WO2018121510A1 - 一种注射用多元甘醇环氧衍生物交联的透明质酸钠凝胶及其制备方法 - Google Patents

一种注射用多元甘醇环氧衍生物交联的透明质酸钠凝胶及其制备方法 Download PDF

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WO2018121510A1
WO2018121510A1 PCT/CN2017/118542 CN2017118542W WO2018121510A1 WO 2018121510 A1 WO2018121510 A1 WO 2018121510A1 CN 2017118542 W CN2017118542 W CN 2017118542W WO 2018121510 A1 WO2018121510 A1 WO 2018121510A1
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sodium hyaluronate
gel
group
cross
epoxy derivative
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PCT/CN2017/118542
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English (en)
French (fr)
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魏真
林美娜
赵宣
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北京键凯科技股份有限公司
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Priority to US16/473,479 priority Critical patent/US11191870B2/en
Publication of WO2018121510A1 publication Critical patent/WO2018121510A1/zh

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    • 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
    • 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
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D301/00—Preparation of oxiranes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/28—Ethers with hydroxy compounds containing oxirane rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/12—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms
    • C07D303/18—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by etherified hydroxyl radicals
    • C07D303/28—Ethers with hydroxy compounds containing oxirane rings
    • C07D303/30—Ethers of oxirane-containing polyhydroxy compounds in which all hydroxyl radicals are etherified with oxirane-containing hydroxy compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G59/00—Polycondensates containing more than one epoxy group per molecule; Macromolecules obtained by polymerising compounds containing more than one epoxy group per molecule using curing agents or catalysts which react with the epoxy groups
    • C08G59/02—Polycondensates containing more than one epoxy group per molecule
    • C08G59/04—Polycondensates containing more than one epoxy group per molecule of polyhydroxy compounds with epihalohydrins or precursors thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/04—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers only
    • C08G65/06—Cyclic ethers having no atoms other than carbon and hydrogen outside the ring
    • C08G65/08—Saturated oxiranes
    • C08G65/10—Saturated oxiranes characterised by the catalysts used
    • 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
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C08J3/246—Intercrosslinking of at least two polymers
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • 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
    • 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
    • C08J2471/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
    • C08J2471/02—Polyalkylene oxides

Definitions

  • the invention relates to the technical field of sodium hyaluronate gel, in particular to a sodium hyaluronate gel crosslinked by a polyglycol epoxy derivative and a preparation method thereof.
  • Hyalouronic acid is a linear polymer viscous polysaccharide composed of glucuronic acid and acetylglucosamine disaccharide units.
  • Hyaluronic acid is an endogenous substance in the human body, with good biocompatibility; high viscoelasticity and non-Newtonian rheological properties; non-toxic, non-immunogenic, non-irritating, high It is widely used as a soft tissue filler in beauty because of its safety and its ability to be eliminated by injection of hyaluronidase.
  • Hyaluronic acid is injected into the skin to increase the volume of soft tissue, which can achieve the purpose of wrinkle removal or shaping.
  • hyaluronic acid itself exists in a liquid form, it is easily decomposed by the action of hyaluronidase and free radicals in the body, so that it is difficult to achieve a plastic effect and the filling effect has a short duration.
  • the cross-linking technology is applied to hyaluronic acid, that is, the hyaluronic acid molecules are connected by a crosslinking agent to form a relatively stable network structure, so that it is no longer easy to be decomposed and the time for filling is maintained. long.
  • the transparent sodium gel After cross-linking modification, the transparent sodium gel has the advantages of good viscoelasticity, insoluble in water, high mechanical strength and long degradation time.
  • the different degree of crosslinking brings different physical properties, which makes it better. Meet the beauty filling needs.
  • the crosslinking agents currently used in commercially available sodium hyaluronate gel products are mainly BDDE (butanediol diglycidyl ether) and DVS (divinyl sulfone).
  • BDDE butanediol diglycidyl ether
  • DVS divinyl sulfone
  • the gel prepared by the two cross-linking agents has distinct physical properties: the DVS cross-linked product has a hard texture and high cross-linking activity; the BDDE cross-linked product has a soft texture, a mild reaction and a large expansion ratio.
  • the above cross-linking agents have toxicity or potential carcinogenic potential, and the cross-linked spatial network structure has a wrapping effect on the unreacted cross-linking agent, and the removal is difficult.
  • the present invention firstly designs and synthesizes a novel type. , low toxicity, high reactivity, water-soluble cross-linking agent, and then using the cross-linking agent to prepare a low toxicity, less residue, small thrust, good shape, good enzyme resistance, long retention time in the body Modified sodium hyaluronate gel for injection. Specifically, in order to overcome the above drawbacks of the prior art, the present invention designs and synthesizes a novel, low toxicity, high reactivity, high water solubility polyglycol epoxy derivative.
  • the polyglycol or polyglycol epoxy derivative of the present invention is a compound of a single molecular weight.
  • the present invention provides a novel, low toxicity, highly reactive, superhydrophilic, low toxicity polyglycol epoxy derivative having the structure of Formula I:
  • n is an integer from 4 to 200;
  • EPOX is Wherein Y is selected from the group consisting of: -(CR 1 R 2 ) m -, -(CH 2 ) m NH-, -NHCO(CH 2 ) m -, -(CH 2 ) m CONH- and -CO(CH 2 ) m - a combination of one or more of
  • n is an integer from 1 to 10;
  • t is selected from 0, 1, and 2;
  • R 3 and R 4 are independently of each other selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heterocyclic, and halogen.
  • n is an integer of 10 to 100; more preferably, n is an integer of 10 to 50, further preferably an integer of 12 to 30; or, preferably, an integer of 10 to 100, more preferably 10 to 30
  • the integer is most preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
  • said Y is -(CR 1 R 2 ) m -.
  • the m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5.
  • said m 1
  • the R 1 and R 2 are independently selected from the group consisting of: H, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, -OR 3 , -NR 3 R 4 , and a halogen.
  • said R 3 and R 4 are independently selected from the group consisting of hydrogen and a C1-C3 alkyl group.
  • both R 1 and R 2 are H.
  • the Y is -CH 2 -.
  • the polyglycol epoxy derivative of the present invention is preferably a compound having a single molecular weight.
  • the polyglycol epoxy derivative of the present invention has the following structure:
  • n is an integer of 4 to 200, preferably an integer of 10 to 100, more preferably an integer of 10 to 50, further preferably an integer of 10 to 30 (specifically, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30).
  • Another aspect of the present invention provides a process for producing a polyglycol epoxy derivative, which comprises the steps of catalyzing a reaction of EPOX-X with a polyglycol in a solvent using a catalyst;
  • n is an integer from 4 to 200;
  • EPOX is Wherein Y is selected from the group consisting of: -(CR 1 R 2 ) m -, -(CH 2 ) m NH-, -NHCO(CH 2 ) m -, -(CH 2 ) m CONH- and -CO(CH 2 ) m - a combination of one or more of them;
  • n is an integer from 1 to 10;
  • t is selected from 0, 1, and 2;
  • R 3 and R 4 are independently of each other selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heterocyclic, and halogen.
  • n is an integer of 10 to 100; more preferably, n is an integer of 10 to 50, further preferably an integer of 12 to 30, or preferably an integer of 10 to 100, more preferably 10 to 30 Integer, most preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
  • the -X is selected from the group consisting of: -F, -Cl, -Br, -I, (-OMs), (-OTs); more preferably, -X is -Cl or -Br;
  • said Y is -(CR 1 R 2 ) m -.
  • the m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5.
  • said m 1
  • the R 1 and R 2 are independently selected from the group consisting of: H, a C1-C6 alkyl group, a C3-C6 cycloalkyl group, -OR 3 , -NR 3 R 4 , and a halogen.
  • said R 3 and R 4 are independently selected from the group consisting of hydrogen and a C1-C3 alkyl group.
  • both R 1 and R 2 are H.
  • the Y is -CH 2 -.
  • the catalyst is a base catalyst, including an organic base or an inorganic base, preferably but not limited to: pyridine, triethylamine, cesium carbonate, sodium carbonate, potassium carbonate, sodium hydrogencarbonate, potassium hydrogencarbonate, sodium hydroxide One or more of potassium hydroxide, sodium alkoxide and potassium alkoxide.
  • the solvent includes, but is not limited to, 1,4-dioxane, tetrahydrofuran, toluene, acetone, ethyl acetate, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide and water.
  • 1,4-dioxane 1,4-dioxane, tetrahydrofuran, toluene, acetone, ethyl acetate, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide and water.
  • the molar ratio of monohydroxyl to EPOX-X in the polyglycol is 1:2-10 (specifically, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7) , 1:8, 1:9 or 1:10).
  • the method for preparing the polyglycol epoxy derivative further comprises a purification step; more preferably, the purification step is selected from one or more of: steaming, washing, extraction, molecular distillation, and column separation. Combination of species.
  • the preparation method comprises the following specific steps: adding a polyhydric glycol, a solvent and a catalyst to the reaction vessel, stirring, and dropwise adding a halogenated or sulfonated propylene oxide to the above mixture.
  • the reaction temperature was controlled to not exceed 35 ° C.
  • the mixture was filtered, the residue was washed, and the filtrate was collected and purified to obtain a polyglycol epoxy derivative.
  • the polyglycol epoxy derivative synthesized by the above preparation method has high purity, and its purity is more than 99% by HPLC.
  • the low toxicity, high reactivity, super hydrophilic polyglycol epoxy derivative can be used as a crosslinking agent for polymer polymerization of natural polymers such as natural polysaccharides and proteins, synthetic polymers such as polyethylene glycol and polyvinyl alcohol. Crosslinking of matter.
  • Another aspect of the present invention provides a crosslinking agent comprising the above polyhydric alcohol epoxy derivative of the present invention.
  • the present invention also provides a high molecular polymer crosslinked by the above polyglycol epoxy derivative.
  • the high molecular polymer may be a natural polymer or a synthetic polymer, more preferably a natural polymer such as a natural polysaccharide or protein.
  • the natural polymer is selected from the group consisting of starch, chitosan, chitin, cellulose, pectin, gelatin, gum arabic, casein, chitin, silk fibroin, albumin, casein, and transparent.
  • Acid glycogen, sericin, gellan gum, xanthan gum, guar gum, dextran, chitosan oligosaccharide, inulin, glucoside glycan, mannooligosaccharide, mannan, galactan, One or more of chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, agar, and derivatives thereof;
  • the synthetic polymer is selected from the group consisting of polyvinylpyrrolidone, polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinyl acetate, polylactic acid, polyglycolic acid, polyacrylic acid, polyacrylamide, One or more of polytetrahydrofuran, polybutylene oxide, polyoxetane, polymaleic anhydride, polyhydroxyethyl methacrylate, polypropylene glycol, polycaprolactone, and derivatives thereof;
  • the natural polymer is a hyaluronic acid derivative, more preferably a hyaluronate, and the hyaluronate is selected from the group consisting of sodium hyaluronate and potassium hyaluronate.
  • the hyaluronate is selected from the group consisting of sodium hyaluronate and potassium hyaluronate.
  • the crosslinked product is sodium hyaluronate crosslinked by the above polyglycol epoxy derivative.
  • the sodium hyaluronate has a molecular weight of 50,000 to 3 million Daltons (specifically, 50,000, 100,000, 500,000, 1,000,000, 1.5 million, 2 million, 2.5 million, or 3 million Daltons). More preferably, it is 300,000 to 1.5 million Daltons.
  • the molar ratio of the polyglycol epoxy derivative to the polymer unit in sodium hyaluronate is 0.01-1:1 (specifically, 0.01:1, 0.1:1, 0.2:1, 0.3:1) 0.4:1, 0.5:1 or 1.0:1).
  • the invention provides a novel low toxicity, high reactivity and water-soluble crosslinking agent, and the invention also provides a low toxicity, less residue, small extrusion thrust, good shapeability and enzyme resistance.
  • a modified sodium hyaluronate gel for injection which has a good retention time in vivo and a preparation method thereof.
  • a method for preparing a cross-linked sodium hyaluronate of a polyglycol epoxy derivative which comprises the step of crosslinking a sodium hyaluronate with the above-mentioned polyglycol epoxy derivative of the present invention under alkaline conditions.
  • the crosslinking reaction step comprises: dissolving the polyglycol epoxy derivative in an alkaline solution, adding sodium hyaluronate powder, 35-45 ° C, preferably 40 ° C The reaction is stirred to form crosslinked sodium hyaluronate.
  • the molar ratio of the polyglycol epoxy derivative to the polymer unit in sodium hyaluronate is 0.01-1:1 (specifically, 0.01:1, 0.1:1, 0.2:1, 0.3:1) 0.4:1, 0.5:1 or 1.0:1).
  • the alkaline solution is selected from the group consisting of sodium hydroxide, potassium hydroxide and sodium carbonate solution; in a preferred embodiment of the invention, the alkaline solution is an aqueous solution of NaOH having a mass concentration of 0.1-10%.
  • the mass ratio of the sodium hyaluronate powder to the alkaline solution is 1:5-30 (specifically, such as 1:5, 1:10, 1:15, 1:20, 1:25 or 1:30) ).
  • the sodium hyaluronate has a molecular weight of 50,000 to 3 million Daltons (specifically, 50,000, 100,000, 500,000, 1,000,000, 1.5 million, 2 million, 2.5 million, or 3 million Daltons). More preferably, it is 300,000 to 1.5 million Daltons.
  • the method for preparing the multi-glycol epoxy derivative cross-linked sodium hyaluronate further comprises a cross-linking agent removing step, comprising sequentially adjusting the pH of the cross-linking reaction product to be neutral, swelling, and buffering with carbonate.
  • the step of liquid washing comprising sequentially adjusting the pH of the cross-linking reaction product to be neutral, swelling, and buffering with carbonate.
  • the cross-linking agent removing step comprises: adjusting the above-mentioned cross-linked sodium hyaluronate to be neutral by adding an acid; adding an appropriate amount of PBS buffer to swell the gel; using a carbonate buffer High temperature washing cross-links sodium hyaluronate gel to change the structure of uncrosslinked small molecules in the gel and reduce toxicity.
  • the acid is hydrochloric acid, more preferably 0.1N-2N hydrochloric acid;
  • the carbonate buffer has a pH of 8-9 and may be Na 2 CO 3 /NaHCO 3 , Na 2 CO 3 /KHCO 3 , K 2 CO 3 /NaHCO 3 , K 2 CO 3 /KHCO 3.
  • the mass concentration of carbonate is 0.02-3%.
  • the crosslinker removal step further comprises a dialysis step.
  • the present invention also provides a crosslinked sodium hyaluronate gel for injection which is obtained by gelation of crosslinked sodium hyaluronate prepared by the above method.
  • the sterilization conditions are sterilized in steam at 121 ° C - 125 ° C for 15-25 minutes.
  • the above method for preparing a crosslinked sodium hyaluronate gel for injection comprises the following specific steps:
  • Step 1 dissolving the polyglycol epoxy derivative in an alkaline solution, adding sodium hyaluronate powder, stirring at 35-45 ° C, preferably 40 ° C to form a crosslinked sodium hyaluronate gel;
  • Step 2 Add acid to adjust the gel to neutral
  • Step 3 adding an appropriate amount of PBS buffer to swell the gel
  • Step 4 Washing the crosslinked sodium hyaluronate gel with a carbonate buffer at a high temperature to change the structure of the uncrosslinked small molecule in the gel to reduce toxicity;
  • Step 5 Repeat the dialysis multiple times to remove the residual small molecule cross-linking agent in the gel;
  • Step 6 Screening with a standard sieve
  • Step 7 The gel particles collected in step 6 are placed in a preparation bottle, the mobile phase is added, filled in a pre-sterilized disposable syringe, and sterilized in steam at 121 ° C - 125 ° C for 15-25 minutes, ie A modified sodium hyaluronate gel for injection can be obtained.
  • J, K, and n are positive integers, n is an integer of 4 to 200, and J and K are determined according to the molecular weight of sodium hyaluronate.
  • the present invention also provides a gel comprising sodium hyaluronate crosslinked by the above polyglycol epoxy derivative.
  • the present invention also provides the use of the above crosslinked sodium hyaluronate in the preparation of a product for pharmaceutical, medical cosmetic and cosmetic use.
  • the application is the use of the above crosslinked sodium hyaluronate in the preparation of a product for medical cosmetic use.
  • the product for medical cosmetic use is a soft tissue filler.
  • the present invention also provides a soft tissue filler comprising the above crosslinked sodium hyaluronate of the present invention.
  • the cross-linking agent used in the cross-linked sodium hyaluronate gel prepared by the invention is a polyglycol epoxy derivative.
  • the water solubility is better than the conventional cross-linking agent BDDE.
  • the polyglycol epoxy derivative is more likely to crosslink with the polysaccharide than the BDDE to form a crosslinked sodium hyaluronate gel; secondly, because of the different polyglycol epoxy derivative crosslinkers
  • polymer polyethylene glycol which is a single molecular weight compound
  • the stability of different batches of sodium hyaluronate gel prepared by cross-linking with a polyglycol epoxy derivative is superior to that of polyethylene glycol epoxy derivative.
  • the short chain polyglycol epoxy derivative has a longer molecular chain length than BDDE, it may be entangled with chain hyaluronic acid molecules, enhancing the softness, viscoelasticity and stability of the gel; The number of repeating units of glycol is easier to adjust, the length is easier to control, and the gel properties prepared as a crosslinking agent are easier to control.
  • the toxicity of the polyglycol epoxy derivative is significantly lower than that of BDDE, the polyglycol epoxy derivative is obtained.
  • the invention integrates the cross-linked sodium hyaluronate into a glue removing and removing process, and can prepare the modified hyaluronic acid for injection with low toxicity, less residue, small pushing force, good shape, good enzyme resistance and long retention time in the body.
  • Example 1 is a graph showing the extrusion thrust curve of a dodecadiol diglycidyl ether crosslinked sodium hyaluronate gel provided in Example 10 of the present invention.
  • Example 2 is a graph showing the extrusion pressure of a polyethylene glycol PEG1000 diglycidyl ether crosslinked sodium hyaluronate gel according to Example 10 of the present invention.
  • alkyl refers to a straight or branched chain and does not a hydrocarbon chain radical containing an unsaturated bond
  • C1-C6 alkyl group in the present invention means an alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, tert-butyl, n-pentyl, n-hexyl, etc., preferably a C1-C3 alkyl group (such as methyl, ethyl, n-propyl and isopropyl);
  • cycloalkyl refers to an alicyclic ring a hydrocarbon having a typical cycloalkyl group having 1 to 4 monocyclic and/or fused rings and having 3 to about
  • a C3-C6 cycloalkyl group means a ring having 3 to 6 carbon atoms.
  • An alkyl group such as a cyclopropyl group, a cyclopentyl group, a cyclohexyl group or the like.
  • Tetraethylene glycol (0.1 mol), tetrahydrofuran (100 mL) and potassium hydroxide (0.4 mol) were added to a three-necked flask, stirred in a water bath, and then epichlorohydrin (0.6 mol) was added dropwise to the reaction system to control the reaction temperature not to exceed The reaction was carried out at 35 ° C overnight at room temperature. After the reaction, the reaction solution was filtered, and the residue was washed with dichloromethane. The crude product is subjected to molecular distillation to obtain pure tetraethylene glycol diglycidyl ether.
  • Mass spectrum MALDI-TOF 1210.0 [M+Na].
  • Mass spectrum MALDI-TOF 901.8, 945.9, 989.9, 1033.9, 1078.0, 1122.0, 1166.1, 1210.1, 1245.1, 1298.2, 1342.2 [M+Na].
  • the cross-linking agent tetraethylene glycol diglycidyl ether (0.2 mol) was dissolved in a NaOH solution, and sodium hyaluronate powder (polymer unit 1 mol) was added, and stirred at 40 ° C to form a crosslinked sodium hyaluronate gel; Add appropriate amount of hydrochloric acid to the gel to adjust the pH to 7.0, then add appropriate amount of PBS buffer gel to swell; then sieve the gel with standard Pharmacopoeia sieve, collect the gel particles, and then wash the crosslinked hyaluronic acid with carbonate buffer at high temperature. Sodium gel, finally, after repeated dialysis, the gel is sieved, compounded, filled, and steam sterilized using a standard Pharmacopoeia sieve to obtain a modified sodium hyaluronate gel for injection.
  • the cross-linking agent, dodecyl diglycidyl diglycidyl ether (0.2 mol) was dissolved in a NaOH solution, added to sodium hyaluronate powder (1 mol of polymer unit), and stirred at 40 ° C to form a crosslinked sodium hyaluronate gel; Add appropriate amount of hydrochloric acid to the gel to adjust the pH to 7.0, then add appropriate amount of PBS buffer gel to swell; then sieve the gel with standard Pharmacopoeia sieve, collect the gel particles, and wash the cross-link transparent with high temperature carbonate buffer. Sodium silicate gel, finally, after repeated dialysis, the gel is sieved, compounded, filled, and steam sterilized using a standard Pharmacopoeia sieve to obtain a modified sodium hyaluronate gel for injection.
  • the cross-linking agent tetraethylene glycol diglycidyl ether (0.2 mol) was dissolved in NaOH solution and added to sodium hyaluronate powder (polymer unit 1 mol), and stirred at 40 ° C to form a crosslinked sodium hyaluronate gel; Next, add appropriate amount of hydrochloric acid to the gel to adjust the pH to 7.0, and then add appropriate amount of PBS buffer gel to swell; then sieve the gel with standard Pharmacopoeia sieve, collect the gel particles, and wash the cross-linking with carbonate buffer at high temperature. Sodium hyaluronate gel. Finally, after repeated dialysis, the gel is sieved, compounded, filled, and steam sterilized using a standard Pharmacopoeia sieve to obtain a modified sodium hyaluronate gel for injection.
  • the cross-linking agent PEG1000 diglycidyl ether (0.2 mol) was dissolved in NaOH solution and added to sodium hyaluronate powder (polymer unit 1 mol), and stirred at 40 ° C to form a crosslinked sodium hyaluronate gel; Add appropriate amount of hydrochloric acid to adjust the pH to 7.0, and then add appropriate amount of PBS buffer gel to swell; then sieve the gel with standard Pharmacopoeia sieve, collect the gel particles, and then wash the crosslinked sodium hyaluronate with carbonate buffer at high temperature. Gel, finally, after repeated dialysis, the gel is sieved, compounded, filled, and steam sterilized using a standard Pharmacopoeia sieve to obtain a modified sodium hyaluronate gel for injection.
  • the cytotoxicity test was obtained by measuring the cell proliferation rate with reference to the "Biological Evaluation of Medical Devices - In Vitro Cytotoxicity Test" standard.
  • the leaching method was used to test the cytotoxicity of L929 cells, and the relative increase rate (RCR) of cells was calculated by enzyme absorbance test. The higher the RCR, the better the biocompatibility and the more toxicity. low.
  • the cytotoxicity test results of the polyglycol glycidyl ether cross-linking agent prepared in Examples 1-4 were as shown in Table 1 using BDDE as a control. Meanwhile, the cross-linking compound (the multi-component shrinkage prepared in Examples 1-3) was used. The half-inhibitory concentration of glyceryl ether and BDDE) was tested.
  • Test group Compound Cell line IC50 ( ⁇ M) 1 Ia L929 127.3 2 Ib L929 2303 3 Ic L929 1883 4 BDDE L929 97.95
  • the status of the cross-linked sodium hyaluronate gel was used as one of the evaluation indicators of product quality.
  • the present invention pushes the push rod at a constant speed of 30 mm/min, and the sample in the syringe is extruded through a 29G needle to obtain a pushing force curve, so that the change in the pushing force during the extrusion of the sample can be seen.
  • the pushing force is small, the sample is easily squeezed out, the pushing force is large, and the sample is not easily extruded; in addition, the height difference of the pushing force is large, indicating that the sample has uneven dispersion or concentration and concentration, which also affects the injection time. The suitability.
  • Table 4 records the change of extrusion thrust during the gel extrusion process.
  • the experimental data of the extrusion thrust of the 12-glycol diglycidyl ether cross-linked sodium hyaluronate gel is shown in Figure 1.
  • the extrusion thrust curve is smoother, indicating that the gel is more Uniform; for the high molecular weight polyethylene glycol PEG1000 diglycidyl ether cross-linked sodium hyaluronate gel extrusion test data shown in Figure 2, the extrusion thrust curve fluctuates greatly, the gel uniformity is poor.

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Abstract

一种注射用多元甘醇环氧衍生物交联的透明质酸钠凝胶及其制备方法，所述的多元甘醇环氧衍生物，其优选为单一分子量的化合物，其分子中存在多个醚键，水溶性好，更易与多糖发生交联反应，同时由于多甘醇重复单元数较易调整，长度较易控制，其作为交联剂制备的透明质酸钠凝胶性能较易调控；所述的交联的透明质酸钠凝胶毒性低、少残留、挤推力小、塑形性好、耐酶性好、体内保留时间长。还公开了一种温和的交联剂灭活技术，将凝胶中未反应的环氧基团在pH＝8-9的碳酸盐缓冲体系中水解反应，可有效降低交联透明质酸钠凝胶的除杂难度，避免现有技术中交联方法使用BDDE的毒性问题。

Description

一种注射用多元甘醇环氧衍生物交联的透明质酸钠凝胶及其制备方法 技术领域
本发明涉及透明质酸钠凝胶技术领域，具体涉及一种多元甘醇环氧衍生物交联的透明质酸钠凝胶及其制备方法。
背景技术
透明质酸(hyalouronic acid，HA)是由葡萄糖醛酸和乙酰氨基葡糖双糖单位重复组成的一种直链高分子粘性多糖。透明质酸是人体内本身具有的内源性物质，具有良好的生物相容性；具有高粘弹性和非牛顿流变学特性；无毒、无免疫源性、无刺激性，具有很高的安全性，以及可通过注射透明质酸酶降解消除等特点，被广泛用作美容中的软组织填充剂。即将透明质酸注射至皮肤内，以增加软组织体积，可达到除皱或塑形等目的。但是由于透明质酸本身以液体形式存在，在体内透明质酸酶和自由基的作用下非常容易被分解，因此难以达到塑性效果且填充效果持续时间短。
为了克服这一缺陷，人们将交联技术运用于透明质酸，即通过交联剂将透明质酸分子连接起来形成相对稳定的网状结构，使其不再容易被分解、填充维持的时间变长。经过交联修饰后透明酸钠凝胶具有粘弹性能好、不溶于水、机械强度高、降解时间长的优点，同时，交联程度的不同带来了不同的物理特性，使其能够更好地满足美容填充需求。
目前市售的透明质酸钠凝胶产品中使用的交联剂主要有：BDDE(丁二醇二缩水甘油醚)和DVS(二乙烯基砜)。如法国高德美公司(Galderma)Restylane瑞蓝系列、美国艾尔健公司(Allergan)Juvederm乔雅登系列、法国维法西公司(Laboratoires Vivacy)Stylage系列、台湾科妍生物海德密丝(Hya-Dermis)系列均采用BDDE交联制备透明质酸钠凝胶，而美国基因酶 公司(Genzyme)Hylaform系列、德国阿杜德姆公司(Adoderm)Varioderm系列、瑞士泰奥赛恩公司(Teoxane)Teosyal系列均采用DVS作为交联剂制备透明质酸钠凝胶。两种交联剂制备的凝胶具有截然不同的物理性能：DVS交联产品质地硬，交联活性高；BDDE交联产品质地柔软，反应较为温和，膨胀率大。但值得注意的是，上述交联剂均具有毒性或潜在致癌隐患，且交联后的空间网状结构对未反应的交联剂具有包裹作用，去除难度较大。
为克服上述缺陷，本发明设计并合成新型低毒、高反应活性、水溶性的多元甘醇环氧衍生物交联剂
Figure PCTCN2017118542-appb-000001
(n＝4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24，…48…200)，并用其作为交联剂制备新型交联透明质酸钠凝胶，该凝胶柔软、粘弹性优良、毒性低、产品均一性好；本发明还涉及该交联透明质酸钠凝胶的具体制备方法。
发明内容
为了克服现有技术中制备的透明质酸钠凝胶存在不能兼具凝胶抗降解性强、交联剂毒性低、可注射性好等优点的问题，本发明首先设计并合成了一种新型、低毒、高反应活性、水溶性的交联剂，然后利用该交联剂制备了一种低毒、少残留、挤推力小、塑形性好、耐酶性好、体内保留时间长的注射用修饰透明质酸钠凝胶。具体而言，为克服上述现有技术的缺陷，本发明设计并合成一种新型、低毒、高反应活性、高水溶性的多元甘醇环氧衍生物
Figure PCTCN2017118542-appb-000002
(n＝4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24，…，48，…，200)，并用其作为交联剂制备新型交联透明质酸钠凝胶，该凝胶柔软、粘弹性优良、毒性低、产品均一性好；本发明还涉及该交联透明质酸钠凝胶的具体制备方法。优选地，本发明所述的多元甘醇或多元甘醇环氧衍生物为单一分子量的化合物。
一方面，本发明提供了一种新型、低毒、高反应活性、超亲水性、低毒性的多元甘醇环氧衍生物，其具有通式I的结构：
Figure PCTCN2017118542-appb-000003
其中，
n为4-200的整数；
EPOX为
Figure PCTCN2017118542-appb-000004
其中Y选自：-(CR 1R 2) m-、-(CH 2) mNH-、-NHCO(CH 2) m-、-(CH 2) mCONH-和-CO(CH 2) m-中的一种或多种的组合，
m为1-10的整数；
R 1和R 2独立地选自：H、烷基、环烷基、环烷基烷基、烯基、芳基、芳烷基、杂环基、杂环基烷基、-COR 3、-C(O)OR 3、-C(O)NR 3R 4、-CH＝NR 3、-CN、-OR 3、-OC(O)R 3、-S(O) t-R 3、-NR 3R 4、-NR 3C(O)R 4和卤素；
t选自0、1和2；
R 3和R 4彼此独立地选自氢、烷基、环烷基、烯基、芳基、杂环基和卤素。
优选地，所述n为10-100的整数；更优选地，n为10-50的整数，进一步优选为12-30的整数；或者，优选为10-100的整数，更优选为10-30的整数，最优选为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30。
优选地，所述Y为-(CR 1R 2) m-。
优选地，所述m为1-5的整数，如1、2、3、4或5。
在本发明的一个优选实施例中，所述m＝1。
优选地，所述R 1和R 2独立地选自：H、C1-C6的烷基、C3-C6的环烷基、-OR 3、-NR 3R 4和卤素。
优选地，所述R 3和R 4独立地选自：氢和C1-C3的烷基。
在本发明的一个优选实施例中，所述R 1和R 2均为H。
在本发明的一个更优选实施例中，所述Y为-CH 2-。
本发明所述的多元甘醇环氧衍生物优选为单一分子量的化合物。
优选地，本发明所述的多元甘醇环氧衍生物具有如下结构：
Figure PCTCN2017118542-appb-000005
其中，n为4-200的整数，优选为10-100的整数，更优选为10-50的整数，进一步优选为10-30的整数(具体如10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30)。
本发明另一方面提供一种多元甘醇环氧衍生物的制备方法，其包括在溶剂中，使用催化剂催化EPOX-X与多元甘醇反应的步骤；
所述的多元甘醇的结构为
Figure PCTCN2017118542-appb-000006
n为4-200的整数；
所述的EPOX-X中，-X为离去基团；
EPOX为
Figure PCTCN2017118542-appb-000007
其中Y选自：-(CR 1R 2) m-、-(CH 2) mNH-、-NHCO(CH 2) m-、-(CH 2) mCONH-和-CO(CH 2) m-中的一种或多种的组合；
m为1-10的整数；
R 1和R 2独立地选自：H、烷基、环烷基、环烷基烷基、烯基、芳基、芳烷基、杂环基、杂环基烷基、-COR 3、-C(O)OR 3、-C(O)NR 3R 4、-CH＝NR 3、-CN、-OR 3、-OC(O)R 3、-S(O) t-R 3、-NR 3R 4、-NR 3C(O)R 4和卤素；
t选自0、1和2；
R 3和R 4彼此独立地选自氢、烷基、环烷基、烯基、芳基、杂环基和卤素。
优选地，所述n为10-100的整数；更优选地，n为10-50的整数，进一步优选为12-30的整数，或者优选为10-100的整数，更优选为10-30的整数，最优选为10、11、12、13、14、15、16、17、18、19、20、21、22、23、 24、25、26、27、28、29或30。
优选地，所述-X选自：-F、-Cl、-Br、-I、
Figure PCTCN2017118542-appb-000008
(-OMs)、
Figure PCTCN2017118542-appb-000009
(-OTs)；更优选地，-X为-Cl或-Br；
在本发明的一个优选实施例中，所述Y为-(CR 1R 2) m-。
优选地，所述m为1-5的整数，如1、2、3、4或5。
在本发明的一个优选实施例中，所述m＝1。
优选地，所述R 1和R 2独立地选自：H、C1-C6的烷基、C3-C6的环烷基、-OR 3、-NR 3R 4和卤素。
优选地，所述R 3和R 4独立地选自：氢和C1-C3的烷基。
在本发明的一个优选实施例中，所述R 1和R 2均为H。
在本发明的一个更优选实施例中，所述Y为-CH 2-。
优选地，所述的催化剂为碱催化剂，包括有机碱或无机碱，优选自但不限于：吡啶、三乙胺、碳酸铯、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、氢氧化钾、醇钠和醇钾中的一种或多种。
优选地，所述的溶剂包括但不限于：1,4-二氧六环、四氢呋喃、甲苯、丙酮、乙酸乙酯、乙腈、N,N-二甲基甲酰胺、二甲基亚砜和水中的一种或多种。
优选地，所述多元甘醇中单羟基与EPOX-X的摩尔比为1：2-10(具体如1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9或1:10)。
优选地，所述多元甘醇环氧衍生物的制备方法还包括纯化步骤；更优选地，所述的纯化步骤选自：旋蒸、洗涤、萃取、分子蒸馏和柱分离中的一种或多种的组合。
在本发明的一个优选实施例中，所述制备方法包括如下具体步骤：向反应 容器中加入多元甘醇、溶剂和催化剂，搅拌，向上述混合物中滴加卤化或磺酸酯化的环氧丙烷，控制反应温度不超过35℃，反应完成后过滤，洗涤滤渣，并收集滤液并纯化，得到多元甘醇环氧衍生物。
上述反应通式如下：
Figure PCTCN2017118542-appb-000010
采用上述制备方法合成的多元甘醇环氧衍生物纯度较高，其纯度HPLC检测大于99％。该低毒性、高反应活性、超亲水性多元甘醇环氧衍生物可作为交联剂用于天然高分子如天然多糖和蛋白质、合成高分子如聚乙二醇和聚乙烯醇等高分子聚合物的交联。
本发明另一方面提供一种交联剂，其包含本发明上述多元甘醇环氧衍生物。
本发明还提供一种上述多元甘醇环氧衍生物交联的高分子聚合物。
优选地，所述的高分子聚合物可为天然高分子或合成高分子，更优选为天然高分子，如天然多糖或蛋白质等。
优选地，所述的天然高分子选自：淀粉、壳聚糖、几丁质、纤维素、果胶、明胶、阿拉伯胶、干酪素、甲壳素、丝素蛋白、白蛋白、酪蛋白、透明质酸、糖原、丝胶、结冷胶、黄原胶、瓜尔胶、葡聚糖、壳寡糖、菊糖、葡苷聚糖、甘露寡糖、甘露聚糖、半乳聚糖、硫酸软骨素、硫酸皮肤素、肝素、硫酸乙酰肝素、琼脂，及其衍生物等中的一种或多种；
优选地，所述的合成高分子选自：聚乙烯吡咯烷酮、聚乙二醇、聚环氧乙烷、聚乙烯醇、聚醋酸乙烯脂、聚乳酸、聚乙醇酸、聚丙烯酸、聚丙烯酰胺、聚四氢呋喃、聚环氧丁烷、聚氧杂环丁烷、聚马来酸酐、聚羟乙基丙烯酸甲酯、聚丙二醇、聚己内酯、及其衍生物等中的一种或多种；
在本发明的一个优选实施例中，所述的天然高分子为透明质酸衍生物，更优选为透明质酸盐，所述的透明质酸盐选自：透明质酸钠、透明质酸钾、透明质酸钙、透明质酸镁、透明质酸锌、透明质酸钴和透明质酸四丁铵中的一种或 多种；更优选为透明质酸钠。
在本发明的一个优选实施例中，所述的交联物为上述多元甘醇环氧衍生物交联的透明质酸钠。
优选地，所述的透明质酸钠的分子量为5万-300万道尔顿(具体如5万、10万、50万、100万、150万、200万、250万或300万道尔顿)，更优选为30万到150万道尔顿。
优选地，所述的多元甘醇环氧衍生物与透明质酸钠中聚合物单元的摩尔比为0.01-1:1(具体如0.01:1、0.1:1、0.2:1、0.3:1、0.4:1、0.5:1或1.0:1)。
本发明在提供了一种新型低毒、高反应活性、水溶性的交联剂的基础上，本发明还提供了一种低毒、少残留、挤推力小、塑形性好、耐酶性好、体内保留时间长的注射用修饰透明质酸钠凝胶及其制备方法。
一种多元甘醇环氧衍生物交联透明质酸钠的制备方法，其包括透明质酸钠与本发明上述多元甘醇环氧衍生物在碱性条件下交联反应的步骤。
在本发明的一个实施例中，所述的交联反应步骤包括：将多元甘醇环氧衍生物溶于碱性溶液中，加入透明质酸钠粉末中，35-45℃，优选为40℃搅拌反应形成交联透明质酸钠。
优选地，所述的多元甘醇环氧衍生物与透明质酸钠中聚合物单元的摩尔比为0.01-1:1(具体如0.01:1、0.1:1、0.2:1、0.3:1、0.4:1、0.5:1或1.0:1)。
优选地，所述碱性溶液选自：氢氧化钠、氢氧化钾和碳酸钠溶液；在本发明的一个优选实施例中，所述碱性溶液为质量浓度为0.1-10％的NaOH水溶液。
优选地，所述的透明质酸钠粉末与碱性溶液的质量比为1:5-30(具体如1:5、1:10、1:15、1:20、1:25或1:30)。
优选地，所述的透明质酸钠的分子量为5万-300万道尔顿(具体如5万、10万、50万、100万、150万、200万、250万或300万道尔顿)，更优选为30万到150万道尔顿。
优选地，所述多元甘醇环氧衍生物交联透明质酸钠的制备方法还包括交联 剂去除步骤，包括依次将上述交联反应产物pH值调中性、溶胀、用碳酸盐缓冲液洗涤的步骤。
在本发明的一个实施例中，所述的交联剂去除步骤包括：加酸将上述交联透明质酸钠调节为中性；加入适量PBS缓冲液使凝胶溶胀；用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，改变凝胶中未交联小分子的结构，降低毒性。
优选地，所述酸为盐酸，更优选为浓度为0.1N-2N盐酸；
优选地，所述的碳酸盐缓冲液的pH为8-9，可为Na 2CO 3/NaHCO 3、Na 2CO 3/KHCO 3、K 2CO 3/NaHCO 3、K 2CO 3/KHCO 3，碳酸盐的质量浓度为0.02-3％。
更优选地，所述的交联剂去除步骤还包括透析步骤。
本发明还提供一种注射用交联透明质酸钠凝胶，其由上述方法制备得到的交联透明质酸钠凝胶灭菌得到。
优选地，所述的灭菌条件为121℃-125℃蒸汽中灭菌15-25分钟。
在本发明的一个优选实施例中，上述注射用交联透明质酸钠凝胶的制备方法包括如下具体步骤：
步骤一：将多元甘醇环氧衍生物溶于碱性溶液中，加入透明质酸钠粉末中，35-45℃，优选为40℃搅拌反应形成交联透明质酸钠凝胶；
步骤二：加酸将凝胶调节为中性；
步骤三：加入适量PBS缓冲液使凝胶溶胀；
步骤四：用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，改变凝胶中未交联小分子的结构，降低毒性；
步骤五：反复透析多次，去除凝胶中残留的小分子交联剂；
步骤六：用标准筛筛分；
步骤七：将步骤六中收集的凝胶颗粒放入配制瓶中，加入流动相，灌装于事先灭菌的一次性注射器中，在121℃-125℃蒸汽中灭菌15-25分钟，即可得到注射用修饰透明质酸钠凝胶。
本发明制得的交联透明质酸钠结构示意图如下：
Figure PCTCN2017118542-appb-000011
其中，J、K、n为正整数，n为4-200的整数，J和K可根据透明质酸钠的分子量确定。本发明还提供一种凝胶，包括上述多元甘醇环氧衍生物交联的透明质酸钠。
本发明还提供一种上述交联透明质酸钠在制备用于药物、医疗美容和化妆品用途的产品中的应用。
优选地，所述的应用为上述交联透明质酸钠在制备用于医疗美容用途的产品中的应用。
优选地，所述的医疗美容用途的产品为软组织填充剂。
本发明还提供一种软组织填充剂，其包括本发明上述交联透明质酸钠。
本发明采用新型交联剂制备交联透明质酸钠的有益效果包括：
本发明制备的交联透明质酸钠凝胶中所用的交联剂为多元甘醇环氧衍生物，首先，由于其分子中存在多个醚键，其水溶性较传统的交联剂BDDE好，同等摩尔数、相同反应条件下，多元甘醇环氧衍生物比BDDE更易与多糖发生交联反应生成交联透明质酸钠凝胶；其次，由于多元甘醇环氧衍生物交联剂不同于高分子的聚乙二醇，其为单一分子量的化合物，采用多元甘醇环氧衍生物交联制备透明质酸钠凝胶的不同批次稳定性均优于聚乙二醇环氧衍生物；再次，由于短链状多元甘醇环氧衍生物的分子链长较BDDE长，其可能与链状透明质酸分子缠绕，增强凝胶的柔软性、粘弹性和稳定性；然后，由于多甘醇重复单 元数较易调整，长度较易控制，其作为交联剂制备的凝胶性能较易调控；最后，由于多元甘醇环氧衍生物毒性较BDDE大幅降低，多元甘醇环氧衍生物交联的透明质酸钠凝胶作为人体软组织填充剂安全性增强，避免了原交联剂BDDE毒性可能引起的不良反应。
本发明整合了交联透明质酸钠成胶除杂工艺，可制备出低毒、少残留、挤推力小、塑形性好、耐酶性好、体内保留时间长的注射用修饰透明质酸钠凝胶；尤其是本发明开发了一种温和的交联剂灭活技术，将凝胶中未反应的环氧基团在pH＝8-9的碳酸盐缓冲体系中水解反应，可有效降低交联透明质酸钠凝胶的除杂难度，避免原交联方法中使用BDDE的毒性问题。
附图说明
图1所示为本发明实施例10提供的十二甘醇二缩水甘油醚交联透明质酸钠凝胶的挤推力曲线。
图2所示为本发明实施例10提供的聚乙二醇PEG1000二缩水甘油醚交联透明质酸钠凝胶挤推力曲线。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义，如：“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，本发明中C1-C6的烷基指含有1-6个碳原子的烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、正己基等，优选C1-C3的烷基(如甲基、乙基、正丙基和异丙基)；“环烷基”指的是脂环烃，典型的环烷基含1至4个单环和/或稠环、含3至约18个碳原子，本发明中C3-C6的环烷基是指含有3-6个碳原子的环烷基，如环丙基、环戊基、环己基等。
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行 清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：合成四甘醇二缩水甘油醚(Ia)
合成如下结构的四甘醇二缩水甘油醚：
Figure PCTCN2017118542-appb-000012
向三口瓶中加入四甘醇(0.1mol)、四氢呋喃(100mL)和氢氧化钾(0.4mol)，水浴搅拌，然后向反应体系中滴加环氧氯丙烷(0.6mol)，控制反应温度不超过35℃，室温反应过夜。反应完后过滤反应液，并用二氯甲烷洗涤滤渣，然后收集滤液，旋蒸除去二氯甲烷得到粗品。粗品经分子蒸馏后得到纯品四甘醇二缩水甘油醚。
1H-NMR(DMSO-d6)：2.52-2.55(m，2H)，2.70-2.73(m，2H)，3.07-3.11(m，2H)，3.22-3.28(m，2H)，3.52-3.56(m，16H)，3.68-3.73(m，2H)；
HPLC检测：产品纯度为99.6％；
质谱ESI：329.6[M+Na]。
实施例2：合成十二甘醇二缩水甘油醚(Ib)
合成如下结构的十二甘醇二缩水甘油醚：
Figure PCTCN2017118542-appb-000013
向三口瓶中加入十二甘醇(0.1mol)、四氢呋喃(100mL)和氢氧化钾(0.4mol)，水浴搅拌，然后向反应体系中滴加环氧氯丙烷(0.6mol)，控制反应温度不超过35℃，室温反应过夜。反应完后过滤反应液，并用二氯甲烷洗涤滤渣，然后收集滤液，旋蒸除去二氯甲烷得到粗品。粗品经分子蒸馏后得到纯品十二甘醇二缩水甘油醚。
1H-NMR(DMSO-d6)：2.51-2.55(m，2H)，2.70-2.73(m，2H)，3.07-3.11(m，2H)，3.22-3.29(m，2H)，3.51-3.57(m，48H)，3.68-3.73(m，2H)；
HPLC检测：产品纯度为99.3％；
质谱ESI：681.9[M+Na]。
实施例3：合成二十四甘醇二缩水甘油醚(Ic)
合成如下结构的二十四甘醇二缩水甘油醚：
Figure PCTCN2017118542-appb-000014
向三口瓶中加入二十四甘醇(0.1mol)、四氢呋喃(100mL)和氢氧化钾(0.4mol)，水浴搅拌，然后向反应体系中滴加环氧氯丙烷(0.6mol)，控制反应温度不超过35℃，室温反应过夜。反应完后过滤反应液，并用二氯甲烷洗涤滤渣，然后收集滤液，旋蒸除去二氯甲烷得到粗品。粗品经柱分离得到纯品二十四甘醇二缩水甘油醚。
1H-NMR(DMSO-d6)：2.51-2.55(m，2H)，2.70-2.73(m，2H)，3.07-3.11(m，2H)，3.22-3.28(m，2H)，3.51-3.56(m，96H)，3.68-3.73(m，2H)；
HPLC检测：产品纯度为99.2％；
质谱MALDI-TOF:1210.0[M+Na]。
实施例4：合成PEG1000二缩水甘油醚(Id)
合成如下结构的PEG1000二缩水甘油醚：
Figure PCTCN2017118542-appb-000015
向三口瓶中加入二HO-PEG1000-OH(0.1mol)、四氢呋喃(100mL)和氢氧化钾(0.4mol)，水浴搅拌，然后向反应体系中滴加环氧氯丙烷(0.6mol)，控制反应温度不超过35℃，室温反应过夜。反应完后过滤反应液，并用二氯甲烷洗涤滤渣，然后收集滤液，旋蒸除去二氯甲烷得到粗品。粗品经柱分离得到纯品 PEG1000二缩水甘油醚。
1H-NMR(DMSO-d6)：2.52-2.55(m，2H)，2.70-2.73(m，2H)，3.06-3.11(m，2H)，3.23-3.29(m，2H)，3.45-3.69(m，-(CH 2CH 2O)-中的氢)，3.72-3.75(m，2H)；
质谱MALDI-TOF:901.8,945.9,989.9,1033.9,1078.0,1122.0,1166.1,1210.1,1245.1,1298.2,1342.2[M+Na]。
实施例5：四甘醇二缩水甘油醚交联透明质酸钠凝胶(IIa)
将交联剂四甘醇二缩水甘油醚(0.2mol)溶于NaOH溶液中加入透明质酸钠粉末(聚合物单元1mol)，40℃搅拌反应形成交联透明质酸钠凝胶；其次向凝胶中加入适量盐酸调节pH为7.0，再加入适量PBS缓冲液凝胶溶胀；然后用标准药典筛将凝胶过筛，收集凝胶颗粒，再用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，最后，凝胶经反复透析后，用标准药典筛筛分、复配、灌装、蒸汽灭菌后即得注射用修饰透明质酸钠凝胶。
1H-NMR(D 2O，NaOD)：1.80(s，3H)，3.26(s，3H)，3.41(s，H)，3.56[s，(H EG4+5H)]，3.79(s，1H)，4.33-4.36(d，2H)。
实施例6：十二甘醇二缩水甘油醚交联透明质酸钠凝胶(IIb)
将交联剂十二甘醇二缩水甘油醚(0.2mol)溶于NaOH溶液中加入透明质酸钠粉末中(聚合物单元1mol)，40℃搅拌反应形成交联透明质酸钠凝胶；其次向凝胶中加入适量盐酸调节pH为7.0，再加入适量PBS缓冲液凝胶溶胀；然后用标准药典筛将凝胶过筛，收集凝胶颗粒，再用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，最后，凝胶经反复透析后，用标准药典筛筛分、复配、灌装、蒸汽灭菌后即得注射用修饰透明质酸钠凝胶。
1H-NMR(D 2O，NaOD)：1.81(s，3H)，3.28(s，3H)，3.40(s，H)，3.56[s，(H EG12+5H)]，3.78(s，1H)，4.32-4.35(d，2H)。
实施例7：二十四甘醇二缩水甘油醚交联透明质酸钠凝胶(IIc)
将交联剂二十四甘醇二缩水甘油醚(0.2mol)溶于NaOH溶液中加入透明质酸钠粉末中(聚合物单元1mol)，40℃搅拌反应形成交联透明质酸钠凝胶；其 次向凝胶中加入适量盐酸调节pH为7.0，再加入适量PBS缓冲液凝胶溶胀；然后用标准药典筛将凝胶过筛，收集凝胶颗粒，再用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，最后，凝胶经反复透析后，用标准药典筛筛分、复配、灌装、蒸汽灭菌后即得注射用修饰透明质酸钠凝胶。
1H-NMR(D 2O，NaOD)：1.80(s，3H)，3.27(s，3H)，3.40(s，H)，3.56[s，(H EG24+5H)]，3.79(s，1H)，4.32-4.36(d，2H)；
实施例8：PEG1000二缩水甘油醚交联透明质酸钠凝胶(IId)
将交联剂PEG1000二缩水甘油醚(0.2mol)溶于NaOH溶液中加入透明质酸钠粉末中(聚合物单元1mol)，40℃搅拌反应形成交联透明质酸钠凝胶；其次向凝胶中加入适量盐酸调节pH为7.0，再加入适量PBS缓冲液凝胶溶胀；然后用标准药典筛将凝胶过筛，收集凝胶颗粒，再用碳酸盐缓冲液高温洗涤交联透明质酸钠凝胶，最后，凝胶经反复透析后，用标准药典筛筛分、复配、灌装、蒸汽灭菌后即得注射用修饰透明质酸钠凝胶。
1H-NMR(D 2O，NaOD)：1.80(s，3H)，3.26(s，3H)，3.41(s，H)，3.55[s，(H PEG1000+5H)]，3.80(s，1H)，4.32-4.35(d，2H)。
实施例9：细胞毒性实验
细胞毒性实验是参照《医疗器械的生物学评价—体外细胞毒性试验》标准，检测细胞增殖率得到的。本实验采用浸提的方法，采用L929细胞进行细胞毒性测试，利用酶联免疫测试仪测定吸光度值来计算细胞的相对增值率(RCR)，RCR越高，表明生物相容性越好，毒性越低。以BDDE为对照，实施例1-4制备的多元甘醇缩水甘油醚交联剂的细胞毒性检测结果如表1；同时，采用L929细胞对交联剂化合物(实施例1-3制备的多元缩水甘油醚和BDDE)的半数抑制浓度进行了测试，实验结果如表2所示；另外，以BDDE交联透明质酸钠凝胶(BDDE-HA)为对照，实施例5-8制备的多元甘醇缩水甘油醚交联透明质酸钠凝胶的细胞毒性检测结果如表3所示。
表1 多元甘醇缩水甘油醚交联剂的细胞毒性检测结果
供试组 Ia Ib Ic Id BDDE
RCR 62％ 89％ 89％ 87％ 30％
表2 多元缩水甘油醚和BDDE半数抑制浓度(IC50)检测结果
供试组 化合物 细胞系 IC50(μM)
1 Ia L929 127.3
2 Ib L929 2303
3 Ic L929 1883
4 BDDE L929 97.95
表3 多元甘醇缩水甘油醚交联透明质酸钠凝胶的细胞毒性检测结果
供试组 IIa IIb IIc IId BDDE-HA
RCR 88％ 91％ 92％ 89％ 85％
从表1和表2可以看出，多元甘醇缩水甘油醚交联剂的生物相容性明显优于BDDE，从表3中可以看出，多元甘醇缩水甘油醚交联透明质酸钠凝胶的生物相容性也较优。
实施例10：挤推力实验
利用万能力学测试仪，通过挤推力实验，了解交联透明质酸钠凝胶实际使用时的状况，作为产品品质的评估指标之一。本发明以30mm/min的恒定速度推动推进杆，注射器中样品经由29G针头被挤出，得到推挤力曲线，从而可以看出样品挤出过程中的推挤力的变化。推挤力小，样品容易被挤出，推挤力大，样品不容易被挤出；另外，推挤力的高低落差大，表明样品有分散不均或聚集浓缩现象，这也会影响注射时的适手性。表4记录凝胶挤推过程中挤推力的变化情况，十二甘醇二缩水甘油醚交联透明质酸钠凝胶的挤推力实验数据见图1，挤推力曲线较为平滑，说明凝胶较为均一；对于高分子量的聚乙二醇PEG1000二缩水甘油醚交联透明质酸钠凝胶挤推力实验数据 见图2，挤推力曲线波动较大，凝胶均一性较差。
表4 元甘醇缩水甘油醚交联透明质酸钠凝胶的挤推力检测结果
供试组 IIa IIb IIc IId BDDE-HA
挤推力 7-8N 7-8N 8-9N 5-9N 7-8N
实施例11：体外酶解稳定性实验
取交联的透明质酸钠凝胶0.5g，加入300U/mL的透明质酸酶溶液2mL，37℃保温降解40小时，加PBS至5mL，取1mL加无水乙醇4mL，10000r/min离心15min，取上清液2mL用PBS定容至5mL，作为甲液；另取交联的透明质酸钠凝胶0.5g，加0.5mol/L硫酸溶液10mL，沸水浴水解15min，加水稀释至100mL，作为乙液。分别取甲液和乙液1mL，用改良咔唑显色法测葡萄糖醛酸含量。凝胶的体外抗酶降解性用系数R表示，R＝1-0.625A/B，其中A为甲液的葡萄糖醛酸含量，B为乙液的葡萄糖醛酸含量。R值越高，说明其体外抗酶降解性能越好，交联的凝胶越稳定。检测结果如表5所示。
表5 多元甘醇缩水甘油醚交联透明质酸钠凝胶的体外酶解稳定性检测结果
供试组 IIa IIb IIc IId BDDE-HA
抗酶降解系数 78％ 82％ 81％ 69％ 71％
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。

Claims (14)

  1. 一种多元甘醇环氧衍生物，其具有通式I的结构：
    Figure PCTCN2017118542-appb-100001
    其中，
    所述的多元甘醇环氧衍生物为单一分子量的化合物，
    n为4-200的整数；
    EPOX为
    Figure PCTCN2017118542-appb-100002
    其中Y选自：-(CR 1R 2) m-、-(CH 2) mNH-、-NHCO(CH 2) m-、-(CH 2) mCONH-和-CO(CH 2) m-中的一种或多种的组合；
    m为1-10的整数；
    R 1和R 2独立地选自：H、烷基、环烷基、环烷基烷基、烯基、芳基、芳烷基、杂环基、杂环基烷基、-COR 3、-C(O)OR 3、-C(O)NR 3R 4、-CH＝NR 3、-CN、-OR 3、-OC(O)R 3、-S(O) t-R 3、-NR 3R 4、-NR 3C(O)R 4和卤素；
    t选自0、1和2；
    R 3和R 4独立地选自氢、烷基、环烷基、烯基、芳基、杂环基和卤素。
  2. 如权利要求1所述的多元甘醇环氧衍生物，其特征在于，所述的n为10-100的整数，优选为10-50的整数，更优选为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30；和/或，
    所述Y为-(CR 1R 2) m-，
    优选地，所述R 1和R 2独立地选自：H、C1-C6的烷基、C3-C6的环烷基、-OR 3、-NR 3R 4和卤素；所述R 3和R 4独立地选自：氢和C1-C3的烷基。
  3. 如权利要求1所述的多元甘醇环氧衍生物，其特征在于，所述的m＝1；和/或，所述R 1和R 2均为H。
  4. 如权利要求1所述的多元甘醇环氧衍生物，其特征在于，所述的多元甘醇环氧衍生物具有如下结构：
    Figure PCTCN2017118542-appb-100003
    其中，n为4-200的整数，优选为10-100的整数，更优选为10-50的整数，进一步优选地，n为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30。
  5. 一种多元甘醇环氧衍生物的制备方法，其包括：在溶剂中，使用催化剂催化EPOX-X与多元甘醇反应的步骤；
    所述的多元甘醇的结构为
    Figure PCTCN2017118542-appb-100004
    n为4-200的整数；
    所述的EPOX-X中，-X为离去基团；
    EPOX为
    Figure PCTCN2017118542-appb-100005
    其中Y选自：-(CR 1R 2) m-、-(CH 2) mNH-、-NHCO(CH 2) m-、-(CH 2) mCONH-和-CO(CH 2) m-中的一种或多种的组合；
    m为1-10的整数；
    R 1和R 2独立地选自：H、烷基、环烷基、环烷基烷基、烯基、芳基、芳烷基、杂环基、杂环基烷基、-COR 3、-C(O)OR 3、-C(O)NR 3R 4、-CH＝NR 3、-CN、-OR 3、-OC(O)R 3、-S(O) t-R 3、-NR 3R 4、-NR 3C(O)R 4和卤素；
    t选自0、1和2；
    R 3和R 4彼此独立地选自氢、烷基、环烷基、烯基、芳基、杂环基和卤素。
  6. 如权利要求5的制备方法，其特征在于，所述的n为6-100的整数，优选为10-100的整数，更优选为10-50的整数，最优选为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30；和/或，
    所述的-X选自：-F、-Cl、-Br、-I、
    Figure PCTCN2017118542-appb-100006
    和/或，
    所述的m＝1；和/或，
    所述的R 1和R 2均为H；和/或，
    所述的催化剂为碱催化剂，包括有机碱或无机碱。
  7. 如权利要求6的制备方法，其特征在于，所述的催化剂选自：吡啶、三乙胺、碳酸铯、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾、氢氧化钠、氢氧化钾、醇钠和醇钾中的一种或多种；和/或，
    所述的溶剂选自：1,4-二氧六环、四氢呋喃、甲苯、丙酮、乙酸乙酯、乙腈、N,N-二甲基甲酰胺、二甲基亚砜和水中的一种或多种；和/或，
    多元甘醇中单羟基与EPOX-X的摩尔比为1:2-10。
  8. 一种如权利要求1-4任一项所述的多元甘醇环氧衍生物交联的高分子聚合物，其中所述的高分子聚合物为天然高分子或合成高分子，优选为天然高分子；
    优选地，所述的天然高分子选自：淀粉、壳聚糖、几丁质、纤维素、果胶、明胶、阿拉伯胶、干酪素、甲壳素、丝素蛋白、白蛋白、酪蛋白、透明质酸、糖原、丝胶、结冷胶、黄原胶、瓜尔胶、葡聚糖、壳寡糖、菊糖、葡苷聚糖、甘露寡糖、甘露聚糖、半乳聚糖、硫酸软骨素、硫酸皮肤素、肝素、硫酸乙酰肝素、琼脂，及其衍生物中的一种或多种。
  9. 如权利要求8所述的多元甘醇环氧衍生物交联的高分子聚合物，其特征在于，所述的天然高分子为透明质酸盐，所述的透明质酸盐选自：透明质酸钠、透明质酸钾、透明质酸钙、透明质酸镁、透明质酸锌、透明质酸钴和透明质酸四丁铵中的一种或多种。
  10. 如权利要求9所述的多元甘醇环氧衍生物交联的高分子聚合物，其特 征在于，所述的多元甘醇环氧衍生物交联的高分子聚合物为多元甘醇环氧衍生物交联的透明质酸钠；优选地，所述交联的透明质酸钠为凝胶。
  11. 一种如权利要求10所述的交联的透明质酸钠在制备用于药物、医疗美容和化妆品用途的产品中的应用；优选地，所述的应用为上述交联透明质酸钠在制备用于医疗美容用途的产品中的应用；更优选地，所述的医疗美容用途的产品为软组织填充剂。
  12. 一种如权利要求10所述的多元甘醇环氧衍生物交联透明质酸钠的制备方法，其包括透明质酸钠与如权利要求1-4任一项所述的多元甘醇环氧衍生物在碱性条件下交联反应的步骤。
  13. 如权利要求12所述的制备方法，其特征在于，所述的制备方法还包括交联剂去除步骤，包括将上述交联反应产物溶胀后用碳酸盐缓冲液洗涤的步骤。
  14. 如权利要求13所述的制备方法，其特征在于，所述多元甘醇环氧衍生物与透明质酸钠中聚合物单元的摩尔比为0.01-1:1；和/或，
    所述的透明质酸钠的分子量为5万-300万道尔顿；和/或，
    所述的碳酸盐缓冲液pH为8-9。
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