WO2008071058A1 - Dérivé macromoléculaire modifié par un groupe mercapto et matériau réticulé - Google Patents

Dérivé macromoléculaire modifié par un groupe mercapto et matériau réticulé Download PDF

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WO2008071058A1
WO2008071058A1 PCT/CN2007/002864 CN2007002864W WO2008071058A1 WO 2008071058 A1 WO2008071058 A1 WO 2008071058A1 CN 2007002864 W CN2007002864 W CN 2007002864W WO 2008071058 A1 WO2008071058 A1 WO 2008071058A1
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polymer
solution
derivative
group
acid
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PCT/CN2007/002864
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English (en)
French (fr)
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Chan Song
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Bioregen Biomedical (Changzhou) Co., Ltd
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Priority to AU2007332031A priority Critical patent/AU2007332031A1/en
Priority to CA2672019A priority patent/CA2672019C/en
Priority to JP2009540576A priority patent/JP5211064B2/ja
Priority to EP07816478.7A priority patent/EP2103631B1/en
Priority to US12/518,473 priority patent/US8124757B2/en
Publication of WO2008071058A1 publication Critical patent/WO2008071058A1/zh

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    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B43/00—Formation or introduction of functional groups containing nitrogen
    • C07B43/06—Formation or introduction of functional groups containing nitrogen of amide groups
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/41—Y being a hydrogen or an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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    • C08B11/00—Preparation of cellulose ethers
    • C08B11/02—Alkyl or cycloalkyl ethers
    • C08B11/04—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals
    • C08B11/14—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals with nitrogen-containing groups
    • C08B11/15—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals with nitrogen-containing groups with carbamoyl groups, i.e. -CO-NH2
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    • 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
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    • 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
    • C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
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    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins

Definitions

  • the present invention relates to a compound, in particular to a polymer thiolated modified derivative; in addition, the present invention also relates to a corresponding disulfide crosslinked material and a sulfhydryl reactive crosslinker crosslinked material of the polymer thiolated modified derivative. .
  • Polymer thiolated modified derivatives have many important biomedical uses, such as chemically active modification of various small molecule drugs and polypeptide protein drugs, preparation of various crosslinked polymer materials, and the like. These novel materials can be used as a cell growth substrate, a wound repair regeneration matrix, a drug sustained release carrier, a wound dressing, an in situ embedded cell matrix, and the like, and have important applications in the biomedical field.
  • thiolated modified macromolecular derivatives there are few kinds of such thiolated modified macromolecular derivatives, and only the polymer thiolated modified derivatives disclosed by Shu et al. in Biomacromolecules, 3, 1304, 2002 have a good application prospect.
  • Such polymeric thiolated modified derivatives have the following structure:
  • P is a polymer compound residue.
  • the structure and properties of the polymer thiolated modified derivatives of these two structures are single and cannot effectively satisfy various biomedical applications.
  • the high molecular thiolated modified derivatives of these two structures have short side chains, which restrict the collision probability of the sulfhydryl groups with other chemical functional groups during further chemical modification and crosslinking, and the chemical reaction performance is not good. Therefore, it has important significance to prepare a polymer thiolated derivative having an adjustable side chain molecular structure and chemical properties.
  • One of the technical problems to be solved by the present invention is to provide a new class of polymer thiolated modified derivatives having important biomedical uses, which have adjustable molecular structure and chemical properties of side chains.
  • the second technical problem to be solved by the present invention is to provide a cross-linking material of a polymer thiolated modified derivative which is cross-linked by disulfide bonds.
  • the third technical problem to be solved by the present invention is to provide a high molecular thiolated modified derivative crosslinked material which is crosslinked by a sulfhydryl reactive crosslinking agent.
  • the invention adopts a polymer compound with a carboxyl group in a side chain as a raw material, and synthesizes a novel method by a chemical preparation method.
  • a thiolated modified macromolecular derivative are adjustable side chain molecular structure and chemical properties and have important applications in the field of biomedicine.
  • the polymer thiolated modified derivative of the present invention is represented by the following formula (I) or ( ⁇ ):
  • ⁇ And! ⁇ may have the same or different chemical structures, and the polymer thiolated modified derivative has a molecular weight of 1,000 to 5,000,000.
  • the above P refers to a polymer compound residue having a carboxyl group in a side chain, and at least one of the polymer compound side chain carboxyl groups is modified to a mercapto group.
  • the polymer compound having a carboxyl group in the side chain includes a polysaccharide, a protein, a synthetic polymer and the like.
  • the polysaccharide includes chondroitin sulfate, dermatan, heparin, heparin, alginic acid, hyaluronic acid, dermatan sulfate, pectin, carboxymethylcellulose, carboxymethyl chitosan, etc.
  • Molecules include polyacrylic acid, polyaspartic acid, polytartaric acid, polyglutamic acid, polyfumaric acid, etc. and their salt forms; proteins include collagen, alkaline gelatin, acid gelatin, basic gene recombinant gelatin Acidic recombinant gelatin protein, elastin, core proteoglycan laminin fiber binding protein, and the like.
  • the polymer compound having a carboxyl group in the side chain is preferably chondroitin sulfate, heparin, heparinoid, alginic acid, hyaluronic acid, polyaspartic acid, polyglutamic acid, and a salt thereof (such as a sodium salt, a potassium salt, etc.).
  • Particularly preferred are chondroitin sulfate, heparin, hyaluronic acid, and their salt forms (e.g., sodium salts, potassium salts, etc.), and basic gelatin proteins, acidic gelatin proteins.
  • the above-mentioned anthracene group means -(CH 2 )-- ( ⁇ is an integer of 1 to 15).
  • ⁇ is an integer of 1-8.
  • the above substituted alkylene group means an alkylene group in which at least one hydrogen atom is substituted with a group such as a lower sulfhydryl group, a hydroxyl group, an amino group, a decyloxy group, a phenyl group or an ester group.
  • aromatic group means an aromatic phenyl group, a naphthyl group or the like. Phenyl is preferred.
  • the above polyether group means -[(CHR)tician0] n -, wherein R is a lower fluorenyl group, n is an integer of 1 to 10, and m is an integer of 1 to 500.
  • R is a hydrogen atom, and n is equal to 2, respectively. , 3 and 4.
  • the above lower sulfhydryl group means a linear or branched fluorenyl group having 1 to 8 carbon atoms.
  • a linear or branched fluorenyl group having 1 to 8 carbon atoms For example: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, neopentyl, hexyl, heptyl, octyl and the like.
  • a linear or branched fluorenyl group having 1 to 4 carbon atoms is preferred, and a methyl group or an ethyl group is particularly preferred.
  • propyl is preferred.
  • the above decyloxy group means a linear or branched alkoxy group having 1 to 6 carbon atoms.
  • a branched or linear alkoxy group having 1 to 4 carbon atoms is preferred, and a methoxy group and an ethoxy group are particularly preferred.
  • the above ester group means -C(0) 0R, wherein R is the above lower fluorenyl group.
  • R is the above lower fluorenyl group.
  • Preferred are a methyl ester group, an ethyl ester group, a propyl ester group and a butyl ester group.
  • a preferred compound of the invention is a neutralized R 2 representing an anthranylene group. Most preferred is! ⁇ and R 2 are an anthracene group having 1 to 8 carbon atoms.
  • the polymer thiolated modified derivative of the present invention has the following chemical structure characteristics -
  • the preparation of the polymer thiolated modified derivative of the formula (I) or (II) of the present invention generally employs a hydrazide/carbodiimide coupling chemistry.
  • the basic principle is that the side chain carboxyl group of the polymer compound forms an active intermediate under the activation of the carbodiimide, and then the amino nucleophilic attacking active intermediate of the dithiodihydrazide forms an adduct, and finally the adduct of the second The sulfur bond is reduced to a free sulfhydryl group and purified to give the product.
  • DGDTPDH dithiodipropionate diacyl glycine dihydrazide
  • DADTPDH dithiodipropionate bisylalanine dihydrazide
  • DHADTPDH Dithiodipropionate bis(hydroxy)aminoacetic acid dihydrazide
  • DPDTPDH dithiodipropionate bisamidopropionic acid dihydrazide
  • DBDTPDH disulfide Didipropionate diamidobutanoic acid dihydrazide
  • DBDTPDH dithiodibutyric acid diacyl glycine dihydrazide
  • DPDTBDH dithiodibutyric acid diacyl Aminopropionate dihydrazide
  • DPDTBDH dithiodibutyric acid diacyl Aminopropionate dihydrazide
  • a preparation method of the generalized thiolated modified derivative of the formula (I) and ( ⁇ ) of the present invention is as follows:
  • the side chain carboxyl group of the polymer compound is in 1-ethyl-3-(3-dimethylaminopropyl)
  • the disulfide bond of the final adduct is hydrogenated with a hydroxy thiol, dithiothreitol or borohydride
  • the reducing agent such as sodium is reduced to a free sulfhydryl group, and the thiolated modified derivative of the formula (I) or (II) of the present invention can be obtained by dialysis purification to remove impurities.
  • the following are chemical synthesis routes and chemical structural formulas in which R, and R 2 are an anthracene group, a substituted anthracen
  • the macromolecular thiolated modified derivative of the formula ⁇ ) or ( ⁇ ) of the present invention when both 1 and 2 are an anthracene group, is a preferred compound of the present invention, and its chemical structure is as follows:
  • (1) represents a structural formula of the polymer thiolated derivative of the formula (I) in which both 1 and 1 are 2 alkylene groups; (2) represents an alkylene group of both 1 and 1 and 2;
  • the general formula (II) of the polymer thiolated derivative of the present invention has a structural formula (i, j, m, n are all integers greater than 1).
  • R 2 is a fluorenylene group having 2 and 3 carbon atoms
  • R is a carbon number of 1 to 5, respectively.
  • the sulfhydryl group, P is a residue of chondroitin sulfate, heparin, hyaluronic acid and their salt forms (such as sodium salt, potassium salt, etc.), alkaline gelatin protein, acid gelatin protein, etc., and its chemical structural formula is as follows :
  • (1) is a dithiodipropionate diacyl glycine dihydrazide modified polymer thiolated derivative (P-DGDTPDH); (2) is dithiodipropionate bisylalanine dihydrazide Modified polymer thiolated derivative (P-DADTPDH); (3) is dithiodibutyric acid diacyl glycine dihydrazide modified macromolecular derivative (P-DGDTBDH); (4) is disulfide Di-n-butyric acid bis-alanine dihydrazide-modified polymer thiolated derivative (P-DADTBDH); (5) is bis-succinic acid bisacyl cystamine dihydrazide modified polymer thiolated derivative (P -DSCDH); (6) is a bis-glutaric acid bisacylamine dihydrazide-modified polymer thiolated derivative (P-DGCDH); (7) is adipic acid acyl cystamine dihydrazide modification
  • the thiolation modification preparation process does not significantly change the molecular weight and its distribution, and generally the molecular weight and distribution of the general formula (I) or ( ⁇ ) polymer thiolated modified derivative of the present invention and the starting material (the carboxyl group in the side chain is high)
  • the molecular weight of the molecular compound and its distribution are substantially the same.
  • the molecular weights of different starting materials vary widely, usually between 1,000 and 5 million; their molecular weight distributions also vary widely, such as the broad molecular weight distribution of gelatin.
  • the molecular weight and distribution of the starting material do not affect the thiolation modification process, and do not affect the polymer thiolated modified derivative of the formula (I) or (II) of the present invention. Preparation.
  • the general formula (I) or ( ⁇ ) polymer thiolated modified derivative of the present invention has many beneficial effects.
  • the invention adopts the chemical bonding mode of the hydrazide bond to carry out the thiolation modification, and has many remarkable advantages such as mild preparation conditions, high yield, high degree of modification and controllability.
  • the invention of the general formula (I) or ( ⁇ ) polymer thiolated modified derivative innovatively introduces an amide bond, the chemical structure of the side chain is flexible and the performance is adjustable. Studies have shown that the general formula (I) or ( ⁇ ) polymer thiolated modified derivatives of the present invention have the following two beneficial effects:
  • the length and structure of the side chain can be flexibly adjusted by introducing an amide bond.
  • the length of the side chains will greatly affect the reactivity of the sulfhydryl groups (Shu et al, Biomaterials 24, 3825, 2003).
  • the amide bond is a strong electron withdrawing group, and the introduction of the side chain amide bond of the compound of the present invention is in a large range
  • the degree of ionization constant (pK a ) of the terminal sulfhydryl group is affected, however this is related to the way the amide bond is connected.
  • the carbonyl group in the side chain amide bond of the macromolecular amide bond of the general formula (I) of the present invention is similar to the terminal sulfhydryl group, and the ionization of the terminal sulfhydryl group is enhanced.
  • the formula (II) of the present invention The nitrogen atom of the side chain amide bond of the polymer thiolated modified derivative is similar to the terminal sulfhydryl group, which weakens the ionization of the terminal thiol group! ⁇ Raised).
  • the side chain linking fragments disclosed by Shu et al. in Biomacromolecules, 3, 1304, 2002 and WO 2004/03716 are polymerized thiolated modified derivatives of the fluorenylene group (carbon number 2 and 3).
  • the thiol radicalization constant of the polymer thiolated modified derivative of the formula (I) of the present invention (pKJ is reduced by about 0.1 to 0.4), and the polymer thiolated modified derivative of the formula (II) of the present invention
  • the thiol ionization constant (pKJ is increased by about 0.2 to 0.7). Therefore, the terminal fluorenyl group of the polymer thiolated modified derivative of the formula (I) of the present invention is more active, and the polymer thiol group of the formula ( ⁇ ) of the invention is more active.
  • the terminal thiol group of the modified derivative is more stable.
  • the macromolecular thiolated modified derivative of the formula (I) or (II) of the present invention has many unique properties compared with the disclosed macromolecular thiolated modified derivative, and can be selected according to the needs of practical applications.
  • the following specific polymer thiolated derivatives of hyaluronic acid (HA) are further exemplified to further illustrate the beneficial effects of the general formula (I) or (II) polymer thiolated modified derivatives of the present invention:
  • the above compounds have the same structure on the terminal oxime side, wherein (a) is a polymer thiolated modified derivative disclosed by Shu et al. in Biomacromolecules, 3, 1304, 2002 and WO 2004/03716; (b) is the present invention
  • the polymer thiolated modified derivative of the formula (I); (c) is a polymer thiolated modified derivative of the formula (II) of the invention.
  • the thiol-reactive activity of the polymer thiolated modified derivative (b) is much higher than that of (a), and its ability to form a disulfide-crosslinked gel is improved by about 50%; and the polymer thiolated modified derivative (c)
  • the sulfhydryl reactivity is much lower than (a), and its sulfhydryl stability can be more than doubled.
  • the polymer thiolated modified derivative of the formula (I) or (II) of the present invention has at least one side chain free sulfhydryl group which can be reoxidized under suitable conditions to form a disulfide bond.
  • Medium-strength oxidants such as oxygen, low-concentration hydrogen peroxide, iodine, and iron trivalent ions can form a disulfide bond by free sulfhydryl groups, thereby preparing a polymer.
  • Cross-linking materials are examples of cross-linking materials.
  • disulfide bonds are usually affected by the pH of the solution: Under alkaline conditions, the sulfhydryl groups are ionized into sulfur anions, reflecting high activity, even if oxygen in the air can rapidly promote the formation of disulfide bonds; The ionization of the sulfhydryl group is inhibited, the reactivity is lowered, and the sulfhydryl group is relatively stable.
  • the thiol group of the polymer thiolated modified derivative of the formula (I) of the present invention can produce a disulfide-bonded cross-linking material even under neutral conditions and weak oxidizing agents; and the formula (II) of the present invention
  • the thiol group of the polymer thiolated modified derivative is relatively stable, and it is required to rapidly form a disulfide crosslinked material under the action of weakly basic or strong oxidizing agent.
  • the preparation method of the disulfide-bonded polymer thiolated modified derivative crosslinked material of the invention is simple and reliable, and the product is flexible and variable.
  • a commonly used preparation method is to prepare one or more polymer thiolated modified derivatives of the general formula (I) or ( ⁇ ) into an aqueous solution or a mixed aqueous solution, and use room temperature air under neutral or weakly alkaline conditions. Oxidation to prepare a disulfide-bonded cross-linking material; or under weakly acidic or acidic conditions, a disulfide-bonded cross-linking material is prepared by oxidation with a stronger oxidizing agent such as low-concentration hydrogen peroxide or iron trivalent ions.
  • the one-component and two-component disulfide-crosslinked polymer thiolated modified derivative crosslinked materials of the present invention are represented by the following general formula (III), (IV) or (V):
  • P 2 is a residue of a polymer compound having a carboxyl group in a side chain, and the definition thereof is the same as defined above.
  • ⁇ and R 2 are as defined above;
  • R 3 and R 4 are the same as ⁇ and R 2 ; and
  • R 2 , R 3 may have the same or different chemical structures.
  • 1 ⁇ and the same it is a one-component cross-linking material, when and! ⁇
  • it is a two-component cross-linked material.
  • the three-component or three-component disulfide-crosslinked polymer thiolated modified derivative cross-linking material of the present invention may be modified by three or more kinds of polymer thiolation of the general formula (I) or (II).
  • the preparation of a derivative is characterized in that three or more polymer compounds are contained by a disulfide bond to form a crosslinked material.
  • the thiol-reactive cross-linking agent cross-linked polymer thiolated modified derivative cross-linking material of the present invention comprises one or more novel polymer thiolated modified derivatives of the formula (I) or (II) and a mercapto group. Reactive activity Preparation of crosslinkers.
  • the thiol-reactive functional groups used in the present invention include maleimide, vinyl sulfone, ⁇ , ⁇ unsaturated acrylate, ⁇ , ⁇ unsaturated methacrylate, halo propionate, halopropionamide, dithio Pyridine, hydrazine-hydroxysuccinimide activated ester, and the like.
  • the thiol-reactive cross-linking agent used in the present invention contains at least two reactive functional groups of polyethylene glycol (referred to as PEG) derivatives, such as two-arm, three-arm, four-arm, eight-arm or more arms.
  • PEG polyethylene glycol
  • Alcohol derivatives which have the following typical chemical structure:
  • Double-arm polyethylene glycol crosslinker Double-arm polyethylene glycol crosslinker
  • G, G 2 , G 3 , G 4 , G 5 , G 6 , G 7 and G 8 are the above-mentioned thiol-reactive functional groups such as maleimide, vinylsulfone, ⁇ , ⁇ -unsaturated acrylate, ⁇ , ⁇ unsaturated methacrylate, halopropionate, halopropionamide, dithiopyridine or hydrazine-hydroxysuccinimide, etc., which may have all the same, partially identical or all different chemical structures ;
  • PEG means a segment having a CH 2 CH 2 0 repeating unit having a molecular weight of 100 to 1,000,000.
  • the general preparation method of the cross-linked polymer thiolated modified derivative cross-linking material of the sulfhydryl reactive cross-linking agent of the present invention comprises the modification of one or more novel polymer thiol groups of the formula (I) or ( ⁇ )
  • the derivative is made into an aqueous solution or a mixed aqueous solution, and the pH of the solution is adjusted to be neutral.
  • the aqueous solution of the above-mentioned thiol-reactive cross-linking agent is added, and after uniformly mixing, it is allowed to stand at room temperature for a while to form a gel, thereby obtaining a cross-linked material.
  • the thiol group of the polymer thiolated modified derivative of the formula (I) of the invention is more active and reacts faster with the crosslinking agent; and the thiol group of the polymer thiolated modified derivative of the formula (II) of the invention is more Stable, the reaction with the crosslinker is relatively slow.
  • the polymer thiolated modified derivative of the formula (I) and the above-mentioned double-armed polyethylene glycol sulfhydryl reactive crosslinking agent as an example, the one-component and two-component sulfhydryl reactive crosslinking agent of the present invention is crosslinked.
  • the polymer thiolated modified derivative crosslinked material has the structure shown below:
  • ⁇ ⁇ 2 is a residue of a polymer compound having a carboxyl group in a side chain, and its definition is the same as defined above; R, R 2 , R 3 and R 4 are as defined above; and R 2 , R 3 , R 4 may be Have the same or different chemical structures.
  • P, and P 2 are the same, it is a one-component cross-linking material, when! ⁇ And! ⁇
  • the polymer thiolated modified derivative crosslinked material may also be prepared by co-crosslinking two or more kinds of the above-mentioned two-arm polyethylene glycol sulfhydryl reactive cross-linking agent.
  • one or more multi-arm polyethylene glycol derivative crosslinkers (such as a three-arm polyethylene glycol derivative crosslinker, a four-arm polyethylene glycol derivative crosslinker, an eight-arm polyether)
  • a diol derivative crosslinking agent or the like is used to prepare a polymer thiolated modified derivative crosslinked material.
  • the cross-linking material of the general formula ( ⁇ ) polymer thiolated modified derivative crosslinked with the thiol reactive crosslinking agent and the crosslinked material of the generalized thiolated modified derivative of the general formula (I) have a similar structure.
  • the cross-linking material of the polymer thiolated modified derivative crosslinked by the three-component or three-component sulfhydryl reactive cross-linking agent of the present invention may be three or more kinds of the general formula (I) or ( ⁇ ) polymer.
  • Preparation of thiolated modified derivatives The usual preparation route is to first prepare a mixed solution of three or more kinds of polymer thiolated modified derivatives of the formula (I) or ( ⁇ ), and then adjust the pH of the solution to be neutral, adding one or more
  • the above polyethylene glycol sulfhydryl reactive derivative crosslinker is prepared to prepare a multicomponent crosslinked material.
  • Figure 1 is a hydrogen atom nuclear magnetic resonance spectrum and an important chemical shift peak of a low-substituted derivative in Example 4 of the present invention (D 2 0 is a solvent);
  • Example 2 is a hydrogen atom nuclear magnetic resonance spectrum and an important chemical shift peak of a highly substituted derivative in Example 4 of the present invention (D 2 0 is a solvent);
  • FIG. 3 is a schematic view showing the morphology of cells on the surface of a blank cell culture plate in Example 20 of the present invention
  • FIG. 4 is a cross-linked hyaluronic acid-gelatin two-component water of cells in polyethylene glycol diacrylate in Example 20 of the present invention. Schematic diagram of the surface of the gel.
  • Example 1 Synthesis of dithiodipropionate diacyl glycine dihydrazide (DGDTPDH) 10 g of dithiodipropionic acid (Aldrich, USA) and 50 ml of anhydrous dimethylformamide were added to a 1000 ml beaker. After stirring at room temperature, 17.0 g of carbonyldiimidazole (Aldrich, USA) was added. At this point the solution produced a large amount of carbon dioxide bubbles and a white precipitate. The reaction was carried out under reduced pressure at room temperature for 3 hours Then, 14.7 g of glycine ethyl ester hydrochloride (Aldrich, USA) was added, and the reaction was stirred for 1 hour.
  • DTDTPDH dithiodipropionate diacyl glycine dihydrazide
  • Sodium hyaluronate (molecular weight 62 to 1.15 million, NovaMatrix FMC BI0P0LYMER, USA) 1 g Dissolved in 200 ml of distilled water to obtain a clear transparent solution.
  • 1.32 g of DGDTPDH prepared in Example 1 was added and dissolved by stirring.
  • the pH of the solution was adjusted to 4.75 with 0.1 mol/L hydrochloric acid, and 0.36 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aldrich, United States), electromagnetic stirring.
  • Sodium hyaluronate (molecular weight 62 to 1.15 million, NovaMatrix FMC BI0P0LYMER, USA) 1 g was dissolved in 200 ml of distilled water to obtain a clear transparent solution.
  • 2.64 g of DGDTPDH prepared in Example 1 was added to the above solution, and dissolved by stirring. Then, the pH of the solution was adjusted to 0.75 by using 0.1 mol/L hydrochloric acid, and 0.9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aldrich, United States), electricity Magnetic stirring. ⁇ The pH of the solution was maintained at 4.75.
  • the viscosity of the solution increased and a gel formed in about 10 minutes. After the gel was formed, the reaction was allowed to stand at room temperature for 2 hours. Then, 20 g of dithiothreitol (Diagnostic Chemical Limited, USA) and a small amount of 0.1 mol/L sodium hydroxide solution were added and stirred. 5 ⁇ The pH of the solution was maintained at 8.5. After the gel was completely dissolved, the reaction was stirred at room temperature for 24 hours. Thereafter, 6 mol/liter of hydrochloric acid was added to the above solution until about pH 3. 0.
  • dithiothreitol Diagnostic Chemical Limited, USA
  • the solution was loaded into a dialysis tube (molecular weight cutoff 3500, Sigma, USA), dialyzed against 10 liters of 0.001 mol/L hydrochloric acid and 0.3 mol/L sodium chloride solution for 5 days, and dialyzed every 8 hours. The solution was then dialyzed against 10 liters of a 0.001 molar/liter hydrochloric acid solution for 3 days, and the dialysate was changed every 8 hours. 5 ⁇ The solution was collected in the form of a white flocculent solid.
  • the gel liquid chromatography (GPC) detection (pure water as mobile phase, UV 210 nm absorption detection) showed no small molecular impurity elution peaks, indicating low substitution and high substitution HA-prepared in Example 2 and Example 3. DGDTPDH is highly purified with impurities below the instrumental level.
  • the side substitution degree of the low-substituted HA-DGDTPDH prepared in Example 2 was calculated to be 27% according to the area of the absorption peak, and the high substitution prepared in Example 3 was used.
  • the side chain substitution degree of HA-DGDTPDH was 59%.
  • the active thiol content of HA-DGDTPDH prepared in Example 2 and Example 3 was tested using the modified Ellman method reported by Shu et al., Biomacromolecules, 3, 1304, 2002.
  • the side chain active thiol content of the low-substituted HA-DGDTPDH prepared in Example 2 was 25. 4 thiol/100 hyaluronic acid disaccharide repeating units, and the side chain active thiol content of the highly substituted HA-DGDTPDH prepared in Example 3 was 55. . 1 thiol/100 hyaluronic acid disaccharide repeating unit. Basically consistent with the results of hydrogen spectroscopy.
  • Example 5 Synthesis of dithiodipropionate bisamidopropionic acid dihydrazide (DADTPDH) 10 g of dithiodipropionic acid (Aldrich, USA), 50 ml of anhydrous dimethyl group in a 1000 ml beaker. Formamide. After stirring at room temperature, 17.0 g of carbonyldiimidazole (Aldrich, USA) was added. At this point the solution produced a large amount of carbon dioxide bubbles and a white precipitate. The reaction was reduced under reduced pressure at room temperature for 3 hours. Then, 14.7 g of ethyl aminopropionate hydrochloride (Aldrich, USA) was added, and the reaction was stirred for 1 hour.
  • DADTPDH dithiodipropionate bisamidopropionic acid dihydrazide
  • Sodium hyaluronate (molecular weight 62.15 million, Nova atrix FC BI0P0LYMER, USA) 1 gram was dissolved in 200 ml of distilled water to obtain a clear transparent solution. 1.43 g of DADTPDH prepared in Example 5 was added to the above solution, and dissolved by stirring. Then, the pH of the solution was adjusted to 4.75 with 0.1 mol/L hydrochloric acid, and added to 0. 48 g of 1-ethyl. -3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aldrich) , United States), electromagnetic stirring. ⁇ The pH of the solution was maintained at 4.75.
  • the viscosity of the solution increased and a gel formed in about 10 minutes. After the gel is formed, it is allowed to stand at room temperature. Reaction for 2 hours. Then, 12 g of dithiothreitol (Diagnostic Chemical Limited, USA) and a small amount of 0.1 mol/L sodium hydroxide solution were added and stirred. 5 ⁇ The pH of the solution was maintained at 8.5. After the gel was completely dissolved, the reaction was stirred at room temperature for 24 hours. Thereafter, 6 mol/liter of hydrochloric acid was added to the above solution until about pH 3. 0.
  • the solution was loaded into a dialysis tube (molecular weight cutoff 3500, Sigma, USA), dialyzed against 10 liters of 0.001 mol/L hydrochloric acid and 0.3 mol/L sodium chloride solution for 5 days, and dialyzed every 8 hours. The solution was then dialyzed against 10 liters of a 0.001 molar/liter hydrochloric acid solution for 3 days, and the dialysate was changed every 8 hours. 5 ⁇ The solution was collected in the form of a white flocculent solid.
  • GPC detection (pure water for mobile phase, UV 210 nm absorption detection) showed no peaks of small molecular impurities, indicating that the synthesized HA-DADTPDH was highly purified, and the impurities were lower than the instrument detection level.
  • HA-DADTPDH Hydrogen nuclear magnetic resonance spectroscopy detection (- NMR) (D 2 0 as solvent).
  • HA-DADTPDH has two new absorption peaks at ⁇ 3. 4, 2. 66 ppm corresponding to CH 2 G3 ⁇ 4NHC (0) CH 2 CH 2 SH and CH 2 CH 2 NHC (0) CH 2 ⁇ 3 ⁇ 4SH Hydrogen atom absorption of chain methylene; G3 ⁇ 43 ⁇ 4NHC (0) CH 2 CH 2 SH and CH 2 CH 2 NHC (0) ⁇ 3 ⁇ 4CH 2 SH
  • the hydrogen absorption peaks of the two side chain methylene groups are around ⁇ 2. 5 ppm Overlapping each other.
  • a small absorption peak with a chemical shift of about ⁇ 2. 8 ppm is an absorption peak of a small amount of side reaction product.
  • the characteristic methyl absorption peak of the hyaluronic acid acetyl group was used as an internal standard, and the side chain substitution degree of the synthesized HA-DGDTPDH was calculated to be 61% based on the area of the absorption peak.
  • the active thiol content of HA-DADTPDH was determined by the modified Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002: 53. 6 thiol/100 hyaluronan disaccharide repeat units, slightly lower than hydrogen spectroscopy Test results.
  • Cysteine dihydrochloride (Aldrich, USA) 100 grams was dissolved in 1500 ml of distilled water to give a clear, clear solution. To the above solution was added 4 mol/liter of sodium hydroxide until the pH of the solution was 10. Then, 133 g of succinic anhydride (Aldrich, USA) was added under electromagnetic stirring while continuously adding 4 mol/l of sodium hydroxide to maintain the pH of the solution at 7-10. After reacting for 2 hours at room temperature, 6 mol/liter of hydrochloric acid was added to the solution. . The white precipitated product was collected by filtration and washed twice with 2000 ml of distilled water. Then vacuum drying under reduced pressure to obtain a white product solid product to synthesize disuccinic bis-succinate (abbreviated as DSC) about 150 Gram, the yield is greater than 90%.
  • DSC disuccinic bis-succinate
  • Example 8 Synthesis and characterization of DSCDH modified hyaluronic acid thiolated derivative (HA-DSCDH) Sodium hyaluronate (molecular weight 62-1.1 million, NovaMatrix FMC BI0P0LYMER, USA) 1 g dissolved in 200 ml of distilled water, Clarify the clear solution. To the above solution, 0.99 g of DSCDH prepared in Example 7 was added and dissolved by stirring. 288 ⁇ 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Aldrich, then added 0.
  • the gel gradually dissolved while continuously adding 0.1 mol/L sodium hydroxide solution to maintain the pH of the solution at 8.5. After the gel was completely dissolved, the reaction was electromagnetically stirred at room temperature for 24 hours. Thereafter, 6 mol/liter of hydrochloric acid was added to the above solution until about pH 3. 0.
  • the solution was loaded into a dialysis tube (molecular weight cutoff 3500, Sigma, USA), dialyzed against 10 liters of 0.001 mol/L hydrochloric acid and 0.3 mol/L sodium chloride solution for 5 days, and dialyzed every 8 hours. The solution was then dialyzed against 10 liters of a 0.001 molar/liter hydrochloric acid solution for 3 days, and the dialysate was changed every 8 hours. 5 ⁇ The solution was finally collected in a lysate.
  • GPC detection (pure water was mobile phase, UV 210 nm absorption detection) showed no peaks of small molecular impurities, indicating that the synthesized HA-DSCDH was highly purified, and the impurities were lower than the instrument detection level.
  • the characteristic methyl absorption peak of the hyaluronic acid acetyl group was used as an internal standard, and the side chain substitution degree of the synthesized HA-DGDTPDH was 38% based on the area of the absorption peak.
  • the active thiol content of HA-DSCDH was determined by the modified Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002: 39. One thiol/100 hyaluronan disaccharide repeat unit, basic and hydrogen spectroscopy The results are consistent.
  • Example 9 Synthesis and characterization of DSCDH modified chondroitin sulfate thiolated derivative (CS-DSCDH) Chondroitin sulfate (type c, from shark cartilage, Sigma, USA) 1 gram dissolved in 100 ml of distilled water for clear and transparent Solution. To the above solution, 0. 704 g of DSCDH prepared in Example 7 was added and dissolved by stirring. 192 ⁇ 1 - Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ⁇ 1 (The pH of the solution was adjusted to 1.75 with 0.1 mol / L hydrochloric acid, added. 11" 11, US), electromagnetic stirring.
  • CS-DSCDH DSCDH modified chondroitin sulfate thiolated derivative
  • Chondroitin sulfate type c, from shark cartilage, Sigma, USA
  • the above solution is charged into a dialysis tube (molecular weight cut off 3,500) , Sigma, United States), dialysis with 10 liters of 0.001 mol / liter of hydrochloric acid and 0.3 mol / liter of sodium chloride solution for 5 days, every 8 hours to change the dialysate; then 10 liters of 0. 001 moles Dialysis of hydrochloric acid solution for 3 days, changing dialysate every 8 hours. 6 ⁇ The solution of the solution was lyophilized to obtain a white flocculent solid about 0.6 g.
  • GPC detection (pure water was mobile phase, UV 210 nm absorption detection) showed no peaks of small molecular impurities, indicating that the synthesized CS-DSCDH was highly purified, and the impurities were lower than the instrument detection level.
  • the characteristic methyl absorption peak of the acetyl group of chondroitin sulfate was used as an internal standard, and the side chain substitution degree of the synthesized CS-DGDTPDH was calculated to be 47% based on the area of the absorption peak.
  • GPC standard curve of monodisperse hyaluronic acid calibration
  • the active thiol content of CS-DSCDH was determined by the modified Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002: 44. 2 thiol/100 chondroitin disaccharide repeat units, slightly lower than hydrogen spectroscopy Test results.
  • EXAMPLES 10 Synthesis and characterization of DSCDH modified gelatin thiolated derivatives (GEL-DSCDH) Gelatin (type B, from pigskin, Sigma, USA) 1 gram was dissolved in 100 ml of distilled water to give a clear, clear solution. To the above solution, 0.75 g of DSCDH prepared in Example 7 was added and dissolved by stirring. Then, the pH of the solution was adjusted to 4.75 with 0.1 mol/L hydrochloric acid, and 1 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aldrich, USA) was added. , electromagnetic stirring.
  • GEL-DSCDH DSCDH modified gelatin thiolated derivatives
  • GPC detection (pure water for mobile phase, UV 210 nm absorption detection) showed no peaks of small molecular impurities, indicating that the synthesized GEL-DSCDH was highly purified, and the impurities were lower than the instrument detection level.
  • the molecular weight and its distribution were determined by GPC (standard curve of standard molecular weight polyethylene glycol): weight average molecular weight (MJ 560,000, number average molecular weight (Mschreib) 2. 10,000, molecular weight distribution 2.67.
  • the active thiol content of GEL-DSCDH was measured using the modified Ellman method reported by Shu et al., Biomacromolecules, 3, 1304, 2002: 0.55 millimoles thiol/gram GEL_DSCDH.
  • Cysteine dihydrochloride (Aldrich, USA) 100 grams was dissolved in 1500 ml of distilled water to give a clear, clear solution. To the above solution, 4 mol / liter of sodium hydroxide was added until the pH of the solution was 10. Then, 152 g of glutaric anhydride (Aldrich, USA) was added under electromagnetic stirring while continuously adding 4 mol / liter of sodium hydroxide to maintain the pH of the solution at 7 to 10. After reacting at room temperature for 2 hours, 6 mol/liter of hydrochloric acid was added to the solution. The white precipitated product was collected by filtration and washed twice with 2000 ml of distilled water. Then, it was dried under vacuum to obtain about 155 g of a white product solid product, glutaric acid bis-succinate (abbreviated as DGC), and the yield was more than 90%.
  • DGC glutaric acid bis-succinate
  • DGCDE diethyl succinic acid diethyl succinate
  • Example 12 Synthesis and characterization of DGCDH modified hyaluronic acid thiolated derivative (HA-DGCDH) Sodium hyaluronate (molecular weight 62-1.1 million, NovaMatrix FMC BI0P0LYMER, USA) 1 g dissolved in 200 ml of distilled water, Clarify the clear solution. 1.53 g of the DGCDH prepared in Example 11 was added to the above solution, and dissolved by stirring. Then, the pH of the solution was adjusted to 4.75 with 0.1 mol/L hydrochloric acid, and 0. 48 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Aldrich, United States), electromagnetic stirring.
  • HA-DGCDH DGCDH modified hyaluronic acid thiolated derivative
  • the above solution was loaded into a dialysis tube (cutting molecular weight 3500, Sigma, USA), dialyzed against 10 liters of 0.001 mol/L hydrochloric acid and 0.3 mol/L sodium chloride solution for 5 days, and dialysis was performed every 8 hours. The solution was then dialyzed against 10 liters of a 0.001 molar/liter hydrochloric acid solution for 3 days, and the dialysate was changed every 8 hours. 5 ⁇ The solution was collected in the form of a white flocculent solid.
  • GPC detection (pure water for mobile phase, UV 210 nm absorption detection) showed no peaks of small molecular impurities, indicating that the synthesized HA-DGCDH was highly purified, and the impurities were lower than the instrument detection level.
  • the characteristic methyl absorption peak of the acetyl group of hyaluronic acid was used as an internal standard, and the degree of side chain substitution of the synthesized HA-DGDTPDH was 52% based on the area of the absorption peak.
  • the active thiol content of HA-DSCDH was determined by the improved Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002: 49. 4 thiol/100 hyaluronan disaccharide repeat units, basic and hydrogen spectroscopy The results are consistent.
  • the disulfide-crosslinked gelatin hydrogel preparation The gelatin thiolated derivative of the present invention (GEL-DSCDH) prepared in Example 10 was dissolved in 10 ml of 0.1 mol / liter of phosphate buffer ( P H 7. 0 ) A clear transparent solution was obtained, and an appropriate amount of 0.1 mol/L sodium hydroxide was added to the above solution until pH 7.4. The solution was then poured into a 25 ml glass beaker and allowed to stand at room temperature for 12 hours. The viscosity of the solution gradually increased and a gel formed.
  • Example 14 Preparation of a multicomponent disulfide crosslinked hydrogel
  • the preparation of the disulfide-crosslinked hyaluronic acid-gelatin two-component hydrogel The hyaluronic acid thiolated derivative of the present invention (HA-DGDTPDH) prepared in Example 3 was dissolved in 10 ml of 0. 1 mol / liter of phosphoric acid 'salt buffer (pH 7. 0) to obtain a clear and transparent solution, an appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4.
  • the gelatin thiolated derivative of the present invention (GEL-DSCDH) prepared in Example 10 was dissolved in 10 ml of 0.1 ml/L phosphate buffer (pH 7.0) to obtain a clear transparent solution.
  • Disulfide-bonded cross-linked chondroitin sulfate-gelatin two-component hydrogel preparation The chondroitin sulfate thiolated derivative of the present invention prepared in Example 9 (CS-DSCDH) 0. 3 g dissolved in 10 ml 0. 1 mol / liter of phosphate buffer (pH 7.0) to obtain a clear transparent solution, the appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4.
  • the gelatin thiolated derivative of the present invention (GEL-DSCDH) prepared in Example 10 was dissolved in 10 ml of 0.1 mol/L phosphate buffer (pH 7.0) to obtain a clear transparent solution.
  • Example 3 prepared hyaluronic acid thiolated derivative of the invention (HA-DGDTPDH) 0. 1g dissolved in 10 ml / liter of phosphate buffer (pH 7. 0) to obtain a clear transparent solution, the appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4.
  • HA-DGDTPDH hyaluronic acid thiolated derivative of the invention
  • the chondroitin sulfate thiolated derivative of the present invention (CS-DSCDH) prepared in Example 9 was dissolved in 10 ml of 0.1 mol/L phosphate buffer (pH 7.0) to obtain a clear transparent solution.
  • an appropriate amount of 0.1 mol/liter of sodium hydroxide was added until pH 7.4.
  • the gelatin thiolated derivative of the present invention (GEL-DSCDH) prepared in Example 10 was dissolved in 10 ml of 0.1 mol/L phosphate buffer (pH 7.0) to obtain a clear transparent solution.
  • An appropriate amount of 0.1 mol/L sodium hydroxide was added until pH 7.4.
  • Example 15 Preparation of a one-component polyethylene glycol divinyl sulfone crosslinked hydrogel
  • the lyophilic thiolated derivative (HA-DGDTPDH) of the present invention prepared in Example 3 was dissolved in 10 ml 0.11. Mol / liter of phosphate buffer (pH 7.0) to obtain a clear transparent solution, the appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4.
  • Polyethylene glycol divinyl sulfone molecular weight 3400, Nektar Therapeutics, USA
  • the sulphate chondroitin thiolated derivative (CS-DSCDH) of the present invention prepared in Example 9 was dissolved in 10 ml 0.11. Mol / liter of phosphate buffer (pH 7.0) to obtain a clear transparent solution, the appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4. Polyethylene glycol divinyl sulfone (molecular weight 3400, Nektar Therapeutics, USA) 0. 1 gram dissolved in 2. 5 ml of 0.1 mol / liter of p-acid buffer (pH 7. 0) to obtain a clear transparent solution.
  • gelatin thiolated derivative of the present invention (GEL-DSCDH) 0. 3g dissolved in 10 ml 0.1 mol / liter Phosphate buffer
  • hyaluronic acid thiolated derivative of the present invention (HA-DGDTPDH) 0. 1 g dissolved in the polyethylene glycol diacrylate crosslinked hyaluronic acid-gelatin two-component hydrogel 10 ml / liter of phosphate buffer (pH 7. 0) to obtain a clear transparent solution, the appropriate amount of 0.1 mol / liter of sodium hydroxide was added to the above solution until pH 7.4.
  • chondroitin sulfate thiolated derivative of the present invention prepared in Example 9 was dissolved in 10 ml of 0.1 mol/L phosphate buffer (pH 7.0) to obtain a clear transparent solution.
  • an appropriate amount of 0.1 mol/liter of sodium hydroxide was added until pH 7.4.
  • Example 10 Preparation of the gelatin thiolated derivative of the present invention (GEL-DSCDH) 0.3 g dissolved in 10 ml of 0.1 mol/L phosphate buffer (pH 7.0) to obtain a clear transparent solution, dissolved in the above Add an appropriate amount of 0.1 mol / liter of sodium hydroxide until ⁇ ⁇ 7. 4.
  • Polyethylene glycol diacrylate (molecular weight 3400, Nektar Therapeutics, USA) 0. 3 g dissolved in 7.5 ml of 0.1 mol/L phosphate buffer (pH 7.0) gave a clear clear solution. Then, the above 10 ml of HA-DGDTPDH solution, 10 ml of CS-DSCDH solution, 10 ml of GEL-DSCDH solution, and 7.5 ml of polyethylene glycol diacrylate solution were simultaneously poured into a 50 ml glass beaker, and electromagnetic stirring was immediately performed for 30 seconds. Allow to stand at room temperature for 30 minutes. The viscosity of the solution gradually increases and forms a gel.
  • Disulfide-bonded cross-linked hyaluronic acid hydrogel for inhibiting cell adhesion A disulfide-crosslinked hyaluronic acid hydrogel was prepared in a 24-well standard cell culture plate according to Example 13, per well. 1 ml. After 12 hours, the whole cell culture plate was immersed in a 75% alcohol solution for 2 hours. Thereafter, the cell culture plate was washed three times with sterile physiological saline. 1 ml of cell culture medium (DMEM, 10% bovine serum) and 20,000 NIH 3T3 fibroblasts were added to each well. Incubate for 24 hours at 37 ° C in a CO2 cell culture incubator.
  • DMEM cell culture medium
  • 20,000 NIH 3T3 fibroblasts were added to each well. Incubate for 24 hours at 37 ° C in a CO2 cell culture incubator.
  • Example 18 Disulfide-bonded cross-linked hyaluronic acid-gelatin two-component hydrogel as a substrate for cell adhesion growth
  • a disulfide-crosslinked hyaluronic acid-gelatin two-component hydrogel was prepared in a 24-well standard cell culture plate according to Example 14 at a volume of 1 ml per well. After 12 hours, the whole cell culture plate was immersed in 75% alcohol solution for 2 hours. Thereafter, the cell culture plate was washed three times with sterile physiological saline. Add 1 ml of cell culture medium (DMEM, 10% bovine serum) and 20,000 NIH 3T3 fibroblasts to each well. Incubate at 37 ° C for 24 hours in a carbon dioxide cell culture incubator.
  • DMEM cell culture medium
  • 20,000 NIH 3T3 fibroblasts 20,000 NIH 3T3 fibroblasts
  • Example 19 Polyethylene glycol divinyl sulfone crosslinked hyaluronic acid hydrogel for inhibiting cell adhesion
  • Polyethylene glycol divinyl sulfone crosslinked hyaluronic acid was prepared according to Example 15 in a 24-well standard cell culture plate. Acid hydrogel, 1 ml per well. After 12 hours, the whole cell culture plate was immersed in 75% alcohol solution for 2 hours. Thereafter, the cell culture plate was washed three times with sterile physiological saline. Add 1 ml of cell culture medium (DMEM, 10% bovine serum) and 20,000 NIH 3T3 fibroblasts to each well. The cells were cultured in a carbon dioxide cell incubator at 37 ° C for 24 hours.
  • DMEM 10% bovine serum
  • Example 20 Polyethylene glycol diacrylate crosslinked hyaluronic acid-gelatin two-component hydrogel as a substrate for cell adhesion growth
  • a polyethylene glycol diacrylate crosslinked hyaluronic acid-gelatin two-component hydrogel was prepared in accordance with Example 16 in a 24-well standard cell culture plate at 1 ml per well. After 12 hours, the whole cell culture plate was immersed in a 75% alcohol solution for 2 hours. Thereafter, the cell culture plate was washed three times with sterile physiological saline. 1 ml of cell culture medium (DMEM, 10% bovine serum) and 20,000 NIH 3T3 fibroblasts were added to each well. Incubate at 37 ° C for 24 hours in a carbon dioxide cell culture incubator.
  • DMEM 10% bovine serum
  • the polymer thiolated modified derivative of the formula (I) or (II) of the present invention has many beneficial effects.
  • the invention adopts the chemical bonding mode of the hydrazide bond to carry out the thiolation modification, and has many remarkable advantages such as mild preparation conditions, high yield, high degree of modification and controllability.
  • the invention can conveniently prepare various disulfide-bonded crosslinked polymer materials: a disulfide cross-linking material of a polysaccharide, a disulfide cross-linking material of a protein, or a complex disulfide cross-linking of two polysaccharides.
  • Materials composite crosslinked materials of two proteins, a composite disulfide crosslinked material of a polysaccharide and a protein, and the like.
  • These disulfide-bonded polymer materials can be formed into various forms such as a film, a sponge, and a gel, and can be used for inhibiting cell adhesion, using as a cell growth substrate, and the like.
  • the cross-linking action of the thiol-reactive cross-linking agent and the polymer thiolated modified derivative of the formula (I) or (II) of the present invention is very fast, and the cross-linking gelation rate is higher than that of the disulfide bond.
  • the speed of the joint is increased by more than 5 times and has important biomedical uses, such as in-situ embedding of cells.
  • the invention can conveniently prepare various sulfhydryl reactive cross-linking cross-linking polymer materials: for example, a cross-linking material of a polysaccharide, a cross-linking material of a protein, or a composite cross-linking material of two polysaccharides, two proteins Composite cross-linking materials, a composite cross-linking material of a polysaccharide and a protein, and the like.
  • thiol-reactive cross-linked polymeric materials can be used in various forms such as films, sponges, and gels, and can be used for inhibiting cell adhesion, using as a cell growth substrate, and the like.

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Description

高分子巯基化改性衍生物及其交联材料
技术领域：
.本发明涉及化合物， 尤其涉及高分子巯基化改性衍生物； 此外， 本发明还涉 及该高分子巯基化改性衍生物相应的二硫键交联材料和巯基反应活性交联剂交 联材料。
背景技术：
高分子巯基化改性衍生物具有许多重要的生物医学用途，如用于各种小分子 药物和多肽蛋白药物的化学活性修饰、制备各种交联高分子材料等等。这些新型 材料可以作为细胞生长基质、创伤修复再生基质、药物缓释载体、伤口敷料， 原 位包埋细胞基质等等，在生物医学领域具有重要用途。然而到目前为止， 这类巯 基化改性高分子衍生物的种类很少， 只有 Shu等人在 Biomacromolecules, 3， 1304, 2002 中公开的高分子巯基化改性衍生物具有较好的应用前景。 这类高分 子巯基化改性衍生物具有下述结构：
Figure imgf000003_0001
其中 P是高分子化合物残基。然而这两种结构的高分子巯基化改性衍生物侧 链结构和性能单一，并不能有效地满足各种生物医学应用。同时这两种结构的高 分子巯基化改性衍生物的侧链较短，制约了巯基在进一步化学修饰和交联时与其 它化学官能团的碰撞几率，化学反应性能不佳。 因此，制备具有可调节的侧链分 子结构和化学性能的高分子巯基化类衍生物具有重要的意义。
发明内容：
本发明要解决的技术问题之一在于提供一类新的具有重要生物医学用途的 高分子巯基化改性衍生物， 该类衍生物具有可调节的侧链分子结构和化学性能。
本发明要解决的技术问题之二是提供一类二硫键交联的高分子巯基化改性 衍生物交联材料。
本发明要解决的技术问题之三是提供一类巯基反应活性交联剂交联的高分 子巯基化改性衍生物交联材料。
本发明以侧链含羧基的高分子化合物为原料，采用化学制备方法合成了新颖 的巯基化改性高分子衍生物。 该类衍生物具有可调节的侧链分子结构和化学性 能， 在生物医药领域具有重要的用途。
本发明的高分子巯基化改性衍生物用下述通式 （I ) 或 （π) 表示：
Figure imgf000004_0001
其中 和 是亚垸基、 取代亚垸基、 芳香基、 聚醚基等; ！^和！^可以具有 相同或不相同的化学结构，所述高分子巯基化改性衍生物的分子量为 1000到 500 万。
上述 P是指侧链含有羧基的高分子化合物残基，其中至少一 高分子化合物 侧链羧基被改性为巯基。侧链含有羧基的高分子化合物包括多糖、蛋白质以及合 成高分子等。 其中多糖包括硫酸软骨素、 皮肤素、 肝素、 类肝素、 海藻酸、 透明 质酸、 硫酸皮肤素、 果胶、 羧甲基纤维素、 羧甲基壳聚糖等以及它们的盐形式； 合成高分子包括聚丙烯酸、 聚天冬氨酸、聚酒石酸、聚谷氨酸、 聚富马酸等以及 它们的盐形式； 蛋白质包括胶原蛋白、碱性明胶蛋白、 酸性明胶蛋白、碱性基因 重组明胶蛋白、酸性基因重组明胶蛋白、弹性蛋白、核心蛋白多糖层粘连蛋白纤 维结合蛋白等。 侧链含有羧基的高分子化合物优选硫酸软骨素、 肝素、 类肝素、 海藻酸、透明质酸、聚天冬氨酸、聚谷氨酸以及它们的盐形式（如钠盐，钾盐等）， 和碱性明胶蛋白、酸性明胶蛋白、碱性基因重组明胶蛋白、酸性基因重组明胶蛋 白。特别优选硫酸软骨素、肝素、透明质酸以及它们的盐形式（如钠盐，钾盐等）， 和碱性明胶蛋白、 酸性明胶蛋白。
上述亚垸基是指- (CH2)„- (η是 1~15的整数）。 优选 η是 1~8的整数。
上述取代亚烷基是指至少一个氢原子被低级垸基、羟基、 氨基、垸氧基、 苯 基、 酯基等基团取代的亚烷基。
上述芳香基是指芳香族的苯基、 萘基等。 优选苯基。
上述聚醚基是指- [ (CHR)„0]n-， 其中 R是低级垸基， n是 1~10的整数， m是 1~500的整数。 优选 R为氢原子， n分别等于 2、 3和 4。
上述低级垸基是指具有 1~8个碳原子的直链或支链的垸基。例如： 甲基、 乙 基、 丙基、 异丙基、 丁基、 异丁基、 叔丁基、 仲丁基、 戊基、 新戊基、 己基、 庚 基、 辛基等。优选具有 1~4个碳原子的直链或支链的垸基， 特别优选甲基、 乙基 和丙基。
. 上述垸氧基是指具有 1~6个碳原子的直链或支链的烷氧基。 例如： 甲氧基、 乙氧基、 丙氧基、异丙氧基、 丁氧基、异丁氧基、叔丁氧基、仲丁氧基、戊氧基、 新戊氧基、 己氧基等。优选具有 1~4个碳原子的支链或直链的烷氧基， 特别优选 甲氧基和乙氧基。
上述酯基是指 -C (0) 0R， 其中 R是上述低级垸基。 优选甲酯基、 乙酯基、 丙 酯基和丁酯基。
优选的本发明化合物是其中 和 R2代表亚垸基。 最优选的是！^和 R2是碳原 子数为 1~8的亚垸基。
以透明质酸为例， 本发明的高分子巯基化改性衍生物具有下列化学结构特 征-
Figure imgf000005_0001
Figure imgf000005_0002
其中 1^和 的定义同前述； i是大于 0的整数， j是大于或等于 0的整数。 本发明通式 （I ) 或 （II ) 高分子巯基化改性衍生物的制备一般采用酰肼 / 碳二亚胺偶合化学方法。其基本原理是高分子化合物的侧链羧基在碳二亚胺的活 化下生成活性中间体， 然后二硫代二酰肼的氨基亲核进攻活性中间体生成加成 物， 最后加成物的二硫键被还原成自由巯基， 纯化即可得到产物。 为了制备本发明通式 （I ) 或 （Π ) 的高分子巯基化改性衍生物， 首先按照 本申请人己申请的发明专利（申请号： 200610118715. 2, 发明名称： 二酰肼化合 物及其制备方法和用途） 合成以下两类新型的二硫代二酰肼：
Figure imgf000006_0001
其中 和^的定义同前述。.这两类新型二硫代二酰肼的特征是含有一个二硫 键、 两个酰胺键和两个酰肼官能团， 可由相应的二硫代二酸和二硫代二氨制备。 以下是可用于合成本发明通式 （I ) 和 （II ) 的高分子巯基化改性衍生物的部分 二硫代二酰肼化合物的化学结构式：
Figure imgf000006_0002
Figure imgf000007_0001
其中， （1)是二硫代二丙酸双酰甘氨酸二酰肼（简称 DGDTPDH); (2)是二 硫代二丙酸双酰丙氨酸二酰肼 （简称 DADTPDH); (3) 是二硫代二丙酸双酰 （羟 基）氨基乙酸二酰肼（简称 DHADTPDH); (4)是二硫代二丙酸双酰氨基丙酸二酰 肼（简称 DPDTPDH)； (5)是二硫代二丙酸双酰氨基丁酸二酰肼（简称 DBDTPDH)； (6)是二硫代二丁酸双酰甘氨酸二酰肼（简称 DGDTBDH); (7)是二硫代二丁酸 双酰氨基丙酸二酰肼（简称 DPDTBDH) ； (8)是双丙二酸双酰胱胺二酰肼（简称 DPCDH) ； (9)是双琥珀酸双酰胱胺二酰肼（简称 DSCDH) ； (10)是双（甲基） 丁二酸双酰胱胺二酰肼（简称 DMPCDH) ； (11)是双戊二酸双酰胱胺二酰肼（简 称 DGCDH) ； (12)是双己二酸双酰胱胺二酰肼 （简称 DACDH) ； (13)是双庚二 酸双酰胱胺二酰肼 （简称 DHCDH) 。
本发明通式 （I) 和 （Π) 高分子巯基化改性衍生物的一种制备方法为： 高 分子化合物的侧链羧基在 1-乙基 -3- (3-二甲胺丙基)碳二亚胺盐酸盐的活化下与 二硫代二酰肼的一个或两个酰肼官能团生成加成物 最后加成物的二硫键被羟基 硫醇、 二硫苏糖醇或硼氢化钠等还原剂还原成自由巯基， 透析纯化除去杂质即可 得到本发明通式 （I) 或 （II) 的高分子巯基化改性衍生物。 以下是其化学合成 路径及化学结构式， 其中， R,和 R2是亚垸基、 取代亚垸基、 芳香基、 聚醚基等。 二亚胺盐^ k
Figure imgf000007_0002
二亚胺盐 ¾it
Figure imgf000008_0001
本发明通式 α)或（ιι)高分子巯基化改性衍生物， 当 和1?2都为亚垸基， 即为本发明的优选化合物， 其化学结构如下所示：
Figure imgf000008_0002
其中， （1 )代表 1^和1?2都为亚烷基的本发明通式 （I ) 高分子巯基化衍生物 结构通式； （2)代表 1^和1?2都为亚烷基的本发明通式（II )高分子巯基化衍生物 结构通式（i、 j、 m、 n都是大于 1的整数） 。
本发明最优选的通式 （I ) 或 （II ) 高分子巯基化改性衍生物， 其中 R2是碳 原子数为 2和 3的亚垸基， R,分别是碳原子数为 1~5的亚垸基， P为硫酸软骨素、 肝 素、 透明质酸和它们的盐形式（如钠盐， 钾盐等）、 碱性明胶蛋白、 酸性明胶蛋 白等的残基， 其化学结构式如下所示：
Figure imgf000008_0003
其中， （1 ) 是二硫代二丙酸双酰甘氨酸二酰肼改性高分子巯基化衍生物 (P-DGDTPDH) ； (2)是二硫代二丙酸双酰丙氨酸二酰肼改性高分子巯基化衍生 物（P-DADTPDH) ； (3 )是二硫代二丁酸双酰甘氨酸二酰肼改性高分子巯基化衍 生物（P-DGDTBDH) ； (4)是二硫代二丁酸双酰丙氨酸二酰肼改性高分子巯基化 衍生物（P-DADTBDH) ； (5)是双琥珀酸双酰胱胺二酰肼改性高分子巯基化衍生 物 （P-DSCDH ) ； ( 6 ) 是双戊二酸双酰胱胺二酰肼改性高分子巯基化衍生物 ( P-DGCDH ) ； (7)是双己二酸双酰胱胺二酰肼改性高分子巯基化衍生物 ( P-DACDH ) ； (8)是双庚二酸双酰胱胺二酰肼改性高分子巯基化衍生物 ( P-DHCDH) 。
巯基化改性制备过程不会显著改变分子量及其分布， 通常本发明通式 （I ) 或（Π )高分子巯基化改性衍生物的分子量及其分布与起始原料（侧链含羧基高 分子化合物）的分子量及其分布基本相同。不同起始原料（侧链含羧基高分子化 合物）的分子量差别较大， 通常在 1000到 500万之间； 其分子量分布也差别较大， 如明胶的分子量分布通常较宽。起始原料（侧链含羧基高分子化合物）.的分子量 及其分布并不会影响巯基化改性过程， 不会影响本发明通式 （I ) 或 （II ) 高分 子巯基化改性衍生物的制备。
本发明通式 （I ) 或 （Π ) 高分子巯基化改性衍生物具有很多有益效果。 本 发明采用酰肼键的化学结合方式进行巯基化改性， 具有制备条件温和、 产率高、 改性程度高且可控等许多显著优点。与 Shu等人在 Biomacromolecules, 3，. 1304, 2002和 W0 2004/03716所公开的高分子巯基化改性衍生物相比， 虽然都采用酰 肼键的化学结合方式进行巯基化改性， 但本发明通式 （I ) 或 （Π ) 高分子巯基 化改性衍生物创新性地引入了一个酰氨键， 侧链化学结构灵活多变、 性能可调。 研究表明本发明的通式 （I ) 或 （Π ) 高分子巯基化改性衍生物具有以下两大有 益效果：
( 1 ) 通过引入酰氨键的方式， 可以灵活调节侧链的长度和结构。 侧链的长 度将在很大程度上影响巯基的反应性能（Shu等， Biomaterials 24, 3825， 2003)。 侧链越长，端巯基和其他反应官能团的碰撞几率越高， 因而进一步使反应改性的 性能得到了很大的提髙。
(2) 酰氨键为强吸电子基团， 本发明化合物侧链酰氨键的引入， 在很大程 度上影响了端巯基的电离常数 （pKa)， 然而这与酰氨键的连接方式有关。 本发明 通式 （I ) 高分子巯基化改性衍生物的侧链酰氨键中的羰基与端巯基相近， 强化 了端巯基的电离 1^降低）； 与此相反， 本发明通式 （II ) 高分子巯基化改性衍 生物的侧链酰氨键的氮原子与端巯基相近， 弱化了端巯基的电离 ^！^升高）。一 般说来， 与 Shu等人在 Biomacromolecules, 3， 1304， 2002和 W0 2004/03716 所公开的侧链连接片断为亚垸基（碳数为 2和 3) 的高分子巯基化改性衍生物相 比， 本发明通式 （I ) 高分子巯基化改性衍生物的巯基电离常数 （pKJ 降低约 0. 1-0. 4, 而本发明通式 （II ) 高分子巯基化改性衍生物的巯基电离常数 （pKJ 则升高约 0. 2~0. 7。 因而本发明通式 （I ) 高分子巯基化改性衍生物的端巯基更 活泼， 而本发明通式 （Π ) 高分子巯基化改性衍生物的端巯基则更稳定。
本发明通式 （I ) 或 （II ) 高分子巯基化改性衍生物与己公开的高分子巯基 化改性衍生物相比， 具有许多独特的性能， 可以根据实际应用的需要， 选择合适 的化学结构。 以透明质酸（HA)的下述具体高分子巯基化衍生物为例来进一步说 明本发明通式 （I ) 或 （II ) 高分子巯基化改性衍生物的有益效果：
Figure imgf000010_0001
上述化合物在端巯基侧具有相同的结构， 其中 （a ) 是 Shu等人在 Biomacromolecules, 3， 1304， 2002和 WO 2004/03716所公开的高分子巯基化改 性衍生物； (b) 是本发明通式 （I ) 高分子巯基化改性衍生物； （c) 是本发明 通式 （II ) 高分子巯基化改性衍生物。 高分子巯基化改性衍生物 （b) 的巯基反 应活性比（a)高很多， 其形成双硫键交联凝胶的能力提高了约 50%; 而高分子巯 基化改性衍生物（c)的巯基反应活性比（a)低很多， 其巯基稳定性能提高了一 倍以上。
本发明通式（I ) 或 （II ) 的高分子巯基化改性衍生物， 具有至少一个侧链 自由巯基， 可以在合适的条件下重新氧化形成二硫键。 氧气、 低浓度过氧化氢、 碘、 铁三价离子等中强度氧化剂均可使自由巯基形成二硫键， 从而制备出高分子 交联材料。 二硫键的形成通常受溶液 pH值的影响： 在碱性条件下， 巯基电离成硫 负离子， 反映活性很高， 即使空气中的氧气也能快速促进二硫键的形成； 而在酸 性条件下， 巯基的电离受到抑制， 反应活性降低， 巯基相对稳定。本发明通式（I ) 的高分子巯基化改性衍生物的巯基较活 即使在中性的条件和弱氧化剂的作用 下也可生成二硫键交联材料； 而本发明通式（I I )的高分子巯基化改性衍生物的 巯基较稳定， 需要在弱碱性或较强氧化剂的作用下才能快速生成二硫键交联材 料。
本发明的二硫键交联的高分子巯基化改性衍生物交联材料的制备方法简单 可靠， 产物灵活多变。 常用的制备方法是将一种或多种牢发明通式（I )或（Π ) 的高分子巯基化改性衍生物制成水溶液或混合水溶液，在中性或弱碱性条件下用 室温空气氧化制备二硫键交联材料； 或者在弱酸性或酸性条件下， 用低浓度过氧 化氢、 铁三价离子等更强的氧化剂氧化制备二硫键交联材料。
本发明单组份和双组份的二硫键交联的高分子巯基化改性衍生物交联材料 用下述通式 （III ) 、 （IV) 或 （V ) 表示：
(V)
Figure imgf000011_0001
其中 和 P2都是侧链含羧基的高分子化合物残基， 其定义同前述。 ^和 R2 的定义同前述； R3和 R4的定义与 ^和 R2相同； 、 R2、 R3、 可以具有相同或不 相同的化学结构。 当 1^和 相同时， 即为单组份交联材料， 当 和！^不相同时， 即为双组份交联材料。
本发明的三组份或三组分以上的二硫键交联的高分子巯基化改性衍生物交 联材料可由三种或三种以上通式 （I ) 或 （I I ) 高分子巯基化改性衍生物制备， 其结构特征是包含三种或三种以上高分子化合物通过二硫键结合形成交联材料。
本发明的巯基反应活性交联剂交联的高分子巯基化改性衍生物交联材料由 一种或多种通式 （I ) 或 （II ) 的新颖高分子巯基化改性衍生物和巯基反应活性 交联剂制备。 本发明采用的巯基反应活性官能团包括马来酰亚胺、 乙烯砜、 α， β 不饱和丙烯酸酯、 α， β 不饱和甲基丙烯酸酯、 卤代丙酸酯、 卤代丙酰胺、 二硫代吡啶、 Ν-羟基丁二酰亚胺活化酯等等。 其中马来酰亚胺、 乙烯砜、 碘代丙 酸酯、碘代丙酰胺、二硫代吡啶等官能团具有很高巯基反应活性。 以本发明通式 ( I ) 高分子巯基化改性衍生物为例， 以下给出了巯基和这些官能团的化学反应 式， 其中， 和 是亚烷基、 取代亚烷基、 芳香基、 聚醚基等：
Figure imgf000012_0001
这些反应可以分为三类： （1 )巯基和活化不饱和双键的加成反应， 属于这类 反应的官能团包括马来酰亚胺、 乙烯砜、 α， β 不饱和丙烯酸酯、 α， β 不饱 和甲基丙烯酸酯等； （2 )巯基和活化卤代烷的取代反应， 属于这类反应的官能团 包括碘代丙酸酯、 溴代丙酸酯、 氯代丙酸酯、 碘代丙酰胺、 溴代丙酰胺、 氯代丙 酰胺、 二硫代吡啶等； （3 )最后一类是硫酯化反应， 这类反应得官能团包括各种 羧酸的活化酯， 如 N-羟基丁二酰亚胺活化酯等等。
本发明采用的巯基反应活性交联剂至少含有两个上述反应官能团的聚乙二 醇 （简称 PEG) 的衍生物， 如双臂、 三臂、 四臂、 八臂或更多臂的聚乙二醇衍生 物， 它们具有如下的典型化学结构：
G,— PEG— G2
双臂聚乙二醇交联剂
Gl、
Figure imgf000013_0001
三臂聚乙二醇交联剂 四臂聚乙二醇交联剂
Figure imgf000013_0002
八臂聚乙二醇交联剂
其中 G,、 G2、 G3、 G4、 G5、 G6、 G7和 G8为上述巯基反应活性官能团如马来酰亚 胺、 乙烯砜、 α , β 不饱和丙烯酸酯、 α， β 不饱和甲基丙烯酸酯、 卤代丙酸 酯、 卤代丙酰胺、 二硫代吡啶或 Ν-羟基丁二酰亚胺等等， 它们可以具有全部相 同、 部分相同或全部不相同的化学结构； PEG是指分子量为 100到 1000000的具 有 CH2CH20重复单元的链段。
以双臂的聚乙二醇为例，以下是本发明采用的常见交联剂——双臂聚乙二醇 巯基反应活性交联剂的化学结构式：
聚
Figure imgf000013_0003
乙二醇二马来跣亚胺 聚乙二醇二乙烯砜
Figure imgf000013_0004
乙二酵二二砥代吡啶
聚乙二醇二 N- 基丁二跣亚胺 本发明的巯基反应活性交联剂交联的高分子巯基化改性衍生物交联材料的 通常制备方法包括将一种或多种通式 （I ) 或 （Π ) 的新颖高分子巯基化改性衍 生物制成水溶液或混合水溶液， 调节溶液的 pH值为中性， 然后加入上述巯基反 应活性交联剂的水溶液， 混合均匀后室温静置片刻即形成凝胶， 得到交联材料。 本发明通式 （I ) 的高分子巯基化改性衍生物的巯基较活泼， 与交联剂的反应较 快; 而本发明通式（II )的高分子巯基化改性衍生物的巯基较稳定， 与交联剂的 反应则相对较慢。
以通式 （I ) 高分子巯基化改性衍生物和上述双臂聚乙二醇巯基反应活性交 联剂为例，本发明的单组份和双组份的巯基反应活性交联剂交联的高分子巯基化 改性衍生物交联材料具有如下所示的结构：
Figure imgf000014_0001
Figure imgf000015_0001
聚乙二醇二 N-羟基丁二酜亚胺交联
其中 Ρ^Π Ρ2都是侧链含羧基的高分子化合物残基， 其定义同前述； R,、 R2、 R3和 R4的定义同前述； 、 R2、 R3、 R4可以具有相同或不相同的化学结构。 当 P, 和 P2相同时， 即为单组份交联材料， 当！^和！^不相同时， 即为双组份交联材料。 也可以采用两种或两种以上的上述双臂聚乙二醇巯基反应活性交联剂共同交联 制备高分子巯基化改性衍生物交联材料。另外也可以采用一种或多种多臂聚乙二 醇衍生物交联剂 （如三臂聚乙二醇衍生物交联剂、 四臂聚乙二醇衍生物交联剂、 八臂聚乙二醇衍生物交联剂等等）来制备高分子巯基化改性衍生物交联材料。巯 基反应活性交联剂交联的通式（Π )高分子巯基化改性衍生物交联材料和通式（I ) 高分子巯基化改性衍生物交联材料具有类似的结构。
本发明的三组份或三组分以上的巯基反应活性交联剂交联的高分子巯基化 改性衍生物交联材料可由三种或三种以上通式 （I ) 或 （Π ) 高分子巯基化改性 衍生物制备。 通常的制备途径是首先制备出三种或三种以上通式 （I ) 或 （Π ) 高分子巯基化改性衍生物的混合溶液， 然后调节溶液的 pH值为中性， 加入一种 或多种上述聚乙二醇巯基反应活性衍生物交联剂， 从而制备出多组份交联材料。 附图说明：
图 1是本发明实施例 4中低取代衍生物的氢原子核磁共振谱图和重要化学位 移峰的归属 （D20为溶剂） ；
图 2是本发明实施例 4中高取代衍生物的氢原子核磁共振谱图和重要化学位 移峰的归属 （D20为溶剂） ；
图 3是本发明实施例 20中细胞在空白细胞培养板表面的形貌示意图； 图 4是本发明实施例 20中细胞在聚乙二醇二丙烯酸酯交联透明质酸-明胶双 组分水凝胶表面的形貌示意图。
实现本发明的最佳方式：
下面的实施例可以使本领域技术人员更全面地理解本发明，但不以任何方式 限制本发明。
实施例 1. 合成二硫代二丙酸双酰甘氨酸二酰肼 （简称 DGDTPDH) 在 1000 毫升烧杯中加入 10克二硫代二丙酸 （Aldrich, 美国）， 50毫升无 水二甲基甲酰胺。 室温搅拌溶解后， 加入 17. 0克羰二咪唑 （Aldrich, 美国） 。 此时溶液产生大量二氧化碳气泡和白色沉淀。 室温减压反应 3 小时。 然后加入 14. 7 克甘氨酸乙酯盐酸盐（Aldrich, 美国）， 搅拌反应 1小时。然后在加入 500 毫升乙醚， 静止 1小时。 小心倒去上层有机相， 然后加入 100毫升乙醇和 10毫 升水合肼。 室温搅拌过夜， 过滤收集沉淀产物。沉淀用 200毫升无水乙醇淋洗两 次， 然后真空减压干燥得到略带黄色固体产物 DGDTPDH约 8. 5克， 产率约 50%。
实施例 2. 低取代 DGDTPDH改性透明质酸巯基化衍生物（HA-DGDTPDH) 的合 成
透明质酸钠 （分子量 62~115万， NovaMatrix FMC BI0P0LYMER, 美国） 1克 溶解于 200毫升蒸馏水中， 得到澄清透明溶液。 在上述溶液中加入 1. 32克实施 例 1制备的 DGDTPDH，搅拌溶解。然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 0. 36克 1-乙基 -3- (3-二甲胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国） ， 电 磁搅拌。 在上述溶液中不断加入适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 15分钟左右形成凝胶。 凝胶形成后， 室温静置 反应 2小时。然后加入 10克二硫苏糖醇 (Diagnostic Chemical Limited, 美国） 和少量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。 凝胶逐渐溶解， 同时不断加入 0. 1 摩尔 /升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电 磁搅拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001摩尔 / 升的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后 再用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收集透 析管内的溶液， 冷冻干燥得到白色絮状固体约 0. 7克。
实施例 3. 高取代 DGDTPDH改性透明质酸巯基化衍生物（HA-DGDTPDH) 的合 成
透明质酸钠 （分子量 62〜115万， NovaMatrix FMC BI0P0LYMER, 美国） 1克 溶解于 200毫升蒸馏水中， 得到澄清透明溶液。 在上述溶液中加入 2. 64克实施 例 1制备的 DGDTPDH,搅拌溶解。然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 0. 96克 1-乙基 -3- (3-二甲胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国） ， 电 磁搅拌。 在上述溶液中不断加入适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 10分钟左右形成凝胶。 凝胶形成后， 室温静置 反应 2小时。然后加入 20克二硫苏糖醇 (Diagnostic Chemical Limited, 美国） 和少量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。 凝胶逐渐溶解， 同时不断加入 0. 1 摩尔 /升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电 磁搅拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001摩尔 / 升的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后 再用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收集透 析管内的溶液， 冷冻干燥得到白色絮状固体约 0. 7克。
实施例 4. DGDTPDH改性透明质酸巯基化衍生物 （HA-DGDTPDH) 的表征
HA-DGDTPDH的化学结构式如下:
Figure imgf000017_0001
对实施例 2和实施例 3制备的 HA-DGDTPDH进行表征：
1、 凝胶液相色谱（GPC)检测（纯水为流动相， 紫外 210纳米吸收检测）均 未发现小分子杂质流出峰， 表明实施例 2 和实施例 3 制备的低取代和高取代 HA-DGDTPDH为高度纯化， 杂质低于仪器检测水平。
2、 氢谱核磁共振检测 （ -NMR) (D20为溶剂）， 谱图和化学位移峰的归属如 图 1和图 2所示， HA- DGDTPDH在 δ 3. 96、 2. 70、 2. 56 ppm出现了三个新的吸 收峰， 分别对应于 fl¾NHC (0) CH2CH2SH、 CH2NHC (0) C¾CH2SH和 CH2NHC (0) CH2<¾SH 三个侧链亚甲基的氢原子吸收。 化学位移为 δ 2. 8 ppm左右的小吸收峰为少量 副反应产物的吸收峰。 以透明质酸的乙酰基的特征甲基吸收峰为内标，根据吸收峰的面积计算出实 施例 2制备的低取代 HA-DGDTPDH的侧链取代度为 27%， 实施例 3制备的高取代 HA-DGDTPDH的侧链取代度为 59%。
3、 分子量及其分布测定 （GPC测定， 以单分散透明质酸校正标准曲线）： 实 施例 2制备的低取代 HA- DGDTPDH 重均分子量（MJ 102万， 数均分子量（M„) 53 万， 分子量分布 1. 92; 实施例 3制备的高取代 HA- DGDTPDH重均分子量（M，） 123 万， 数均分子量 （Mn) 58万， 分子量分布 2. 12。
4、采用 Shu等人在 Biomacromolecules, 3， 1304, 2002中报道的改进 Ellman 方法检测实施例 2和实施例 3制备的 HA- DGDTPDH的活性巯基含量。 实施例 2制 备的低取代 HA-DGDTPDH的侧链活性巯基含量 25. 4个巯基 /100个透明质酸二糖 重复单元，实施例 3制备的高取代 HA- DGDTPDH的侧链活性巯基含量为 55. 1个巯 基 /100个透明质酸二糖重复单元。 基本与氢谱核磁共振检测结果相符。
实施例 5. 合成二硫代二丙酸双酰氨基丙酸二酰肼 （简称 DADTPDH) 在 1000 毫升烧杯中加入 10 克二硫代二丙酸（Aldrich, 美国）， 50毫升无 水二甲基甲酰胺。 室温搅拌溶解后， 加入 17. 0 克羰二咪唑（Aldrich, 美国） 。 此时溶液产生大量二氧化碳气泡和白色沉淀。 室温减压反应 3 小时。 然后加入 14. 7 克氨基丙酸乙酯盐酸盐 （Aldrich, 美国）， 搅拌反应 1小时。 然后再加入 500 毫升乙醚， 静止 1 小时。 小心倒去上层有机相， 然后加入 100毫升乙醇和 10毫升水合肼。 室温搅拌过夜， 过滤收集沉淀产物。 沉淀用 200毫升无水乙醇 淋洗两次，然后真空减压干燥得到略带黄色固体产物 DADTPDH约 7. 3克，产率约 40%。
实施例 6. DADTPDH改性透明质酸巯基化衍生物 （HA-DADTPDH) 的合成和表 征
透明质酸钠 (分子量 62 115万， Nova atrix F C BI0P0LYMER, 美国） 1克 溶解于 200毫升蒸馏水中， 得到澄清透明溶液。 在上述溶液中加入 1. 43克实施 例 5制备的 DADTPDH,搅拌溶解。然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 0. 48克 1-乙基. -3- (3-二甲胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国） ， 电 磁搅拌。 在上述溶液中不断加入适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 10分钟左右形成凝胶。 凝胶形成后， 室温静置 反应 2小时。然后加入 12克二硫苏糖醇 (Diagnostic Chemical Limited, 美国） 和少量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。 凝胶逐渐溶解， 同时不断加入 0. 1 摩尔 /升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电 磁搅拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001摩尔 / 升的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后 再用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收集透 析管内的溶液， 冷冻干燥得到白色絮状固体约 0. 7克。
GPC检测 （纯水为流动相， 紫外 210纳米吸收检测）均未发现小分子杂质流 出峰， 表明合成的 HA-DADTPDH为高度纯化， 杂质低于仪器检测水平。
氢谱核磁共振检测（ - NMR) (D20为溶剂）。 HA- DADTPDH在 δ 3. 4、 2. 66 ppm 出现了两个新吸收峰分别对应于 CH2G¾NHC (0) CH2CH2SH 和 CH2CH2NHC (0) CH2<¾SH 两个侧链亚甲基的氢原子吸收； G¾¾NHC (0) CH2CH2SH和 CH2CH2NHC (0) <¾CH2SH两 个侧链亚甲基的氢原子吸收峰在 δ 2. 5 ppm左右相互重叠。 化学位移为 δ 2. 8 ppm左右的小吸收峰为少量副反应产物的吸收峰。
以透明质酸的乙酰基的特征甲基吸收峰为内标，根据吸收峰的面积计算出合 成的 HA-DGDTPDH的侧链取代度为 61%。
用 GPC测定分子量及其分布测定 （以单分散透明质酸校正标准曲线）： 重均 分子量 （M„) 122万， 数均分子量 （MJ 67万， 分子量分布 1. 82。
采用 Shu等人在 Biomacromolecules, 3， 1304， 2002中报道的改进 Ellman 方法检测 HA-DADTPDH的活性巯基含量： 53. 6个巯基 /100个透明质酸二糖重复单 元， 稍低于氢谱核磁共振检测结果。
实施例 7. 合成双琥珀酸双酰胱胺二酰肼 （简称 DSCDH)
胱胺二盐酸盐（Aldrich, 美国） 100克溶解于 1500毫升蒸馏水， 得到澄清 透明溶液。 在上述溶液中加入 4 摩尔 /升的氢氧化钠直至溶液 pH值为 10。 然后 在电磁搅拌下加入 133 克琥珀酸酐（Aldrich, 美国）， 同时不断加入 4摩尔 /升 的氢氧化钠使溶液的 pH值保持在 7~10。 室温反应 2小时后， 在溶液中加入 6摩 尔 /升的盐酸。。 过滤收集白色沉淀产物， 用 2000毫升蒸熘水洗两次。 然后真空 减压干燥， 得到白色产物固体产物合成双琥珀酸双酰胱胺酸 （简称 DSC) 约 150 克， 产率大于 90%。
在 250毫升三颈圆底烧瓶中加入 100克 DSC， 1200毫升无水乙醇和 100滴浓 硫酸。氮气保护下回流 2小时， 然后减压浓缩至小于 200毫升。剩余溶液转移到 2500毫升分液漏斗， 然后加入 600毫升乙酸乙酯。 有机相用 500毫升水洗三次， 弃去水相， 有机相减压蒸馏得到白色腊状固体产物双琥珀酸双酰胱胺酸二乙酯 (简称 DSCDE) 约 93克， 产率大于 80%。
在 150毫升烧杯中加入 10克 DSCDE, 80毫升乙醇。 室温搅拌溶解后再加入 10 毫升水合肼 （Aldrich, 美国）， 反应过夜。 过滤收集白色沉淀产物， 然后用 40 毫升乙醇淋洗四次。 室温通风厨中挥发有机溶剂后， 真空减压干燥， 得到白 色产物固体产物 DSCDH约 8克， 产率大于 75%。
实施例 8. DSCDH改性透明质酸巯基化衍生物 （HA-DSCDH) 的合成和表征 透明质酸钠 （分子量 62~115万， NovaMatrix FMC BI0P0LYMER, 美国） 1克 溶解于 200毫升蒸馏水中， 得到澄清透明溶液。 在上述溶液中加入 0. 95克实施 例 7制备的 DSCDH,搅拌溶解。然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75, 加入 0. 288克 1-乙基 -3- (3-二甲胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国） ， 电 磁搅拌。 在上述溶液中不断加入适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 10分钟左右形成凝胶。 凝胶形成后， 室温静置 反应 2小时。然后加入 10克二硫苏糖醇 (Diagnostic Chemical Limited, 美国） 和少量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。 凝胶逐渐溶解， 同时不断加入 0. 1 摩尔 /升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电 磁搅拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001摩尔 / 升的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后 再用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收集透 析管内的溶液， 冷冻干燥得到白色絮状固体约 0. 7克。
GPC检测 （纯水为流动相， 紫外 210纳米吸收检测）均未发现小分子杂质流 出峰， 表明合成的 HA- DSCDH为高度纯化， 杂质低于仪器检测水平。
氢谱核磁共振检测 (Ή-NMR) (D20为溶剂）。 HA- DSCDH在 δ 3. 26、 2. 5 ppm 出现了两个新吸收峰。其中 δ 3. 26 111的新吸收峰对应于(： (¾ (0) 1^<¾(¾5^1 一个侧链亚甲基氢原子吸收； <¾CH2C (0) NHCH2CH2SH、 CH2fi¾C (0) NHCH2CH2SH 和 CH2CH2C (0) NHCH2G¾SH的三个侧链亚甲基的氢原子吸收则重叠在 δ 2. 5 ppm。
以透明质酸的乙酰基的特征甲基吸收峰为内标，根据吸收峰的面积计算出合 成的 HA- DGDTPDH的侧链取代度为 38%。
采用 Shu等人在 Biomacromolecules, 3， 1304， 2002中报道的改进 Ellman 方法检测 HA-DSCDH的活性巯基含量: 39. 1个巯基 /100个透明质酸二糖重复单元， 基本与氢谱核磁共振检测结果相符。
实施例 9. DSCDH改性硫酸软骨素巯基化衍生物 （CS- DSCDH) 的合成和表征 硫酸软骨素（c型， 来自鲨鱼软骨， Sigma, 美国） 1克溶解于 100毫升蒸馏 水中， 得到澄清透明溶液。 在上述溶液中加入 0. 704克实施例 7制备的 DSCDH, 搅拌溶解。 然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 0. 192克 1 - 乙基 -3- (3-二甲胺丙基)碳二亚胺盐酸盐^1(11" 11， 美国） ， 电磁搅拌。 在上述 溶液中不断加入适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75， 室温电磁 搅拌反应 2小时。 然后加入 10克二硫苏糖醇 (Diagnostic Chemical Limited, 美国)和少量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。凝胶逐渐溶解， 同时不断加入 0. 1摩尔 /升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室 温电磁搅拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001 摩尔 /升的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后再用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收 集透析管内的溶液， 冷冻干燥得到白色絮状固体约 0. 6克。
GPC检测 （纯水为流动相， 紫外 210纳米吸收检测）均未发现小分子杂质流 出峰， 表明合成的 CS- DSCDH为高度纯化， 杂质低于仪器检测水平。
氢谱核磁共振检测 CH-NMR) (D20为溶剂）。 CS- DSCDH在 δ 3. 27、 2. 54 ppm 出现了两个新吸收峰。其中 δ 3. 27 111的新吸收峰对应子（¾(¾ (0) 6¾012511 侧链亚甲基氢原子吸收； fl¾CH2C (0) NHCH2CH2SH、 CH2i¾C (0) NHCH2CH2SH 和 CH2CH2C (0) NHCH2C¾SH的三个侧链亚甲基的氢原子吸收则重叠在 δ 2. 54 ppm。
以硫酸软骨素的乙酰基的特征甲基吸收峰为内标，根据吸收峰的面积计算出 合成的 CS-DGDTPDH的侧链取代度为 47%。 用 GPC测定分子量及其分布测定 （以单分散透明质酸校正标准曲线）： 重均 分子量 （M„) 3. 8万， 数均分子量 （Mn) 1. 7万， 分子量分布 2. 23。
采用 Shu等人在 Biomacromolecules, 3， 1304， 2002中报道的改进 Ellman 方法检测 CS- DSCDH的活性巯基含量： 44. 2个巯基 /100个硫酸软骨素二糖重复单 元， 稍低于氢谱核磁共振检测结果。
实施例 10. DSCDH改性明胶巯基化衍生物 （GEL-DSCDH) 的合成和表征 明胶（B型， 来自猪皮， Sigma, 美国） 1克溶解于 100毫升蒸馏水中， 得到 澄清透明溶液。 在上述溶液中加入 0. 75克实施例 7制备的 DSCDH, 搅拌溶解。 然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 1克 1-乙基 -3- (3-二甲 胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国) ， 电磁搅拌。 在上述溶液中不断加入 适量 0. 1摩尔 /升盐酸， 使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 10分钟左右形成凝胶。 凝胶形成后， 室温静置反应 2小时。 然后加入 10克二硫 苏糖醇 (Diagnostic Chemical Limited, 美国）和少量 0. 1摩尔 /升的氢氧化钠 溶液， 搅拌。 凝胶逐渐溶解， 同时不断加入 0. 1摩尔 /升的氢氧化钠溶液使溶液 的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电磁搅拌反应 24小时。 此后， 在 上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上述溶液装入透析管 （截除分 子量 3500, Sigma, 美国）， 用 10升 0. 001摩尔 /升的盐酸和 0. 3摩尔 /升的氯化 钠溶液透析 5天，每 8小时换一次透析液；然后再用 10升 0. 001摩尔 /升的盐酸 溶液透析 3天， 每 8小时换透析液。最后收集透析管内的溶液， 冷冻干燥得到白 色絮状固体约 0. 6克。
GPC检测 （纯水为流动相， 紫外 210纳米吸收检测）均未发现小分子杂质流 出峰， 表明合成的 GEL-DSCDH为高度纯化， 杂质低于仪器检测水平。
氢谱核磁共振检测 CH-NMR) (D20为溶剂）。 GEL- DSCDH在 δ 3. 27、 2. 54 ppm 出现了两个新的强吸收峰。 其中 δ 3. 28 ppm 的新吸收峰对应于 CH2CH2C (0) NHG¾CH2SH 侧链亚甲基氢原子吸收； （¾CH2C (0) NHCH2CH2SH、 CH2<¾C (0) NHCH2CH2SH和 CH2CH2C (0) NHCH2<¾SH的三个侧链亚甲基的氢原子吸收则 重叠在 δ 2. 53 ppm。
用 GPC测定分子量及其分布测定 （以标准分子量聚乙二醇校正标准曲线）： 重均分子量 （MJ 5. 6万， 数均分子量 （M„) 2. 1万， 分子量分布 2. 67。 采用 Shu等人在 Biomacromolecules, 3， 1304， 2002中报道的改进 Ellman 方法检测 GEL- DSCDH的活性巯基含量： 0. 57毫摩尔巯基 /克 GEL_DSCDH。
实施例 11. 合成双戊二酸双酰胱胺酸二酰肼 （简称 DGCDH)
胱胺二盐酸盐 （Aldrich, 美国） 100克溶解于 1500毫升蒸熘水， 得到澄清 透明溶液。 在上述溶液中加入 4 摩尔 /升的氢氧化钠直至溶液 pH值为 10。 然后 在电磁搅拌下加入 152克戊二酸酐 （Aldrich, 美国）， 同时不断加入 4摩尔 /升 的氢氧化钠使溶液的 pH值保持在 7〜10。 室温反应 2小时后， 在溶液中加入 6摩 尔 /升的盐酸。过滤收集白色沉淀产物， 用 2000毫升蒸馏水洗两次。然后真空减 压干燥， 得到白色产物固体产物双戊二酸双酰胱胺酸（简称 DGC) 约 155克， 产 率大于 90%。
在 2500毫升三颈圆底烧瓶中加入 100克 DGC， 1200毫升无水乙醇和 100滴 浓硫酸。氮气保护下回流 2小时， 然后减压浓缩至小于 200毫升。剩余溶液转移 到 2500毫升分液漏斗， 然后加入 600毫升乙酸乙酯。 有机相用 500毫升水洗三 次， 然后减压蒸馏得到白色腊状固体产物双戊二酸双酰胱胺酸二乙酯 （简称 DGCDE) 约 94克， 产率大于 80%。
在 150毫升烧杯中加入 10克 DGCDE, 80毫升乙醇。 室温搅拌溶解后再加入 10毫升水合肼， 反应过夜。 过滤收集白色沉淀产物， 然后用 40毫升乙醇淋洗四 次。 室温通风厨中挥发有机溶剂后， 真空减压干燥， 得到白色产物固体产物双戊 二酸双酰胱胺酸二酰肼 （简称 DGCDH) 约 7. 1克， 产率大于 75%。
实施例 12. DGCDH改性透明质酸巯基化衍生物 （HA-DGCDH) 的合成和表征 透明质酸钠 （分子量 62~115万， NovaMatrix FMC BI0P0LYMER, 美国） 1克 溶解于 200毫升蒸馏水中， 得到澄清透明溶液。 在上述溶液中加入 1. 53克实施 例 11制备的 DGCDH，搅拌溶解。然后溶液的 pH值用 0. 1摩尔 /升盐酸调节至 4. 75， 加入 0. 48克 1-乙基- 3- (3-二甲胺丙基)碳二亚胺盐酸盐 (Aldrich, 美国）， 电磁 搅拌。在上述溶液中不断加入适量 0. 1摩尔 /升盐酸，使溶液的 pH值保持在 4. 75。 溶液黏度不断增加， 并在 10分钟左右形成凝胶。 凝胶形 后， 室温静置反应 2 小时。 然后加入 10克二硫苏糖醇 （Diagnostic Chemical Limited, 美国）和少 量 0. 1摩尔 /升的氢氧化钠溶液， 搅拌。凝胶逐渐溶解， 同时不断加入 0. 1摩尔 / 升的氢氧化钠溶液使溶液的 pH值保持在 8. 5。 待凝胶全部溶解后， 室温电磁搅 拌反应 24小时。 此后， 在上述溶液中加入 6摩尔 /升的盐酸直至约 pH 3. 0。 上 述溶液装入透析管 （截除分子量 3500， Sigma, 美国）， 用 10升 0. 001摩尔 /升 的盐酸和 0. 3摩尔 /升的氯化钠溶液透析 5天， 每 8小时换一次透析液； 然后再 用 10升 0. 001摩尔 /升的盐酸溶液透析 3天，每 8小时换透析液。最后收集透析 管内的溶液， 冷冻干燥得到白色絮状固体约 0. 7克。
GPC检测 （纯水为流动相， 紫外 210纳米吸收检测）均未发现小分子杂质流 出峰， 表明合成的 HA-DGCDH为高度纯化， 杂质低于仪器检测水平。
氢谱核磁共振检测 （ - NMR) (D20为溶剂）。 HA-DGCDH在 δ 3. 23、 2. 56、 2. 3 ppm出现了三个新吸收峰。 其中 δ 3. 23、 2. 56 ppm的新吸收峰分别对应于 CH2CH2CH2C (0) NHfl¾CH2SH和 CH2CH2CH2C (0) NHCH2G¾SH两个侧链亚甲基氢原子吸收； <¾CH2CH2C (0) NHCH2CH2SH和 CH2CH2G¾ (0) NHCH2CH2SH两个侧链亚甲基的氢原子吸 收峰则重叠在 δ 2. 3 ppm左右； CH26¾CH2C (0) NHCH2CH2SH链亚甲基的氢原子吸收 峰则和透明质酸的乙酰基的特征甲基吸收峰在 δ 3. 23 ppm附近重叠。
以透明质酸的乙酰基的特征甲基吸收峰为内标，根据吸收峰的面积计算出合 成的 HA- DGDTPDH的侧链取代度为 52%。 、
采用 Shu等人在 Biomacromolecules, 3， 1304, 2002中报道的改进 Ellman 方法检测 HA-DSCDH的活性巯基含量： 49. 4个巯基 /100个透明质酸二糖重复单元， 基本与氢谱核磁共振检测结果相符。
实施例 13. 单组分二硫键交联水凝胶的制备
1、 二硫键交联透明质酸水凝胶的制备： 实施例 3制备的本发明透明质酸巯 基化衍生物（HA-DGDTPDH)O. 1克溶解于 10 毫升 0. 1摩尔 /升的憐酸盐缓冲液（pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1 摩尔 /升的氢氧化钠直至 pH 7. 4。 然后上述溶液倒入 25毫升玻璃烧杯， 室温静置 12小时， 溶液黏度逐渐 增加并形成凝胶。
2、二硫键交联明胶水凝胶的制备： 实施例 10制备的本发明明胶巯基化衍生 物 （GEL-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液 （PH 7. 0 ) 得到澄清透明溶液，在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 然后上述溶液倒入 25毫升玻璃烧杯，室温静置 12小时，溶液黏度逐渐增加并形 成凝胶。 实施例 14. 多组分二硫键交联水凝胶的制备
1、二硫键交联透明质酸-明胶双组分水凝胶的制备： 实施例 3制备的本发明 透明质酸巯基化衍生物（HA- DGDTPDH) 0. 1克溶解于 10 毫升 0. 1摩尔 /升的磷酸' 盐缓冲液 （pH 7. 0 ) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的 氢氧化钠直至 pH 7. 4。 实施例 10制备的本发明明胶巯基化衍生物 （GEL-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液（pH 7. 0 )得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 然后上述两种溶液 同时倒入 50毫升玻璃烧杯， 电磁搅拌 10分钟。 此后室温静置 12小时， 溶液黏 度逐渐增加并形成凝胶。
2、二硫键交联硫酸软骨素-明胶双组分水凝胶的制备： 实施例 9制备的本发 明硫酸软骨素巯基化衍生物（CS- DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷 酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升 的氢氧化钠直至 pH 7. 4。实施例 10制备的本发明明胶巯基化衍生物 (GEL- DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液（pH 7. 0)得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 然后上述两种溶液 同时倒入 50毫升玻璃烧杯， 电磁搅拌 10分钟。 此后室温静置 12小时， 溶液黏 度逐渐增加并形成凝胶。
3、 二硫键交联透明质酸-硫酸软骨素-明胶三组分水凝胶的制备： 实施例 3 制备的本发明透明质酸巯基化衍生物 （HA-DGDTPDH) 0. 1克溶解于 10 毫升 0. 1 摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 实施例 9制备的本发明硫酸软骨素巯基化 衍生物（CS- DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液（pH 7. 0) 得到澄清透明溶液，在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 实施例 10制备的本发明明胶巯基化衍生物 （GEL- DSCDH) 0. 3克溶解于 10 毫升 0. 1 摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入 适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 然后上述三种溶液同时倒入 50毫升 玻璃烧杯， 电磁搅拌 10分钟。此后室温静置 12小时， 溶液黏度逐渐增加并形成 凝胶。
实施例 15. 单组分聚乙二醇二乙烯砜交联水凝胶的制备 1、 聚乙二醇二乙烯砜交联透明质酸水凝胶的制备： 实施例 3制备的本发明 透明质酸巯基化衍生物 （HA- DGDTPDH) 0. 1克溶解于 10 毫升 0. 1摩尔 /升的磷 酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升 的氢氧化钠直至 pH 7. 4。聚乙二醇二乙烯砜（分子量 3400， Nektar Therapeutics, 美国） 0. 1克溶解于 2. 5毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清 透明溶液。然后上述 2. 5毫升聚乙二醇二乙烯砜溶液加入到 10毫升 HA-DGDTPDH 溶液， 立即电磁搅拌 30秒， 室温静置 30分钟。 溶液黏度逐渐增加并形成凝胶。
2、 聚乙二醇二乙烯砜交联硫酸软骨素水凝胶的制备： 实施例 9制备的本发 明硫酸软骨素巯基化衍生物（CS-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷 酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升 的氢氧化钠直至 pH 7. 4。聚乙二醇二乙烯砜（分子量 3400, Nektar Therapeutics, 美国） 0. 1克溶解于 2. 5毫升 0. 1摩尔 /升的憐酸盐缓冲液 （pH 7. 0 ) 得到澄清 透明溶液。 然后上述 2. 5毫升聚乙二醇二乙烯砜溶液加入到 10毫升 CS-DSCDH 溶液， 立即电磁搅拌 30秒， 室温静置 30分钟。 溶液黏度逐渐增加并形成凝胶。
3、聚乙二醇二乙烯砜交联明胶水凝胶的制备： 实施例 10制备的本发明明胶 巯基化衍生物 （GEL-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液
(pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠 直至 pH 7. 4。 聚乙二醇二乙烯砜 （分子量 3400， Nektar Therapeutics, 美国） 0. 1克溶解于 2. 5毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶 液。然后上述 2. 5毫升聚乙二醇二乙烯砜溶液加入到 10毫升 CS- DSCDH溶液，立 即电磁搅拌 30秒， 室温静置 30分钟。 溶液黏度逐渐增加并形成凝胶。
实施例 16. 多组分聚乙二醇二丙烯酸酯交联水凝胶的制备
1、 聚乙二醇二丙烯酸酯交联透明质酸-明胶双组分水凝胶的制备： 实施例 3 制备的本发明透明质酸巯基化衍生物 （HA- DGDTPDH) 0. 1克溶解于 10 毫升 0. 1 摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。实施例 10制备的本发明明胶巯基化衍生物
(GEL-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0 ) 得 到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 聚乙二醇二丙烯酸酯 （分子量 3400， Nektar Therapeutics, 美国） 0. 2克溶解 于 5毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液。 然后上述 10毫升 HA- DGDTPDH溶液、 10毫升 GEL- DSCDH溶液、 5毫升聚乙二醇二丙烯酸酯 溶液同时倒入 50毫升玻璃烧杯， 立即电磁搅拌 30秒。 室温静置 30分钟。 溶液 黏度逐渐增加并形成凝胶。
2、聚乙二醇二丙烯酸酯交联硫酸软骨素-明胶双组分水凝胶的制备：实施例 9制备的本发明硫酸软骨素巯基化衍生物（CS-DSCDH) 0. 3克溶解于 10 毫升 0. 1 摩尔 /升的磷酸盐缓冲液 （pH 7. 0 ) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。实施例 10制备的本发明明胶巯基化衍生物
(GEL-DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0 ) 得 到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 聚乙二醇二丙烯酸酯 （分子量 3400, Nektar Therapeutics, 美国） 0. 2克溶解 于 5毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0 ) 得到澄清透明溶液。 然后上述 10毫升 CS-DSCDH溶液、 10毫升 GEL- DSCDH溶液、 5毫升聚乙二醇二丙烯酸酯溶 液同时倒入 50毫升玻璃烧杯， 立即电磁搅拌 30秒。 室温静置 30分钟。 溶液黏 度逐渐增加并形成凝胶。
3、 聚乙二醇二丙烯酸酯交联透明质酸 -硫酸软骨素-明胶三组分水凝胶的制 备： 实施例 3制备的本发明透明质酸巯基化衍生物（HA-DGDTPDH) 0. 1克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液（pH 7. 0)得到澄清透明溶液， 在上述溶液 中加入适量 0. 1摩尔 /升的氢氧化钠直至 pH 7. 4。 实施例 9制备的本发明硫酸软 骨素巯基化衍生物（CS- DSCDH) 0. 3克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲 液 （pH 7. 0 ) 得到澄清透明溶液， 在上述溶液中加入适量 0. 1摩尔 /升的氢氧化 钠直至 pH 7. 4。 实施例 10 制备的本发明明胶巯基化衍生物 （GEL-DSCDH) 0. 3 克溶解于 10 毫升 0. 1摩尔 /升的磷酸盐缓冲液 （pH 7. 0) 得到澄清透明溶液， 在上述溶 中加入适量 0. 1摩尔 /升的氢氧化钠直至 ΡΗ 7. 4。 聚乙二醇二丙烯酸 酯 (分子量 3400， Nektar Therapeutics, 美国） 0. 3克溶解于 7. 5毫升 0. 1摩 尔 /升的磷酸盐缓冲液（pH 7. 0 )得到澄清透明溶液。然后上述 10毫升 HA- DGDTPDH 溶液、 10毫升 CS-DSCDH溶液、 10毫升 GEL- DSCDH溶液、 7. 5毫升聚乙二醇二丙 烯酸酯溶液同时倒入 50毫升玻璃烧杯，立即电磁搅拌 30秒。室温静置 30分钟, . 溶液黏度逐渐增加并形成凝胶。 实施例 17. 二硫键交联透明质酸水凝胶用于抑制细胞的粘附 按照实施例 13在 24孔标准细胞培养板内制备二硫键交联透明质酸水凝胶， 每孔为 1 毫升。 12小时后整块细胞培养板浸泡在 75%的酒精溶液消毒 2小时。 此后细胞培养板用无菌生理盐水浸洗三次。 每孔加入 1毫升细胞培养液 （DMEM, 10%牛血清） 和 2万个 NIH 3T3成纤细胞。 37摄氏度、 二氧化碳细胞培养箱培养 24 小时。 显微镜观察发现绝大部分成纤细胞悬浮在二 键交联透明质酸水凝胶 表面， 不能黏附铺展； 而在空白细胞培养板孔内， 成纤细胞黏附在底部， 呈纺锤 形。这个结果表明二硫键交联透明质酸水凝胶可以抑制细胞的黏附，可用于外科 手术后粘连的预防和治疗。
实施例 18. 二硫键交联透明质酸 -明胶二组份水凝胶作为细胞粘附生长的 基质
按照实施例 14在 24孔标准细胞培养板内制备二硫键交联透明质酸-明胶二 组份水凝胶， 每孔为 1 毫升。 12小时后整块细胞培养板浸泡在 75%的酒精溶液 消毒 2小时。此后细胞培养板用无菌生理盐水浸洗三次。每孔加入 1毫升细胞培 养液 （DMEM， 10%牛血清） 和 2万个 NIH 3T3成纤细胞。 37摄氏度、 二氧化碳细 胞培养箱培养 24小时。显微镜观察发现细胞在二硫键交联透明质酸 -明胶二组份 水凝胶表面的黏附铺展与空白细胞培养板相似，细胞呈纺锤形。这个结果表明二 硫键交联透明质酸-明胶二组份水凝胶是细胞粘附生长的良好基质。
实施例 19. 聚乙二醇二乙烯砜交联透明质酸水凝胶用于抑制细胞的粘附 按照实施例 15在 24孔标准细胞培养板内制备聚乙二醇二乙烯砜交联透明质 酸水凝胶， 每孔为 1 毫升。 12小时后整块细胞培养板浸泡在 75%的酒精溶液消 毒 2小时。此后细胞培养板用无菌生理盐水浸洗三次。每孔加入 1毫升细胞培养 液 （DMEM, 10%牛血清） 和 2万个 NIH 3T3成纤细胞。 37摄氏度、 二氧化碳细胞 培养箱培养 24小时。 显微镜观察发现绝大部分成纤细胞悬浮在聚乙二醇二乙烯 砜交联透明质酸水凝胶， 不能黏附铺展； 而在空白细胞培养板孔内， 成纤细胞黏 附在底部，呈纺锤形。这个结果表明聚乙二醇二乙烯砜交联透明质酸水凝胶可以 抑制细胞的黏附。
实施例 20. 聚乙二醇二丙烯酸酯交联透明质酸 -明胶双组分水凝胶作为细 胞粘附生长的基质 按照实施例 16在 24孔标准细胞培养板内制备聚乙二醇二丙烯酸酯交联透明 质酸-明胶双组分水凝胶， 每孔为 1 毫升。 12小时后整块细胞培养板浸泡在 75% 的酒精溶液消毒 2小时。 此后细胞培养板用无菌生理盐水浸洗三次。 每孔加入 1 毫升细胞培养液 （DMEM, 10%牛血清） 和 2万个 NIH 3T3成纤细胞。 37摄氏度、 二氧化碳细胞培养箱培养 24小时。 显微镜观察发现细胞在聚乙二醇二丙烯酸酯 交联透明质酸 -明胶双组分水凝胶表面的黏附铺展与空白细胞培养板相似， 细胞 呈纺锤形（如图 3和图 4所示）。这个结果表明二硫键交联透明质酸 -明胶二组份 水凝胶是细胞粘附生长的良好基质。
工业实用性：
本发明通式 （I ) 或 （II ) 高分子巯基化改性衍生物具有很多有益效果。 本 发明采用酰肼键的化学结合方式进行巯基化改性， 具有制备条件温和、 产率高、 改性程度高且可控等许多显著优点。
本发明可以方便地制备各种二硫键交联高分子材料：如一种多糖的二硫键交 联材料、 一种蛋白质的二硫键交联材料、 或者两种多糖的复合二硫键交联材料、 两种蛋白质的复合交联材料、 一种多糖和一种蛋白质的复合二硫键交联材料等 等。这些二硫键交联高分子材料可以制成薄膜、海绵、 凝胶等各种形态， 可以用 于抑制细胞的粘附， 用作细胞生长基质等等。
通常说来， 巯基反应活性交联剂和本发明通式 （I ) 或 （II ) 高分子巯基化 改性衍生物的交联作用非常快速， 其交联凝胶化速度比二硫键的交联速度提高 5 倍以上， 具有重要的生物医学用途， 如细胞的原位包埋等等。本发明可以方便地 制备出各种巯基反应活性交联剂交联高分子材料： 如一种多糖的交联材料、一种 蛋白质的交联材料、或者两种多糖的复合交联材料、两种蛋白质的复合交联材料、 一种多糖和一种蛋白质的复合交联材料等等。这些巯基反应活性交联高分子材料 可以制成薄膜、海绵、 凝胶等各种形态， 可以用于抑制细胞的粘附， 用作细胞生 长基质等等。

Claims

权利要求书
1、 下述通式（I )或 （II ) 的高分子巯基化改性衍生物:
Figure imgf000030_0001
(Π)
其中 和1^是亚垸基、取代亚垸基、芳香基或聚醚基； P是侧链含有羧基的 高分子化合物残基， 所述高分子巯基化改性衍生物的分子量为 1000到 500万。
2、 按照权利要求 1所述的通式（I )或（II )高分子巯基化改性衍生物， 其 特征在于，所述的侧链含有羧基的高分子化合物包括多糖、蛋白质以及合成高分 子。
3、 按照权利要求 2所述的通式（I )或（II )高分子巯基化改性衍生物， 其 特征在于， 所述的多糖包括硫酸软骨素、 皮肤素、肝素、类肝素、海藻酸、透明 质酸、 硫酸皮肤素、 果胶、 羧甲基纤维素、 羧甲基壳聚糖及其盐。
4、 按照权利要求 2所述的通式（I )或（II )高分子巯基化改性衍生物， 其 特征在于，所述的合成高分子包括聚丙烯酸、聚天冬氨酸、聚酒石酸、聚谷氨酸、 聚富马酸及其盐。
5、 按照权利要求 2所述的通式（I )或（II )高分子巯基化改性衍生物， 其 特征在于， 所述的蛋白质包括胶原蛋白、碱性明胶蛋白、酸性明胶蛋白、碱性基 因重组明胶蛋白、酸性基因重组明胶蛋白、弹性蛋白、核心蛋白多糖层粘连蛋白 纤维结合蛋白。
6、 按照权利要求 1所述的通式（I )或（II )高分子巯基化改性衍生物， 其 中 1^和1¾2是亚垸基。
7、 按照权利要求 6所述的通式（I )或（II )高分子巯基化改性衍生物， 其 中！^和 R2都是碳原子数 1到 8的亚垸基。
8、 一种或一种以上如权利要求 1-7任一项所述的高分子巯基化改性衍生物 通过二硫键交联生成的高分子交联材料。
9、 一种或一种以上如权利要求 1-7任一项所述的高分子巯基化改性衍生物 通过与至少含有两个相同或不相同巯基反应活性官能团交联剂交联生成的高分 子交联材料。
10、按照权利要求 9所述的高分子交联材料， 其特征在于， 所述的巯基反应 . 活性官能团交联剂包括巯基反应活性官能团的双臂、三臂或更多臂的聚乙二醇衍 生物， 所述的聚乙二醇衍生物的分子量为 100到 1000000。
11、 按照权利要求 10所述的高分子交联材料， 其特征在于， 所述的巯基反 应活性官能团包括马来酰亚胺、 乙烯砜、 α， β 不饱和丙烯酸酯、 α， β 不饱 和甲基丙烯酸酯、 碘代丙酸酯、 溴代丙酸酯、 碘代丙酰胺、 溴代丙酰胺、 二硫代 吡啶、 N-羟基丁二酰亚胺活化酯。
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