温度为60℃，反应时间为30分钟。取出部分所得到的化学交联凝胶放置于真空干 燥箱内，50℃条件下干燥至恒重，得到UHBCS-96IA干凝胶膜。
制备实施例14：制备温敏凝胶
本实施例提供了采用实施例7提供的UHBCS-38IA通过光固化制备温敏凝胶的方法。将1.0g UHBCS-38IA溶解于10mL去离子水中，采用质量分数为0.1％的光引发剂I2959引发UHBCS-96IA的化学交联，升温至37℃待凝胶固化之后，采用紫外光照射强度为5mW·cm -2，照射时间为5分钟。UHBCS-38IA水溶液在37℃时发生温敏相转变，由透明溶胶转变为白色凝胶，继续经过紫外光照射，侧链的双键发生自由基交联反应而形成交联凝胶。
制备实施例15：制备温敏凝胶
本实施例提供了采用实施例7提供的UHBCS-38IA通过热固化制备温敏凝胶的方法。将1.0g UHBCS-38IA溶解于10mL去离子水中，采用质量分数为1％的热引发剂AIBA引发UHBCS-38IA的化学交联，交联温度为60℃，反应时间为30分钟。取出部分所得到的化学交联凝胶放置于真空干燥箱内，50℃条件下干燥至恒重，得到UHBCS-38IA干凝胶膜。
实验例1：水凝胶的体外降解性能测试
对实施例12所得的UHBCS-96IA水凝胶进行体外降解性能测试。由于降解试验耗时较长，在选用的HBCS分子量相同的情况下，侧链接枝的不饱和基团越多，则交联形成的水凝胶交联度越大，交联度越大的结果就是降解时间越长，因此在降解试验时，选用的是取代度最大的UHBCS-96IA水凝胶作为标识水凝胶。
称取1.0g的UHBCS-96IA干凝胶膜，浸泡于pH 7.4的PBS缓冲液中，加入溶菌酶，使溶液中的溶菌酶的浓度为100单位·mL -1，实验初始的5天每天补加相同浓度的溶菌酶一次，第六天起停止加入溶菌酶。再另称取1.0g干凝胶膜浸泡于pH 4.0的抗坏血酸-抗坏血酸钠(AH 2/AH -)缓冲溶液中，在实验起始的五天内每天加入0.2mL质量分数为3％的双氧水溶液以诱发ROS的生成。在浸泡一段时间之后，取出残留的凝胶，用去离子水冲洗，于50℃条件下真空干燥至恒重，再次称重以确定凝胶在降解过程中的质量损失。降解结果如图5所示，图中A为溶菌酶降解曲线，B为ROS降解曲线。
从图中曲线A可以看出，溶菌酶主要通过破坏UHBCS-96IA中部分残留的N-乙酰氨基葡糖之间的β-1,4糖苷键而达到分解壳聚糖的目的。由于溶菌酶是直接破坏壳聚糖主链，所以在降解过程中会发现原本完整的凝胶膜会***为碎片，然后这些碎片再在溶菌酶的作用下继续降解。图中可以看出在添加溶菌酶14小时后，酶活性降低，因此在14-24小时的时间区间内，残存的UHBCS-96IA凝胶膜降解缓慢。24小时后补入溶菌酶，残存的UHBCS-96IA凝胶膜则基本被降解完全。通过补加溶菌酶的方法，UHBCS-96IA的降解时间在36小时之内。图中曲线B可以发现在降解初期，凝胶膜的质量损失速率较慢。这是由于在降解初期，凝胶膜中致密的交联网络结构阻碍了溶菌酶或者ROS对凝胶膜内部结构进行破坏，只能从外部层层推进。一旦凝胶膜的内部结构被破坏，凝胶膜的网络结构则会迅速瓦解，质量损失的开始加速，直至最终降解完全。同样地，虽然ROS的体积比溶菌酶要小很多，但ROS并不像溶菌酶那样的特异性降解，可供ROS进攻的位点较多，包括β-1,4糖苷键、 糖环中的缩醛键、羟丁基与主链相连的醚键以及衣康酸酯键均是ROS的进攻目标。ROS会优先进攻空间位阻相对较小的目标，所以在ROS降解的过程中，只是偶尔出现像溶菌酶降解那样的凝胶膜碎片，大部分情况下就是凝胶膜体积逐渐减小最终完全消失。同溶菌酶的情况相类似，在降解反应发生12小时之后，ROS几乎被消耗殆尽，所以在14-24小时的时间区段内，UHBCS-96IA凝胶膜的降解缓慢。但由于体系的酸性环境，UHBCS-96IA将会继续缓慢地降解。24小时之后补加双氧水，则凝胶膜的降解加速，经过第二个ROS失活过程(36-48小时)，在第三次补加双氧水之后，UHBCS-96IA完全降解，整个降解过程在60小时之内。
实验例2：水凝胶的机械强度测试
对实施例13和实施例15所得的UHBCS-96IA水凝胶和UHBCS-38IA水凝胶进行机械强度测试，方法如下：将实施例13和实施例15中通过热固化之后得到的厚度均匀的UHBCS-96IA干凝胶膜和UHBCS-38IA干凝胶膜裁剪成标准样条，样条宽度2mm，厚度0.1mm。通过万能试验机在室温条件下检测其机械性能，主要研究凝胶膜的极限强度(MPa)，断裂伸长率(％)以及杨式模量(MPa)，万能试验机的拉伸速率为200mm·min -1。检测结果如图6所示，图中a为UHBCS-96IA干凝胶膜的应变曲线，图中b为UHBCS-38IA干凝胶膜的应变曲线。
从图6中可以看到，UHBCS-96IA凝胶膜在拉伸过程中经历了一个应***化与应***化的过程。应***化是由于凝胶膜中存在的线性聚合物链的动态再结晶作用引起的，而应***化则由刚性的凝胶交联网络导致。同时UHBCS-96IA凝胶膜的断裂伸长率要低于UHBCS-38IA凝胶膜，而断裂强度与杨式模量却均比UHBCS-38IA凝胶膜高。这些现象都说明了UHBCS-96IA凝胶膜并不是典型的弹性体，而更类似一种互穿的网络结构。因为在交联过程中形成聚衣康酸(PIA)，凝胶结构中同时存在了壳聚糖与聚衣康酸的分子链，而不是单纯的两个衣康酸官能团的交联。让UHBCS-96IA水凝胶溶胶先发生温敏凝胶化之后再化学交联这样的方法为聚衣康酸分子链的形成提供了的条件。在温敏凝胶化之后，UHBCS-96IA分子链紧密缠绕，这使得分子链中的IA官能团团聚在一起，当自由基反应引发之后，相邻的多个IA都能发生自由基聚合反应形成聚衣康酸，而不是两个衣康酸基团的交联反应。UHBCS-38IA中由于IA基团含量较低，难以形成PIA，所以UHBCS-38IA凝胶膜则属于是典型的弹性体。这也论证了UHBCS-96IA凝胶膜的杨式模量要远高于UHBCS-38IA凝胶膜的结果。
实验例3水凝胶的体外细胞毒性测试
对实施例13和实施例15所得的UHBCS-96IA水凝胶和UHBCS-38IA水凝胶进行体外细胞毒性测试，方法如下：
将L-929细胞种植在细胞培养基中，加入1％的盘尼西林-链霉素溶液以及10％的胎牛血清溶液。L-929细胞在潮湿的含有5％二氧化碳的细胞培养箱中，37℃下恒温孵化3天。然后，再将UHBCS-IA水凝胶转移进入96孔板中，紫外灯固化并灭菌。再次，将L-929细胞培养液加入到放置了UHBCS-IA凝胶的孔中，并加入1mL含有0.1％EDTA的胰蛋白酶溶液，每孔中的L-929细胞数量为1×10 5个。并继续放置在细胞培养箱中以促进细胞生长。
UHBCS-IA交联水凝胶的细胞毒性由MTT法测定。在分别培养了24，48以及72小时之后，向 每个孔中加入100μL的MTT水溶液(浓度为5mg·mL -1)并置于培养箱中继续培养4小时。然后移除MTT溶液，加入150μL的二甲亚砜以溶解甲瓒结晶，采用酶标仪在490nm波长处检测溶液的吸光度以确定该凝胶对L-929的细胞毒性。细胞存活率根据下式计算：
细胞存活率(％)＝(As/Ac)×100％
其中，As为样品溶液在490nm处的吸光度，Ac为空白对照在490nm处的吸光度。
细胞毒性结果如图7所示：从图中可以看出UHBCS-96IA水凝胶与UHBCS-38IA水凝胶均显示出了良好的细胞增值率，UHBCS-96IA在24，48以及72小时的细胞存活率分别为91％，93％和97％，细胞毒性为I级，说明HBCS-96IA水凝胶没有细胞毒性。UHBCS-38IA水凝胶在24，48以及72小时的细胞存活率为94％，98％与102％，其在72小时的细胞毒性为零级，其余的为I级。从这两类凝胶的细胞存活率可以看出，UHBCS-IA水凝胶均没有细胞毒性，其细胞存活率随时间增长而升高的现象表明UHBCS-IA水凝胶在一定程度上会起到促进细胞增长的作用。
实验例4水凝胶的防黏连性能测试
对实施例13所得的UHBCS-96IA水凝胶进行防黏连性能测试，方法如下：
1.动物模型制作与动物分组
将受试动物分为两组，UHBCS-96IA受试组、模型对照组。每组各5只大鼠。实验设计方案如下：在椎板切除手术中，只切除大鼠右侧椎板，左侧不予处理。UHBCS-96IA受试组右侧取出椎板止血后给予UHBCS-96IA溶胶(溶胶pH 7.2)，溶胶接触到体内组织后，先由动物体的体温触发溶胶的凝胶化，待凝胶凝固后缝合伤口。模型对照组右侧取出椎板止血后给予羧甲基壳聚糖溶液并缝合伤口。此实验自造模给药后8周处死进行病理组织形态学观察。
正式实验手术流程如下：腹腔注射麻醉4.5％戊巴比妥钠(1ml/kg)，大鼠取俯卧位，剔除背部下2/3鼠毛，触摸确定脊柱位置后，于脊柱正上方切口，切口长度6-7cm，皮层切开后，在脊柱两旁分别切开肌肉层，去除脊柱上覆盖的多余肌肉。暴露大鼠腰椎第4、5椎板，右侧神经根损伤，移除黄韧带和硬膜外脂肪，尽量使留下干净的硬脊膜更大程度上暴露。UHBCS-96IA受试组给予UHBCS-96IA溶胶待凝固后缝合伤口，模型对照组止血后给予羧甲基壳聚糖溶液并缝合伤口。
2.动物模型标本的制备与评定
初始校准手术区，包括椎板切除点，椎骨骼和周围的肌肉，整块取出然后将其存储在4％多聚甲醛溶液，经甲酸-甲醛液脱钙，梯度乙醇脱水，EG1150H自动生物组织包埋机包埋，Leica RM2255切片机制片，HE染色。在显微镜下对染色切片进行分析，以量化存在不同的炎症、纤维化及相关组织变性。参照Hooker等评分标准，由病理科医师盲法对神经组织外膜胶原纤维粘合性，瘢痕组织的厚度进行评分。结果记录为：0分＝无瘢痕粘连(0级)，1分＝离散的，少量的瘢痕(I级)，2分＝中度，较多离散瘢痕组织(II级)，3分＝显著，较多致密瘢痕(III级)，4分＝严峻，大量的致密瘢痕组织(IV级)。HE染色的结果如图8所示。粘连评分结果如表2所示。同时，采用MASSON三色染色观察椎板间隙：常规石蜡切片脱蜡至水，采用Masson三色染色法半定量分析胶原纤维。MASSON染色结果如图9所示。
表2大鼠硬膜外组织粘连及瘢痕评分及统计结果
Figure PCTCN2021073923-appb-000007
通过观察图8，发现术后8周模型对照组10/10只动物神经织组周围纤维***增生，见轻度至中度以淋巴细胞为主炎细胞浸润反应，并见少量多核巨细胞及新生毛细血管。偶见水肿及脂肪浸润，未见明显变性坏死。UHBCS-96IA凝胶受试试验组中，5/10只动物神经织组周围反应与模型对照组相近，其余5/10未见明显病理形态学改变。纤维化评分UHBCS-96IA凝胶受试组与模型对照组比较有显著差异，(P<0.05)，数据见表2。模型对照组中的瘢痕发生率竟高达100％，而UHBCS-96IA凝胶受试组的瘢痕发生率仅为50％，发生的瘢痕均为轻微瘢痕。
图9中的脊髓组织与神经根组织呈红色，瘢痕组织呈蓝绿色。从图9中可以发现，椎板切除术后8周时间，模型对照组中的神经根周围已被纤维组织包裹，瘢痕与粘连的发生非常明显。而在UHBCS-96IA凝胶给药组的神经跟周围几乎没有发现有蓝绿色的粘连以及瘢痕组织，可见UHBCS-96IA凝胶的防粘连效果非常明显。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开实施例提供一种不饱和羟丁基壳聚糖(UHBCS)、其制备方法、其在制备温敏凝胶中的应用、其在制备温敏凝胶的方法、由其制备的温敏凝胶以及由其制备的温敏凝胶在制备软组织填充材料、软组织修复材料、体表创伤敷料、软组织、肌腱与神经组织手术后防粘连材料中的应用。其中，所制备的羟丁基壳聚糖能够提高其凝胶的机械强度从而能够应用于生物软组织材料的修复和替换；并且在制备不饱和羟丁基壳聚糖中，该制备方法简单、易操作。同时，不饱和羟丁基壳聚糖在制备温敏凝胶的方法中，反应条件简单、快速；使得制备得到的温敏凝胶机械强度更高，具有更高的弹性模量。

Claims (17)

  1. 一种不饱和羟丁基壳聚糖，所述不饱和羟丁基壳聚糖为侧链含有不饱和基团的羟丁基壳聚糖分子，所述不饱和基团包括不饱和酸酐基团和/或不饱和酰氯基团，所述不饱和基团的接枝率为10％～100％；
    优选地，所述不饱和基团的接枝率为38％～98％；
    进一步优选地，所述不饱和基团的接枝率为96％。
  2. 根据权利要求1所述的不饱和羟丁基壳聚糖，其中，所述不饱和羟丁基壳聚糖含有如下单体(A)～(C)中的一种或两种以上组合：
    单体(A)为
    Figure PCTCN2021073923-appb-100001
    单体(B)为
    Figure PCTCN2021073923-appb-100002
    单体(C)为
    Figure PCTCN2021073923-appb-100003
    其中R 1包括不饱和酸酐基团和/或不饱和酰氯基团；R 2包括不饱和酸酐基团和/或不饱和酰氯基团；R 3包括不饱和酸酐基团和/或不饱和酰氯基团；优选地，所述不饱和羟丁基壳聚糖含有数量比为1:5～9的单体(A)和单体(C)；
    优选地，所述不饱和羟丁基壳聚糖含有所述(A)～(C)三种单体，且单体(A)的数量：单体(B)的数量：单体(C)的数量为1：0.01～4：5～9。
  3. 根据权利要求2所述的不饱和羟丁基壳聚糖，其中，所述不饱和酸酐基团包括甲基丙烯酸酐、丙烯酸酐、马来酸酐或衣康酸酐中的一种或两种以上组合；所述不饱和酰氯基团包括丙烯酰氯和/或甲基丙烯酰氯；
    优选地，所述不饱和酸酐基团为衣康酸基团，且单体(A)的数量：单体(B)的数量：单体(C)的数量为1:1:8；
    优选地，所述不饱和酸酐基团为甲基丙烯酸酐，且单体(A)的数量：单体(B)的数量：单体(C)的数量为1：1.5：7.5；
    优选地，所述不饱和酸酐基团为丙烯酰氯，且单体(A)的数量：单体(B)的数量：单体(C)的数量为1：1.5：7.5。
  4. 制备权利要求1～3任一项所述的不饱和羟丁基壳聚糖的方法，所述方法包括：将羟丁基壳聚糖水溶液与不饱和酰化试剂的有机溶液混合后，经过酰化反应制得不饱和羟丁基壳聚糖；
    优选地，所述羟丁基壳聚糖水溶液中羟丁基壳聚糖的质量分数为0.1％～10％；
    优选地，所述羟丁基壳聚糖水溶液中羟丁基壳聚糖的质量分数为1～10％；
    优选地，所述不饱和酰化试剂的有机溶液的质量分数为1％～50％；
    优选地，所述酰化反应在自然pH下进行，反应完成后调节pH至7.0；
    优选地，所述酰化反应的温度为20～60℃；
    优选地，所述酰化反应的温度为40～50℃；
    优选地，所述酰化反应的时间为2～24h；
    优选地，所述酰化反应的时间为15～20h；
    优选地，所述酰化反应在催化剂作用下进行；
    优选地，所述催化剂包括弱碱性催化剂；
    优选地，所述弱碱性催化剂包括醋酸钠、磷酸钠、三乙胺或碳酸钠中的一种或两种以上组合；
    优选地，所述催化剂的加入量为羟丁基壳聚糖加入量的1～50％；
    优选地，所述酰化反应在惰性气体保护气氛下进行；
    优选地，所述惰性气体包括氮气；
    优选地，所述酰化反应完成并调节pH至7.0之后还依次包括过滤和干燥的步骤；
    优选地，所述干燥的方法包括冷冻干燥法。
  5. 根据权利要求4所述的方法，其中，所述不饱和酰化试剂的有机溶液所使用的溶剂为溶于水的非活性氢试剂；
    优选地，所述溶剂包括四氢呋喃、液体聚乙二醇、N,N-二甲基甲酰胺、二甲基亚砜或N-甲基吡咯烷酮。
  6. 根据权利要求4所述的方法，其中，所述过滤的方法包括超滤，所述超滤的时长为2～24h；
    优选地，所述超滤的步骤所用的器材包括膜包或中空纤维；
    优选地，所述膜包或中空纤维的截留分子量为1000～30000Da。
  7. 权利要求1-3任一项所述的不饱和羟丁基壳聚糖或采用权利要求4-6任一项所述的方法制备得到的不饱和羟丁基壳聚糖在制备温敏水凝胶中的应用。
  8. 一种制备温敏水凝胶的方法，所述方法包括：
    利用权利要求1～3任一项所述的不饱和羟丁基壳聚糖或采用权利要求4～6任一项所述的法制备得到的不饱和羟丁基壳聚糖在交联引发剂的存在下通过自由基交联反应制得温敏水凝胶；
    优选地，所述交联引发剂包括光交联引发剂或热交联引发剂；
    优选地，所述交联引发剂为光交联引发剂，所述方法包括：将所述不饱和羟丁基壳聚糖与所述光交联引发剂溶于水，然后在35℃以上条件下，采用光强度为0.05～50mW·cm -2的紫外光或蓝光照射1～20min， 得到光交联温敏水凝胶；所述不饱和羟丁基壳聚糖质量分数为0.5％～20％，所述光交联引发剂的质量分数为0.01％～1％；所述的光交联引发剂与不饱和羟丁基壳聚糖的质量比为1:1～200；
    进一步优选地，所述光交联引发剂包括I2959；
    进一步优选地，所述紫外光的波长为200～400nm；
    进一步优选地，所述蓝光的波长为400～480nm；
    优选地，所述交联引发剂为热交联引发剂，所述方法包括：将所述不饱和羟丁基壳聚糖与热交联引发剂溶于水，所述不饱和羟丁基壳聚糖质量分数为0.5％～10％，所述热交联引发剂的质量分数为0.01％～5％，然后在35℃条件下使不饱和羟丁基壳聚糖发生胶化后，再升温至60～80℃，持续反应10～120min，得到热交联温敏水凝胶；所述的热交联引发剂与所述不饱和羟丁基壳聚糖的质量比为1:2～200；
    进一步优选地，所述热交联引发剂包括水溶性过氧引发剂或水溶性偶氮引发剂；
    进一步优选地，所述水溶性过氧引发剂包括过氧苯甲酸，过硫酸钾或过氧乙酸中的一种或两种以上组合；
    进一步优选地，所述水溶性偶氮引发剂包括偶氮二异丁脒盐酸盐和/或偶氮二异丁咪唑啉盐酸盐。
  9. 一种采用权利要求8所述的方法制备得到的温敏水凝胶。
  10. 权利要求9所述的温敏水凝胶在制备软组织替换材料，软组织修复材料，体表创伤敷料，软组织、肌腱与神经组织手术后防粘连材料中的应用。
  11. 一种温敏水凝胶，包括或由相互交联的根据权利要求1-3任一项所述的不饱和羟丁基壳聚糖组成。
  12. 一种生物医用材料，包括权利要求9或11所述的温敏水凝胶。
  13. 根据权利要求12所述的生物医用材料，其中所述生物医用材料选自软组织修复材料，体表创伤敷料，或软组织、肌腱与神经组织手术后防粘连材料。
  14. 根据权利要求12或13所述的生物医用材料，用于修复软组织，治疗体表创伤，或软组织、肌腱与神经组织手术后防粘连的用途。
  15. 一种对受试者中创伤处理的方法，包括：
    向所述有此需要的受试者施用根据权利要求12或13所述的生物医用材料。
  16. 根据权利要求15的方法，其中，所述受试者患有软组织损伤，体表创伤，或经历了手术。
  17. 根据权利要求15的方法，其中，所述手术是与软组织、肌腱与神经组织相关的手术。
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