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WO2012031515A2 - 低巯基化改性度生物相容高分子巯基化衍生物及其交联材料和用途 - Google Patents

低巯基化改性度生物相容高分子巯基化衍生物及其交联材料和用途 Download PDF

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WO2012031515A2
WO2012031515A2 PCT/CN2011/077985 CN2011077985W WO2012031515A2 WO 2012031515 A2 WO2012031515 A2 WO 2012031515A2 CN 2011077985 W CN2011077985 W CN 2011077985W WO 2012031515 A2 WO2012031515 A2 WO 2012031515A2
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biocompatible
disulfide
crosslinked
degree
derivative
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PCT/CN2011/077985
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French (fr)
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WO2012031515A3 (zh
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舒晓正
仲伟平
王云云
俞美霞
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常州百瑞吉生物医药有限公司
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Application filed by 常州百瑞吉生物医药有限公司 filed Critical 常州百瑞吉生物医药有限公司
Priority to CA2810590A priority Critical patent/CA2810590C/en
Priority to ES11823038.2T priority patent/ES2601812T3/es
Priority to JP2013527457A priority patent/JP6063867B2/ja
Priority to AU2011301069A priority patent/AU2011301069B2/en
Priority to EP11823038.2A priority patent/EP2614828B1/en
Priority to US13/821,666 priority patent/US9446067B2/en
Publication of WO2012031515A2 publication Critical patent/WO2012031515A2/zh
Publication of WO2012031515A3 publication Critical patent/WO2012031515A3/zh
Priority to US15/078,389 priority patent/US10064889B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/738—Cross-linked polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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
    • 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
    • C08B37/0072—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
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/34—Introducing sulfur atoms or sulfur-containing groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L101/00—Compositions of unspecified macromolecular compounds
    • C08L101/02—Compositions of unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups
    • 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
    • C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives

Definitions

  • the invention relates to a low-modification biocompatible polymer derivative, in particular to a biodegradable polymer thiolated derivative with low thiolization degree, and the invention relates to a bisulfide cross-linked biological phase with low crosslinking degree.
  • the macromolecular cross-linking material, and the invention also relates to the use of the cross-linked material in the field of medicine.
  • Biocompatible polymers have many important physiological functions, such as hyaluronic acid, which has significant effects in the adhesion-promoting treatment of arthritis and the promotion of wound healing.
  • biocompatible polymers are rapidly metabolized or soluble in body fluids, which greatly limits their use in many clinical medicines, such as hyaluronic acid for the treatment of arthritis.
  • the weekly five-week joint injection is not only inconvenient for patients and medical workers, but also increases the risk of infection.
  • Chemical modification, chemical crosslinking, or chemical modification of biocompatible polymers is an effective means to delay the metabolism of biocompatible polymers in the body and reduce solubility, and significantly expand biocompatible polymers. Application in clinical medicine.
  • cross-linked sodium hyaluronate for the adhesion therapy of arthritis, a single joint injection of cross-linked sodium hyaluronate can achieve the effect of five-joint injection of non-crosslinked sodium hyaluronate; at the same time, cross-linked hyaluronic acid is also widely used in dermal fillers, etc. Cosmetic use.
  • biocompatible polymers greatly expands their application in clinical medicine, there are still contradictions in the theoretical and practical processes.
  • the chemical modification and/or cross-linking of biocompatible polymers must reach a certain level in order to delay the metabolism of biocompatible polymers in vivo and reduce the solubility. Therefore, the chemical modification widely used in clinical medicine is currently being applied.
  • Both biocompatible polymer derivatives or cross-linked materials have high or relatively high degree of modification or cross-linking.
  • the FYA company's HYAFF series is highly esterified hyaluronic acid.
  • biocompatible polymers are a new type of chemical modification and crosslinking method, which have many advantages and have many important potential uses in clinical medicine.
  • biocompatible macromolecular thiolated derivatives have been used for chemically active modification of various small molecule drugs and polypeptide protein drugs, etc., and crosslinked materials prepared based on these biocompatible macromolecular thiolated derivatives can be used as cell growth.
  • biocompatible polymers The degree of thiolation modification and/or degree of crosslinking is high.
  • 26.8 to 66.8% of the groups are modified and crosslinked (Shu et al., Biomacromolecules, 3: 1304, 2002).
  • One of the technical problems to be solved by the present invention is to provide a class of biocompatible macromolecular thiolated derivatives having low thiolization degree of modification.
  • Low thiol modification Biocompatible macromolecular thiolated derivatives maximize the initial structure, physiological function and biocompatibility of biocompatible polymers, and the introduced sulfhydryl groups can be effectively cross-linked to prepare biocompatibility with low cross-linking degree. Polymer cross-linking materials.
  • the second technical problem to be solved by the present invention is to provide a type of disulfide bond cross-linking.
  • Biocompatible polymer cross-linking material the cross-linking material has a low degree of cross-linking of disulfide bonds, not only maximally maintaining the initial structure, physiological function and biocompatibility of the biocompatible polymer, but also effectively It delays the metabolism of biocompatible polymers in the body and reduces the solubility, which satisfies the requirements of various medical applications.
  • cross-linking process can be completed in an injectable container, has injectable properties, is convenient to use, contains no impurities, has good biocompatibility, and has no toxic and side effects, and has a very broad application prospect in the medical field.
  • the third technical problem to be solved by the present invention is to provide the use of the above-mentioned disulfide-bonded cross-linked biocompatible polymer cross-linking material in the field of medicine.
  • Biocompatible polymer It refers to polymers with good biocompatibility, including polysaccharides, proteins, and synthetic polymers.
  • polysaccharides include sulfuric acid Osteosynthesis, dermatan, heparin, heparanoid, alginic acid, hyaluronic acid, dermatan sulfate, pectin, carboxymethylcellulose, chitosan, carboxymethyl chitosan, etc. and their salt forms (such as sodium Salt, potassium salt, etc.) and derivative forms
  • synthetic polymers include polyacrylic acid, polyaspartic acid, polytartaric acid, polyglutamic acid, polyfumaric acid, etc.
  • proteins include collagen, alkaline gelatin, acid gelatin, elastin, Core proteins, polysaccharide laminins, fibronectin, and the like, as well as their salt forms (e.g., sodium salts, potassium salts, etc.) and derivative forms.
  • Biocompatible polymer is preferably sulfuric acid Osteone, heparin, heparin, alginic acid, hyaluronic acid, polyaspartic acid, polyglutamic acid, chitosan, carboxymethyl chitosan, alkaline gelatin protein and acid gelatin protein and their salt forms (eg sodium salt, potassium salt, etc.) and derivative forms Particularly preferred are calcitonin and hyaluronic acid and their salt forms (e.g., sodium salt, potassium salt, etc.) and derivative forms.
  • the biocompatible macromolecular thiolated derivative refers to a derivative of a biocompatible polymer side chain group which is chemically introduced into a thiol group.
  • the degree of thiolation modification means that the amount of sulfhydryl group introduced accounts for the modification of the biocompatible polymer.
  • the percentage of the number of side chain groups For example, when the side chain carboxyl group of hyaluronic acid is subjected to thiolation modification, the degree of thiolation modification means the percentage of the amount of thiol group introduced to the total number of carboxyl groups in the side chain of hyaluronic acid.
  • Disulfide cross-linking refers to the formation of a three-dimensional network structure by a biocompatible macromolecular thiolated derivative in the form of a disulfide bond, and the degree of cross-linking of the disulfide bond means
  • the number of sulfhydryl groups forming a disulfide bond in the biocompatible macromolecular thiolated derivative is a percentage of the number of modified side chain groups available for the biocompatible polymer.
  • a hydrogel is a substance having a three-dimensional crosslinked network structure containing a large amount of water, between a liquid state and a solid state, and having no fluidity. Gelation refers to the process from a liquid state with fluidity to a gel state with fluidity.
  • Dynamic viscosity is the force required to produce a unit flow rate per unit area of a liquid layer per unit distance, in centipoise (mPa ⁇ s ) or poise ( Pa ⁇ s).
  • the dynamic viscosity is an index for evaluating the viscosity. The smaller the dynamic viscosity, the better the fluidity, and the worse the fluidity.
  • One aspect of the present invention provides a class of low thiol modification degree biocompatible macromolecular thiolated derivatives, these low thiolization degrees of modification
  • the biocompatible macromolecular thiolated derivative maximizes the initial structure, physiological function and biocompatibility of the biocompatible polymer, and the introduced sulfhydryl group can be effectively chemically crosslinked to prepare a biological phase with low cross-linking degree.
  • Capacitive polymer cross-linking materials are examples of thiol modification degree biocompatible macromolecular thiolated derivatives, these low thiolization degrees of modification.
  • the degree of low thiolization modification biocompatible polymer thiolated derivatives can generally be prepared by the following methods, which have been The patent document WO2009006780 is described.
  • Method one is the side chain carboxyl group Amino (hydrazide) / carbodiimide coupling chemistry.
  • the usual way is that the carboxyl group forms an intermediate product under the activation of carbodiimide, the disulfide bond containing diamine or dihydrazide is nucleophilicly substituted to form an intermediate product, and finally the disulfide bond is reduced to a thiol group to obtain a biocompatible polymer.
  • a biocompatible macromolecular thiolated derivative, or an intermediate product obtained may also be obtained by substituting a primary amide containing a free sulfhydryl group (or a thiol group) with a diamine or a dihydrazide containing a disulfide bond. Removal of a thiol protecting group results in a biocompatible macromolecular thiolated derivative (Gianolio et al, Bioconjugate Chemistry, 16, 1512, 2005).
  • the above carbodiimide generally means 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.
  • the second method is that the side chain carboxyl group is directly bonded to the carbodiimide containing a disulfide bond (eg Prepared by the reaction of 2,2'-dithiobis(N-ethyl(N'-ethylcarbodiimide)), etc., and the prepared biocompatible polymer thiolated modified derivative has the following Structure of formula (III) Bulpitt et al., US 6884788).
  • the third method is the modification of the side chain amino group, which is generally divided into direct and indirect modification.
  • Direct modification means direct modification of the side chain amino group to introduce a thiol group.
  • a thiol group for example, bis-succinic acid bisacyl cystamine double carbonyl diimidazole activated ester thiolation of collagen amino groups (Yamuchi et al, Biomaterials, 22, 855, 2001; Nicolas et al, Biomaterials, 18, 807, 1997).
  • the indirect thiolation modification method of the amino group in the third method is generally divided into two steps. The first step is the carboxylation of the amino group, and the second step is the thiolation modification of the carboxyl group using one or two of the foregoing methods.
  • Method four is Modification of side chain hydroxyl groups.
  • the usual method is the carboxylation of a hydroxyl group under a strong base condition, and then the carboxyl group is further subjected to the aforementioned method 1 and method 2.
  • Thiolation for example, side chain hydroxyl groups of polymers such as cellulose, hyaluronic acid, chitin and chitosan can be carboxymethylated and then thiolated using an amino (hydrazide) / carbodiimide carbon chemical reaction. modified.
  • the one or more of the above methods can be used to prepare the low thiolization degree biocompatible macromolecular thiolated derivative of the present invention. .
  • biocompatible macromolecular thiolated derivative is prepared by the above preparation method, and the invention can be realized by adjusting parameters such as the feed ratio, reaction time and reaction temperature of the reaction raw materials.
  • low thiol modification degree Purification of biocompatible macromolecular thiolated derivatives is very important. Residual impurities may not only cause toxic side effects such as inflammation in the living body, but also interfere with subsequent cross-linking of disulfide bonds. In the present invention, residual impurities can pass through dialysis and / or organic solvent (such as ethanol) precipitation method.
  • the molecular weight of the biocompatible polymer used is usually between 1000 and 10000000, preferably 10000 ⁇ 3000000, especially preferably 20000 ⁇ 1500000.
  • the initial structure of most of the biocompatible polymer is retained, and the degree of thiolation modification is low.
  • Low sulfhydryl modification degree of the invention The side chain of the biocompatible macromolecular thiolated derivative contains at least three fluorenyl groups, and the degree of thiolation modification is ⁇ 4.5%, the degree of thiolation modification is preferably 0.5% to 3.0%, and the degree of thiolation modification is particularly preferred. 0.75% ⁇ 2.5%.
  • biocompatible macromolecular thiolated derivatives researchers generally have a technical bias: A higher degree of thiolation modification is necessary for the preparation of cross-linking materials for subsequent biocompatible macromolecular thiolated derivatives and for clinical medical use.
  • Prestwich Studies have shown that biocompatible polymer derivatives with higher degree of thiolation modification can achieve cross-linking better (Prestwich et al., WO2008/008857 Therefore, researchers generally tend to increase the degree of thiolation modification of biocompatible polymers.
  • Sparer et al. disclosed glycosaminoglycans (hyaluronic acid and chondroitin sulfate) in 1983.
  • a derivative of cysteine methyl ester, a cysteine methyl ester and a glycosaminoglycan are coupled via an amide bond, and 5% to 87% of the side chain carboxyl group of the glycosaminoglycan is modified to a sulfhydryl group (Sparer et al., Chapter 6, Page 107-119 , Controlled Release Delivery System, Edited by Theodore J. Roseman, S.Z. Mansdorf, Marcel Dekker Inc.).
  • Gianolio equals the disclosure of hyaluronic acid in 2005 - Cysteamine derivatives, cysteine and side chain carboxyl groups of hyaluronic acid are coupled via amide bonds, and 22% of hyaluronic acid side chain carboxyl groups are modified to thiol groups (Gianolio et al., Bioconjugate) Chemistry, 16: 1512-1518, 2005). India is equal to the disclosure of hyaluronic acid in 2008 - The cysteamine derivative, which is coupled with the side chain carboxyl group of hyaluronic acid via an amide bond, contains 10 to 200 ⁇ mol/g thiol and 120 to 500 ⁇ mol/g.
  • the disulfide bond is calculated according to the molecular weight of the disaccharide repeating segment of hyaluronic acid of 400, and the degree of thiolation modification is 10% to 48%, respectively (ie, 10% to 48% of the hyaluronic acid side chain carboxyl group is thiolated). ) (Print, etc., CN 101367884).
  • the degree of thiolation modification of hyaluronic acid thiolated derivatives disclosed by Shu et al. by hydrazide bond is 26.8% ⁇ 66.8% (Sh et al. Biomacromolecules, 3: 1304, 2002).
  • the prepared low thiolation degree of modification The biocompatible macromolecular thiolated derivatives are highly purified by one or more of the foregoing methods, and the residual impurities are usually present in an amount of less than one thousandth or even one ten thousandth.
  • the beneficial effects of the present invention are the low thiol modification degree of the present invention.
  • the biocompatible polymer thiolated derivative has a low degree of thiolation modification, which maximizes the initial structure, physiological function and biocompatibility of the biocompatible polymer, and also has a small amount of raw materials and a short reaction time.
  • Low thiol modification degree Biocompatible macromolecular thiolated derivatives can be conveniently used to prepare crosslinked materials and meet various clinical medical applications.
  • the present invention overcomes the aforementioned technical bias: A higher degree of thiolation modification is necessary for the preparation of cross-linking materials for subsequent biocompatible macromolecular thiolated derivatives and for clinical medical use.
  • Another aspect of the invention provides a class of disulfide crosslinks Biocompatible polymer cross-linking materials, these biocompatible polymer cross-linking materials have a low degree of cross-linking of disulfide bonds, which not only maintains the initial structure, physiological function and biocompatibility of biocompatible polymers to the utmost extent. At the same time, it also effectively delays the metabolism of biocompatible polymers in the body and reduces the solubility, which satisfies the requirements of various clinical medical applications.
  • the disulfide-crosslinked biocompatible macromolecular cross-linking material is usually in the form of a hydrogel, preferably in the form of a hydrogel having a water content of 95% (weight/volume percent, g/ml) or more, particularly preferably water content. for 98% (w/v, g/ml) above the hydrogel form.
  • Disulfide crosslinked of the present invention The biocompatible polymer crosslinked hydrogel can be dried or freeze-dried to form a solid form such as a film or a sponge.
  • the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention adopts the low thiol modification degree of the present invention
  • the biocompatible macromolecular thiolated derivative is prepared as a raw material, and an implementation route is as follows: the low thiolization degree biocompatible macromolecular thiolated derivative of the present invention is dissolved in water to obtain a solution of a suitable concentration ( Usually 0.2% ⁇ 5.0%), adjust the pH value of the solution to a specific value (usually neutral, ie pH is about 7) Then, the sulfhydryl group gradually forms a disulfide bond under the oxidation of oxygen in the air and dissolved oxygen in the solution, the solution gradually gelatinizes, the viscosity of the solution gradually increases, and finally the solution loses fluidity to form a three-dimensional network crosslinked structure. It is also possible to add an oxidizing agent (such as hydrogen peroxide or the like) to the above solution to accelerate the crosslinking process.
  • the second way to achieve the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention is to adopt the method disclosed by Shu et al. WO2010043106).
  • the gelation process can be completed in an injectable container, with injectable properties, easy to use, no impurities, good biocompatibility, no toxic side effects.
  • the specific process of the implementation path is as follows: Low thiol modification degree Biocompatible polymer thiolated derivative is dissolved in water to obtain a suitable concentration of solution (usually 0.2% ⁇ 5.0%) to adjust the pH of the solution. The value is a specific value (usually neutral), and then the solution is poured into an injectable container and sealed.
  • the sulfhydryl group gradually forms a disulfide bond under the oxidation of dissolved oxygen in the solution, the solution gradually gels, and the dynamic viscosity of the solution gradually increases. Finally, the solution loses fluidity to form a three-dimensional network crosslinked structure. It is also possible to add an oxidizing agent (such as hydrogen peroxide or the like) to the above solution to accelerate the crosslinking process.
  • an oxidizing agent such as hydrogen peroxide or the like
  • the second realization route of the disulfide-bonded biocompatible polymer crosslinked hydrogel of the present invention can be produced by aseptic process or terminal sterilization process (such as wet heat sterilization process commonly used in the pharmaceutical industry) to meet different clinical medical requirements.
  • Large-scale industrial production can be realized by the canned production line commonly used in the pharmaceutical industry, and the hourly output can be easily reached. More than 3000.
  • Canned production line is available Gaoninger's linear automatic syringe pre-filling line or honeycomb type automatic pre-canning and stoppering equipment, as well as pre-sterilized syringe liquid potting machine of Bosch and other companies in Germany.
  • Injectable container It can be a glass or plastic syringe, such as the BD Hypak SCF pre-sterilized syringe, or an extrudable container such as a soft plastic bag.
  • the degree of low thiolation modification of the present invention is employed
  • the biocompatible macromolecular thiolated derivative is used as a raw material, and the degree of crosslinking of the disulfide bond depends on the degree of thiolation modification of the biocompatible polymer thiolated derivative of the low thiolization degree of the present invention ( ⁇ 4.5%) Therefore, the present invention
  • the degree of cross-linking of disulfide bonds in the biocompatible polymer crosslinked hydrogels crosslinked by disulfide bonds is also low ( ⁇ 4.5%).
  • the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention generally has a kinematic viscosity of more than 50 times, and can be improved by more than 500 times under optimized conditions.
  • the disulfide bond of the present invention is crosslinked.
  • the high dynamic viscosity properties of biocompatible macromolecular crosslinked hydrogels have unique advantages in important clinical and medical applications such as postoperative adhesion prevention and arthritis adhesion therapy.
  • the dynamic viscosity of the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention is usually greater than 10,000 centipoise at a shear rate of not less than 0.25 Hz and 25 ⁇ 0.1 °C ( mPa ⁇ s), the dynamic viscosity is preferably more than 25,000 (mPa ⁇ s), and the dynamic viscosity is particularly preferably more than 40,000 (mPa ⁇ s).
  • the disulfide-crosslinked biocompatible macromolecular crosslinked material of the present invention may contain one or more of the low thiolization degree of the present invention.
  • the biocompatible polymeric thiolated derivative may additionally contain one or more other materials. These substances can be polysaccharides, proteins or synthetic polymers such as sulfuric acid.
  • corticosteroids which are
  • polypeptide protein drugs such as various growth factors (basic growth factor, acid growth factor, vascular growth factor, osteogenic growth factor, etc.), nucleic acids (such as RNA) and the like.
  • the active pharmaceutical ingredient can be dispersed in the form of solid particles and / or dissolved in the disulfide-bonded cross-linking biocompatible polymer cross-linking material of the present invention.
  • biocompatible macromolecular crosslinked hydrogel has a suitable storage time and its performance needs to be stable.
  • disulfide-bonded cross-linking The highly modified biocompatible macromolecular crosslinked hydrogel is unstable. As the storage time increases, the hydrogel gradually shrinks, and a large amount of water is extruded from the hydrogel, resulting in a greatly reduced dynamic viscosity, which seriously affects Gel properties, not compatible
  • the practical application of clinical medicine seriously restricts the application of biocompatible polymer crosslinked hydrogels with disulfide cross-linking in the field of medicine.
  • a disulfide-crosslinked hyaluronic acid crosslinked hydrogel (13.5%) The degree of thiolation modification) After storage for 6 months at room temperature, the volume of the hydrogel shrinks by about 30%.
  • the disulfide-bonded biocompatible macromolecular crosslinked hydrogel of the present invention adopts the low thiol modification degree of the present invention
  • Biocompatible polymer thiolated derivatives are used as raw materials, and unexpected technical effects have been obtained, which solves the problem of instability of the above-mentioned disulfide-bonded biocompatible polymer crosslinked hydrogel.
  • the stability test of the stability of the month shows that the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention has excellent stability and will be further described in connection with the examples.
  • the beneficial effects of the present invention are the disulfide cross-linking of the present invention.
  • the biocompatible polymer cross-linking material has a low degree of cross-linking, which not only maintains the initial structure, physiological function and biocompatibility of the biocompatible polymer to the utmost extent, but also has a high dynamic viscosity, effectively delaying
  • the biocompatible polymer is metabolized in the body and reduces solubility, which satisfies the requirements of various clinical medical applications.
  • the beneficial effects of the present invention also include the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention having excellent stability.
  • Disulfide-bonded cross-linked biocompatible polymer cross-linking material of the invention Applications in medicine include: wound dressings that can be used to promote wound healing, can be applied to skin or other wounds; can also be used to prevent adhesions, including prevention of fibrotic adhesion between tissues or organs after surgery (eg, sinus surgery) It can also be used as a joint lubricant for the treatment of arthritis adhesion.
  • the use of the disulfide-bonded biocompatible macromolecular cross-linking material prepared by the present invention in pharmacy includes a sustained release carrier as a variety of active therapeutic substances to achieve slow release properties.
  • the active therapeutic substance can be a chemical drug or a biologically active factor, including an anti-inflammatory agent, an antibiotic, an analgesic, an anesthetic, a wound healing promoter, a cell growth promoting or inhibiting factor, an immunostimulating agent, an antiviral drug, and the like.
  • the beneficial effect of the invention is the low thiol modification degree of the invention
  • the biocompatible polymer thiolated derivative has a low degree of thiolation modification, which maximizes the initial structure, physiological function and biocompatibility of the biocompatible polymer, and also has a small amount of raw materials and a short reaction time.
  • Low thiol modification degree Biocompatible macromolecular thiolated derivatives can be conveniently used to prepare crosslinked materials and meet various clinical medical applications.
  • the present invention overcomes the aforementioned technical bias: A higher degree of thiolation modification is necessary for the preparation of cross-linking materials for subsequent biocompatible macromolecular thiolated derivatives and for clinical medical use.
  • the beneficial effects of the invention are that the double-sulfur cross-linked biocompatible polymer cross-linking material of the invention has a low degree of cross-linking, and not only maintains the initial structure, physiological function and biological phase of the biocompatible polymer to the utmost extent. Capacitive, at the same time has a high dynamic viscosity, effectively delaying the metabolism of biocompatible polymers in the body and reducing the solubility, and better meet the requirements of various clinical medical applications.
  • the beneficial effects of the present invention also include the disulfide-crosslinked biocompatible macromolecular crosslinked hydrogel of the present invention having excellent stability.
  • Fig. 1 is a schematic view showing the results of an experiment of Example 12 of the present invention (i.e., a weight distribution diagram of the left hind foot).
  • the above solution was loaded into a dialysis tube (molecular weight cutoff 3500, Sigma, USA) and dialyzed against a large amount of 0.001 mol/L hydrochloric acid and 0.2 mol/L sodium chloride solution. Days, dial the dialysate every 8 hours; then dialysis with a large amount of 0.001 mol / l hydrochloric acid solution for 3 days, every 8 Change the dialysate every hour. Finally, the solution in the dialysis tube is collected, and the prepared solution is directly used or freeze-dried to obtain a white flocculent solid.
  • the thiol content was determined by the improved Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 and the degree of thiolation modification was calculated or determined by hydrogen spectroscopy ( 1 H-NMR ) (D 2 O as solvent).
  • the degree of thiolation modification (the characteristic methyl absorption peak of the acetyl group of hyaluronic acid is an internal standard).
  • the degree of thiolation modification refers to the percentage of the number of thiol groups introduced to the total number of carboxyl groups in the side chain of hyaluronic acid. The results are as follows:
  • the above solution was loaded into a dialysis tube (molecular weight cutoff 2000, Sigma, USA) and dialyzed against 2 liters of 0.001 mol/L hydrochloric acid and 0.3 mol/L sodium chloride solution. Days, dial the dialysate every 8 hours; then dialicate with 2 liters of 0.001 mol / liter of hydrochloric acid for 3 days, every 8 Change the dialysate every hour. Finally, the solution in the dialysis tube is directly used to prepare the solution or freeze-dried to obtain a flocculent solid.
  • the thiol content was determined by the improved Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 and the degree of thiolation modification was calculated or determined by hydrogen spectroscopy ( 1 H-NMR ) (D 2 O as solvent).
  • the degree of thiolation modification (the characteristic methyl absorption peak of the acetyl group of chondroitin sulfate is the internal standard).
  • the degree of thiolation modification refers to the percentage of the amount of thiol groups introduced to the total number of carboxyl groups in the side chain of chondroitin sulfate. The results are as follows:
  • the reaction was stopped by adding 1.0 mol/L sodium hydroxide solution to pH 8.5. Then add 50 grams of dithiothreitol ( Diagnostic Chemical Limited (USA) and an appropriate amount of 1.0 mol / liter of sodium hydroxide solution, stirred. The pH of the mediation solution is 8.5 . The reaction was stirred at room temperature for 24 hours. Thereafter, 1 mol/liter of hydrochloric acid was added to the above solution until about pH 3.0.
  • the above solution was loaded into a dialysis tube (cutting molecular weight 3500, Sigma , USA), dialyzed with a large amount of 0.001 mol / l hydrochloric acid and 0.5 mol / l sodium chloride solution for 5 days, change dialysate every 8 hours; then use a large amount of 0.001 mol / Literic hydrochloric acid solution was dialyzed for 3 days, and the dialysate was changed every 8 hours. Finally, the solution in the dialysis tube is collected, and the prepared solution is directly used or freeze-dried to obtain a white flocculent solid.
  • the thiol content was determined by the improved Ellman method reported by Shu et al. in Biomacromolecules, 3, 1304, 2002 and the degree of thiolation modification was calculated or determined by hydrogen spectroscopy ( 1 H-NMR ) (D 2 O as solvent).
  • the degree of thiolation modification (the characteristic methyl absorption peak of the acetyl group of hyaluronic acid is an internal standard).
  • the degree of thiolation modification refers to the percentage of the number of thiol groups introduced to the total number of carboxyl groups in the side chain of hyaluronic acid. The results are as follows:
  • Example 1 Two hyaluronic acid thiolated derivatives prepared (the degree of thiolation modification was 2.33% and 4.18%, respectively) In Table 1, No. 4 and 6) were prepared into 10, 15 and 20 mg/ml solutions respectively, and the pH of the solution was adjusted to 7.4. Transfer 2 ml of the above solution to 10 The milliliter glass bottle was sealed and stored at room temperature for 1 week. The solution lost fluidity and formed a crosslinked hydrogel. The water content (g/ml) of the hydrogel was 99%, 98.5% and 98%, respectively.
  • Example 2 The prepared chondroitin sulfate thiolated derivative (the degree of thiolation modification is 4.50%, number 5 in Table 2 ) Prepare a solution of 50 and 80 mg/ml, respectively, and adjust the pH of the solution to 7.4. Transfer 2 ml of the above solution to a 10 ml glass bottle, seal it, and store at room temperature. 1 Week, the solution lost fluidity and formed a crosslinked hydrogel with a water content (g/ml) of 95% and 92%, respectively.
  • a water content g/ml
  • Example 3 Four kinds of hyaluronic acid thiolated derivatives prepared (the degree of thiolation modification is 1.28%, 1.54%, 2.14%, 3.97%, No. 3, 7, 8, and 9 in Table 3 are prepared as 5, 7.5, and 10 mg/ml solutions, respectively, to adjust the pH of the solution. 7.4. 2 ml of the above solution was transferred to a 10 ml glass bottle, sealed, and left to stand at room temperature for 10 days. The solution lost fluidity and formed a crosslinked hydrogel. The water content of the hydrogel (g/ml) was 99.5 %, 99.25% and 99%.
  • Example 3 The hyaluronic acid thiolated derivative prepared (the degree of thiolation modification was 2.14%, number 7 in Table 3 Prepare a solution of 10 mg/ml, then add hyaluronic acid solution (5 mg/ml) and chondroitin sulfate solution (10 mg/ml) according to a volume ratio of 2:1 to adjust the solution. The pH is 7.4. 2 ml of the above solution was transferred to a 10 ml glass bottle, sealed, and left to stand at room temperature for 10 days, and the solution lost fluidity and formed a crosslinked hydrogel.
  • the shear rate is not less than 0.25 Hz, 25 ⁇ 0.1 °C.
  • the preparation prepared in Example 6 was determined.
  • the kinetic viscosity of the crosslinked hydrogel was 408-547 times higher than that of the corresponding unmodified hyaluronic acid solution.
  • Hyaluronic acid concentration (mg/ml) Thiolation modification degree (%) 0 Thiolation modification degree (%) 1.28 Thiolation modification degree (%) 1.54 Thiolation modification degree (%) 2.14 Thiolation modification degree (%) 3.97 5 137 75000 78000 81000 88000 7.5 198 >100000 >100000 >100000 >100000 10 245 >100000 >100000 >100000 >100000
  • Hydrogel 1 The hyaluronic acid thiolated derivative prepared in Example 3 (the degree of thiolation modification is 13.5%, numbered in Table 3 1) Prepare a 10 mg/ml solution and adjust the pH of the solution to 7.4. 2 ml of the above solution was transferred to a 10 ml glass bottle and sealed, and allowed to stand at room temperature for 10 days. The solution loses fluidity and forms a crosslinked hydrogel.
  • Hydrogel 2 The hydrogel prepared in Example 6 (using the hyaluronic acid thiolated derivative prepared in Example 3, The concentration of hyaluronic acid was 10 mg/ml, and the degree of thiolation modification was 1.54%, numbered 3 in Table 3.
  • the degree of thiolation modification of the raw material of hydrogel 2 (ie 1.54%) is significantly lower than that of the raw material of hydrogel 1.
  • the degree of thiolation modification i.e., 13.5%
  • the degree of crosslinking of hydrogel 2 is significantly lower than that of hydrogel 1.
  • Stability test According to the Chinese Pharmacopoeia 2010 edition of the second XIX C Guideline for the stability test of APIs and pharmaceutical preparations. Accelerated stability test of hydrogels. The test conditions are 6 months at a temperature of 40 ⁇ 2 ° C, at the end of the 0, 1, 2, 3 and 6 months of the test. The samples were measured for hydrogel dynamic viscosity and shrinkage (%), and the results are shown in Table 5. For high Cross-linking Degree of hydrogel 1, with the increase of stability accelerated test, the dynamic viscosity decreased sharply, and the volume of the gel continued to shrink, the gel volume shrinkage rate was 10.2 at 1, 2, 3 and 6 months, respectively. 35.1, 39.2 and 41.4%, a large amount of water was extruded from the gel. Low for the present invention The cross-linked hydrogel 2 remained very stable.
  • Example 10 Disulfide-crosslinked hyaluronic acid crosslinked hydrogel to prevent restenosis of sinus mouth after sinusitis
  • the millimeter spherical cutting drill opens the middle wall of the maxillary sinus to the nasal cavity to form a circular channel with a 4 mm diameter edge without mucosa. Fill in both sides of the tunnel of 5 rabbits.
  • Example 6 The prepared hydrogel (hyaluronic acid concentration 10 mg/ml, thiolation modification degree 1.54%) (treatment group), at 5 No rabbits were filled in both sides of the well (control group). The periosteum is then sutured intermittently with absorbable sutures, and the skin is then sutured with absorbable sutures to close the maxillary sinus. Do not do other dressings. The animals were given normal drinking water after surgery.
  • the rabbits were sacrificed. After healing, the wound was healed and the sinus cavity was exposed. The residue in the sinus cavity is gently sucked out by the suction device while flushing with water.
  • a 30 degree nasal endoscope was used to examine the inner wall of the sinus and to perform imaging. Each aperture is measured with a millimeter scale ruler. The double-blind method is used to observe and measure the aperture.
  • the sinus orifice diameter of the treatment group was 2.78 ⁇ 1.17 mm
  • the diameter of the sinus of the control group was 0.7 ⁇ 0.52.
  • the restenosis of the sinus ostium is an important problem in the clinical operation of sinusitis, which will affect the curative effect and even lead to the recurrence of sinusitis.
  • the above results indicate The low cross-linking disulfide-crosslinked hyaluronic acid crosslinked hydrogel of the present invention can significantly prevent the narrowing of the sinus ostium and is expected to be widely used clinically.
  • Example 11 Application of a disulfide-crosslinked hyaluronic acid crosslinked hydrogel in the prevention and treatment of postoperative adhesions.
  • hyaluronic acid solution (10 mg/ml) was used for treatment; the third group used the hydrogel prepared in Example 6 (hyaluronic acid concentration 10 mg/ml, thiolation modification degree 1.54%) The treatment was performed; the wound on the surface of the rat was finally sutured, and the rats were sacrificed two weeks later to observe the adhesion status.
  • Example 12 Use of a disulfide-crosslinked hyaluronic acid crosslinked hydrogel in the treatment of arthritis adhesion.
  • the rabbit arthritis model reported is as follows: intramuscular injection of ketamine (35 mg / kg) and tropazosin (5 mg / Km) was anesthetized, the rabbit's left knee joint cut a 2 cm incision in the lateral side of the kneecap, and then the exposed lateral collateral ligament was cut; the end of the tendon was cut, the lateral meniscus was exposed, and the lateral meniscus was removed. 3.0-4.0 mm; suture the subcutaneous muscle layer and skin layer, and intramuscularly inject about 0.2 ml of ampicillin.
  • the rabbits after partial meniscectomy were divided into three groups: the first group was the saline control group, respectively, after surgery 0, 3, 6, 9 12 days of intra-articular injection of 0.2 ml saline (5 injections in total); the second group was hyaluronic acid treatment group, respectively, 0, 3, 6, 9, 12 after surgery Intra-articular injection of 0.2 ml of commercially available hyaluronic acid solution (10 mg/ml) for treatment (5 in total)
  • the third group was the disulfide cross-linked hyaluronic acid cross-linked hydrogel treatment group of the present invention, and the hydrogel prepared in Example 6 was intra-articularly injected on the 0th day after surgery (hyaluronic acid).
  • the weight distribution of the left hind paw showed that on days 8, 11 and 14 after surgery,
  • the disulfide-crosslinked hyaluronic acid cross-linked hydrogel treatment group (third group) of the present invention is significantly superior to the saline control group (first group) (p ⁇ 0.05); at all postoperative observation time points, Hyaluronic acid treatment group ( The second group) had no statistically significant difference (p>0.05) with the saline control group (first group), and the disulfide cross-linked hyaluronic acid cross-linked hydrogel treatment group of the present invention (third group) )versus There was no statistically significant difference in the efficacy of the hyaluronic acid treatment group (Phase 2) (p>0.05) (see Figure 1).
  • corticosteroids for example, beclomethasone Beclomethasone, beclomethasone dipropionate Beclomethasone dipropionate, budesonide Budesonide, dexamethasone Dexamethasone, prednisolone Prednisolone, prednisone, etc.
  • corticosteroids are uniformly dispersed in the prepared crosslinked hydrogel.
  • the measurement wavelength is as follows: beclomethasone Beclomethasone 246 nm, beclomethasone dipropionate Beclomethasone dipropionate 240 nm , budesonide Budesonide 248 nm, dexamethasone Dexamethasone 242 nm, prednisolone Prednisolone 248 nm, prednisone Prednisone 244 nm.
  • the disulfide-crosslinked hyaluronic acid crosslinked hydrogel is a good drug sustained-release carrier and has a good sustained-release effect on all six corticosteroids.
  • the behavior of the drug released from the hydrogel varies widely. The more hydrophobic the drug, the slower the release.
  • the relatively hydrophilic Prednisolone Prednisolone has been substantially released within 7 days; for the highly hydrophobic beclomethasone dipropionate Beclomethasone Dipropionate, almost no release detected.
  • Disulfide-bonded cross-linked biocompatible polymer cross-linking material of the invention Applications in medicine include: wound dressings that can be used to promote wound healing, can be applied to skin or other wounds; can also be used to prevent adhesions, including prevention of fibrotic adhesion between tissues or organs after surgery (eg, sinus surgery) It can also be used as a joint lubricant for the treatment of arthritis adhesion.
  • the use of the disulfide-bonded biocompatible macromolecular cross-linking material prepared by the present invention in pharmacy includes a sustained release carrier as a variety of active therapeutic substances to achieve slow release properties.
  • the active therapeutic substance can be a chemical drug or a biologically active factor, including an anti-inflammatory agent, an antibiotic, an analgesic, an anesthetic, a wound healing promoter, a cell growth promoting or inhibiting factor, an immunostimulating agent, an antiviral drug, and the like.

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Abstract

本发明公开了一类低改性生物相容高分子巯基化改性衍生物，这些衍生物具有很低的巯基化改性度，最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时引入的巯基可有效地进行化学交联以制备低交联度的生物相容高分子交联材料。本发明还公开了一类双硫键交联的生物相容高分子交联材料，这些交联材料具有很低的交联度，不仅最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时也有效地延缓了生物相容高分子在体内的代谢和降低了溶解性，能较好地满足各种临床医学应用的要求。本发明还涉及该双硫键交联的生物相容高分子交联材料在医学和药学中的用途。

Description

低巯基化改性度生物相容高分子巯基化衍生物及其交联材料和用途 Technical Field
本发明涉及低改性度生物相容高分子衍生物，尤其涉及低巯基化改性度生物相容高分子巯基化衍生物，本发明还涉及低交联度的双硫键交联的生物相容高分子交联材料，此外本发明还涉及该交联材料在医药领域中的应用。
Background Art
生物相容高分子具有许多重要的生理功能，例如透明质酸在关节炎的增粘治疗、促进创伤愈合等方面具有显著的效果。然而，通常生物相容高分子在体内的代谢很快或者易溶于体液，这在很大程度上限制了其在很多临床医学中的应用，例如透明质酸用于关节炎的增粘治疗的疗程为连续五周的每周一次关节注射，不仅给患者和医务工作者带来不便，同时还增加了感染的风险。对生物相容高分子进行化学改性、化学交联、或者化学改性后进行交联是延缓生物相容高分子在体内的代谢和降低溶解性的有效手段，显著拓展了生物相容高分子在临床医学中的应用。例如对于关节炎的增粘治疗，交联透明质酸钠的一次关节注射即可达到非交联透明质酸钠五次关节注射的疗效；同时交联透明质酸还被广泛应用于真皮填料等美容用途。
虽然生物相容高分子的化学改性和 / 或交联大大拓展了其在临床医学中的应用，然而在理论和实际过程中仍然存在相互矛盾之处。一方面，生物相容高分子的化学改性和 / 或交联必须达到一定程度才能延缓生物相容高分子在生物体内的代谢和降低溶解性，因此目前在临床医学中得到广泛应用的化学改性和 / 或交联的生物相容高分子衍生物或交联材料都具有很高或者比较高的改性度或交联度，例如意大利 Fidia 公司的 HYAFF 系列产品是高度酯化的透明质酸钠衍生物（改性度可高达 100% ）；另一方面，化学改性和 / 或交联改变了生物相容高分子的化学结构， 影响和降低了生物相容高分子的生理功能和生物相容性能，甚至引起一定的副反应，例如 Jacob 等人的研究结果表明基于高度改性 HYAFF 的 MeroGel® 引起了炎性反应和成骨化反应（ Jacob 等， Laryngoscope 112: 37-42, 2002 ）。
然而，目前大部分的研究都集中于提高改性度和 / 或交联度以延缓生物相容高分子在体内的代谢和降低溶解性。在我们看来，相当多的情况下高度改性和 / 或交联的生物相容高分子并不能较好地满足临床医学应用的需要，甚至还可能引起炎症反应等副作用。因此，生物相容高分子的化学改性和 / 或交联必须兼顾两方面的因素：即尽可能降低化学改性和 / 或交联程度以保持生物相容高分子的初始结构、生理功能和生物相容性，同时化学改性和 / 或交联程度需要适当地延缓生物相容高分子在体内的代谢和降低溶解性以满足临床医学应用的要求。然而同时兼顾上述两方面因素的生物相容高分子化学改性和 / 或交联是一个技术难题。
生物相容高分子的巯基化改性和双硫键交联是一种新型的化学改性和交联方法，具有很多优点，在临床医学中具有许多重要的潜在用途 。例如生物相容高分子巯基化衍生物已经被用于各种小分子药物和多肽蛋白药物的化学活性修饰等等，基于这些生物相容高分子巯基化衍生物制备的交联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（Bernkop-Schnurch，WO2000/025823；Shu等，Biomacromolecules，3:1304，2002；Bulpitt等，WO2002/068383；Prestwich等，WO2004/037164；Prestwich等，WO2005/056608；Prestwich等，WO2008/008857；Song，WO2008/071058；Song，WO2008/083542； Gonzalez等，WO2009/132226）。通常研究人员认为较高的巯基化改性度对于制备后续的生物相容高分子巯基化改性衍生物的交联材料是必须的，因此在上述公开的文献报道中，生物相容高分子的巯基化改性度和/或交联度都很高，例如在Shu等的报道中26.8~66.8%的基团被改性和交联（Shu等，Biomacromolecules，3: 1304，2002）。
 Technical Problem
本发明要解决的技术问题之一是提供一类低巯基化改性度的 生物相容高分子巯基化衍生物，这些 低巯基化改性度的 生物相容高分子巯基化衍生物最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时引入的巯基可有效地进行化学交联以制备低交联度生物相容高分子交联材料。
本发明要解决的技术问题之二是提供一类双硫键交联的 生物相容高分子交联材料，该交联材料具有很低的双硫键交联度，不仅最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时也有效地延缓了生物相容高分子在体内的代谢和降低了溶解性，较好地满足各种医药学应用的要求。另外， 这些双硫键交联 生物相容高分子交联材料 的交联过程可以在可注射容器内完成，具有可注射的性能，使用方便、不含杂质、生物相容性好、无毒副作用，在医药领域具有非常广泛的应用前景。
本发明要解决的技术问题之三是提供上述双硫键交联生物相容高分子交联材料在医药领域中的用途。
 Technical Solution
在本发明中所使用的部分术语定义如下所述。
生物相容高分子 是指具有良好生物相容性的高分子，包括多糖、蛋白质以及合成高分子等等。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、壳聚糖、羧甲基壳聚糖等以及它们的盐形式（如钠盐，钾盐等）和衍生物形式；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（如钠盐，钾盐等）和衍生物形式；蛋白质包括胶原蛋白、碱性明胶蛋白、酸性明胶蛋白、弹性蛋白、 核心蛋白、多糖层粘连蛋白、纤维结合蛋白等以及它们的盐形式 （如钠盐，钾盐等）和 衍生物形式。 生物相容高分子优选硫酸 软 骨素、肝素、类肝素、海藻酸、透明质酸、聚天冬氨酸、聚谷氨酸、壳聚糖、羧甲基壳聚糖、碱性明胶蛋白和酸性明胶蛋白以及它们的盐形式（如钠盐，钾盐等）和衍生物形式 ；特别优选 硫酸 软 骨素和透明质酸以及它们的盐形式（如钠盐，钾盐等）和衍生物形式 。
生物相容高分子巯基化衍生物是指生物相容高分子侧链基团通过化学方式引入巯基的衍生物，巯基化改性度是指引入巯基的数量占生物相容高分子可供改性侧链基团数量的百分比。例如，当透明质酸的侧链羧基进行巯基化改性时，巯基化改性度是指引入巯基的数量占透明质酸侧链羧基总数量的百分比。
双硫键交联是指生物相容高分子巯基化衍生物 通过双硫键的形式形成三维网状结构，双硫键交联度是指 生物相容高分子巯基化衍生物中形成双硫键的巯基数量占生物相容高分子可供改性侧链基团数量的百分比。
水凝胶是指含有大量水的具 有三维交联网络结构的物质，介于液态和固态之间，没有流动性， 凝胶化是指从具有流动性的液态到失去流动性的凝胶态的过程。
动力粘度是指使单位距离的单位面积液层产生单位流速所需之力，其单位为厘泊（ mPa·s ）或泊（ Pa·s ）。动力粘度是评定粘度的指标，动力粘度越小，流动性越好，反之则流动性越差。
本发明的一方面是提供了一类低巯基化改性度 生物相容高分子巯基化衍生物，这些低巯基化改性度 生物相容高分子巯基化衍生物最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时引入的巯基可有效地进行化学交联以制备低交联度的生物相容高分子交联材料。
在本发明中， 低巯基化改性度 生物相容高分子巯基化衍生物通常可以通过下述方法制备，这些方法已经在 专利文件 WO2009006780 进行了描述。 方法一是侧链羧基的 氨基（酰肼）/碳二亚胺偶合化学方法。其通常方式是羧基在碳化二亚胺的活化下形成中间产物，含有双硫键的二氨或二酰肼亲核取代生成中间产物，最后双硫键还原为巯基即可得到生物相容高分子巯基化衍生物 （ Shu 等， Biomacromolecules ， 3 ， 1304 ， 2002 ； Aeschlimann 等， US 7,196,180 B1 ）。 也可用含有自由巯基（或 巯基保护）的伯氨代替含有双硫键的二氨或二酰肼，即可得到生物相容高分子巯基化衍生物，或 所得到的中间产物 脱除巯基保护基团即可得到生物相容高分子巯基化衍生物 （ Gianolio 等， Bioconjugate Chemistry ， 16 ， 1512 ， 2005 ） 。 上 述的碳化二亚胺通常是 指1-乙基-3-(3-二甲胺丙基)碳二亚胺盐酸盐 。 方法二是侧链羧基直接与含有双硫键的 碳二亚胺 （如 2,2'- 二硫代双（ N- 乙基（ N'- 乙基碳二亚胺））等等）反应 制备，所制备的生物相容高分子巯基化改性衍生物具有下述通式（ III ）的结构（ Bulpitt 等， US 6884788 ）。 方法三是对侧链氨基的改性， 一般分为直接和间接改性两种方式。直接改性方式是指对侧链氨基的直接修饰，引入巯基。 例如 双丁二酸双酰胱胺双 羰二咪唑活化 酯对 胶原氨基的巯基化改性（ Yamauchi 等， Biomaterials ， 22 ， 855 ， 2001 ； Nicolas 等， Biomaterials ， 18 ， 807 ， 1997 ） 。 方法三中氨基的间接巯基化改性方式一般分为两个步骤。第一步是氨基的羧基化，第二步是采用前述方法一或二进行羧基的巯基化改性。方法四是 侧链羟基的改性。通常的方法是羟基在强碱条件下的羧基化，然后羧基再按照前述 方法一和方法二 巯基化，例如，纤维素、透明质酸、甲壳素和壳聚糖等高分子的侧链羟基都可以羧甲基化，然后采用 氨基（ 酰肼） / 碳二亚胺 碳化学反应进行巯基化改性。
对于具有一种或多种官能团（羧基、氨基和羟基）的生物相容高分子，可采用上述一种或多种方法制备本发明的低巯基化改性度 生物相容高分子巯基化衍生物。
在本发明中， 低巯基化改性度 生物相容高分子巯基化衍生物采用前述制备方法制备，通过调节反应原料的投料比、反应时间、反应温度等参数，即可实现本发明。
在本发明中， 低巯基化改性度 生物相容高分子巯基化衍生物的纯化是非常重要的。残余杂质不仅可能在生物体内产生炎症等毒副作用，同时还会干扰后续的双硫键交联。在本发明中，残余杂质可以通过透析和 / 或有机溶剂（如乙醇等）沉淀等方法去除。
在本发明中，所采用的生物相容高分子的分子量通常在 1000~10000000 之间，优选 10000~3000000 ，特别优选 20000~1500000 。
在本发明中，保留了大部分的生物相容高分子的初始结构，巯基化改性度很低。本发明的 低巯基化改性度 生物相容高分子巯基化衍生物侧链至少含有三个巯基，且巯基化改性度 ≤ 4.5% ，巯基化改性度优选 0.5%~3.0% ，巯基化改性度特别优选 0.75%~2.5% 。
对于生物相容高分子巯基化衍生物， 研究人员普遍具有一种技术偏见：即 较高的巯基化改性度对于制备后续的生物相容高分子巯基化衍生物的交联材料以及满足临床医学用途是必须的。例如， Prestwich 等的研究表明巯基化改性度较高的生物相容高分子衍生物才能较好地实现交联（Prestwich等，WO2008/008857 ），因此研究人员普遍倾向于提高生物相容高分子的巯基化改性程度。 Sparer 等在 1983 年公开了糖胺多糖（透明质酸和硫酸软骨素 - 半胱氨酸甲酯的衍生物，半胱氨酸甲酯与糖胺多糖通过酰胺键偶合， 5%~87% 的糖胺多糖侧链羧基被改性为巯基（ Sparer 等， Chapter 6, Page 107-119 ， Controlled Release Delivery System, Edited by Theodore J. Roseman, S.Z. Mansdorf ， Marcel Dekker Inc. ）。 Gianolio 等于 2005 年公开了透明质酸 - 半胱胺衍生物，半胱氨与透明质酸的侧链羧基通过酰胺键偶合， 22% 的透明质酸侧链羧基被改性为巯基（ Gianolio 等， Bioconjugate Chemistry, 16: 1512-1518, 2005 ）。印等于 2008 年公开了透明质酸 - 半胱胺衍生物，半胱氨与透明质酸的侧链羧基通过酰胺键偶合，衍生物同时含有 10~200 µmol/g 巯基和 120~500 µmol/g 双硫键，按照透明质酸的二糖重复链段分子量为 400 计算，巯基化改性度分别为 10%~48%（即10%~48% 的透明质酸侧链羧基被巯基化改性）（印等， CN 101367884 ）。 Shu 等公开的通过酰肼键偶合的透明质酸巯基化衍生物的巯基化改性度为 26.8%~66.8% （ Shu 等， Biomacromolecules ， 3: 1304 ， 2002 ）。
然而，当巯基化改性度较高时，生物相容高分子的初始结构被显著改变，这会对生物相容高分子的生理功能和生物相容性产生不利的影响。例如王等的研究结果表明壳聚糖巯基化衍生物在高巯基化改性度时产生了显著的细胞毒性（王等，高等学校化学学报， 29: 206-211, 2008 ），我们的研究也表明高度的巯基化改变了透明质酸的结构，干扰了与受体（如 CD44 等）的结合。
在本发明中，所制备的 低巯基化改性度 生物相容高分子巯基化衍生物采用前述一种或多种方法进行了高度纯化，残余杂质的重量含量通常小于千分之一甚至万分之一。
本发明的有益效果是 本发明的 低巯基化改性度 生物相容高分子巯基化衍生物的巯基化改性度很低，最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时还具有原料用量少、反应时间短等特点，此外本发明的 低巯基化改性度 生物相容高分子巯基化衍生物可以方便地用于制备交联材料和满足多种临床医学用途的要求。另外 本发明 还克服了前述技术偏见： 即 较高的巯基化改性度对于制备后续的生物相容高分子巯基化衍生物的交联材料以及满足临床医学用途是必须的。
本发明的另一方面是提供一类双硫键交联的 生物相容高分子交联材料，这些生物相容高分子交联材料具有很低的双硫键交联度，不仅最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时也有效地延缓了生物相容高分子在体内的代谢和降低了溶解性，较好地满足各种临床医学应用的要求。本发明的 双硫键交联的 生物相容高分子交联材料通常为水凝胶的形式，其优选含水量为 95% （重量 / 体积百分比， g/ml ）以上的水凝胶形式，特别优选含水量为 98% （重量 / 体积百分比， g/ml ）以上的水凝胶形式。 本发明的双硫键交联的 生物相容高分子交联水凝胶干燥或冷冻干燥后即可制成薄膜、海绵等多种固态形式。
本发明的双硫键交联的 生物相容高分子交联水凝胶采用本发明的 低巯基化改性度 生物相容高分子巯基化衍生物为原料制得，一种实现途径如下所述：将本发明的 低巯基化改性度 生物相容高分子巯基化衍生物溶解于水得到合适浓度的溶液（通常为 0.2%~5.0% ），调节溶液 pH 值为特定值（通常为中性，即 pH 值约为 7 ），然后巯基在空气中的氧气和溶液中的溶解氧气的氧化下逐渐形成双硫键，溶液逐渐凝胶化，溶液动力粘度逐渐增加，最终溶液失去流动性形成三维网状交联结构。也可以在上述溶液中加入氧化剂（如过氧化氢等）加快交联过程。
本发明的双硫键交联的 生物相容高分子交联水凝胶的第二种实现途径是采用 Shu 等公开的方法（ WO2010043106 ）。在该方法中 凝胶化过程可以在可注射容器内完成，具有可注射的性能，使用方便、不含杂质、生物相容性好、无毒副作用等特点。该实现途径的具体过程如下所述： 将本发明的 低巯基化改性度 生物相容高分子巯基化衍生物溶解于水得到合适浓度的溶液（通常为 0.2%~5.0% ），调节溶液 pH 值为特定值（通常为中性），然后将溶液灌注于可注射容器内并密封，巯基主要在溶液中溶解氧气的氧化下逐渐形成双硫键，溶液逐渐凝胶化，溶液动力粘度逐渐增加，最终溶液失去流动性形成三维网状交联结构。也可以在上述溶液中加入氧化剂（如过氧化氢等）加快交联过程。
本发明 的 双硫键交联的 生物相容高分子交联水凝胶的第二种实现途径 可采用无菌工艺或终端灭菌工艺（如医药工业常用的湿热灭菌工艺等）的方式生产，以满足不同临床医学要求。可采用医药工业常用的罐封生产线实现大规模工业化生产，每小时产量可方便地达 3000 支以上。罐封生产线可选用 高宁格公司的直线式全自动注射器预灌封生产线或蜂巢式注射器全自动预罐装和加塞设备、以及德国博世等公司的预消毒灭菌注射器液体灌封机等等。可注射容器 可以是玻璃材质或塑料材质注射器，如 BD 公司的 Hypak SCF 类预消毒灭菌注射器，也可以用软胶袋等可挤出容器代替注射器。
在本发明 的 双硫键交联的 生物相容高分子交联水凝胶中，由于采用本发明的 低巯基化改性度 生物相容高分子巯基化衍生物为原料，双硫键的交联度取决于本发明的 低巯基化改性度 生物相容高分子巯基化衍生物的巯基化改性度（ ≤ 4.5% ），因此本发明的 双硫键交联的 生物相容高分子交联水凝胶中的 双硫键交联度也很低（ ≤ 4.5% ）。通常情况下在 本发明的 双硫键交联的 生物相容高分子交联水凝胶中一半以上的巯基被氧化成了双硫键，形成了三维网状交联结构，失去了液体溶液的流动性，具有很高的动力粘度。与未形成交联的初始溶液相比较， 本发明 的 双硫键交联的 生物相容高分子交联水凝胶的动力粘度通常提高了 50 倍以上，在优化条件下甚至可提高 500 倍以上， 本发明 的 双硫键交联的 生物相容高分子交联水凝胶的高动力粘度的特性使其在术后粘连防治、关节炎增粘治疗等重要临床医学用途方面具有独特的优势。
采用旋转粘度计，按照《中华人民共和国药典》（2005年版）二部附录VI G第二法测定，在剪切速率不小于0.25Hz、25±0.1℃条件下， 本发明 的 双硫键交联的 生物相容高分子交联水凝胶的动力粘度通常大于10000 厘泊（ mPa ·s） ，动力粘度优选大于25000 （ mPa ·s） ，动力粘度特别优选大于40000 （ mPa ·s） 。
本发明的 双硫键交联的 生物相容高分子交联材料可以含有一种或多种 本发明的 低巯基化改性度 生物相容高分子巯基化衍生物，此外 还可以含有一种或多种其它物质。这些物质可以是多糖、蛋白质或合成高分子，如 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、壳聚糖、羧甲基壳聚糖、胶原蛋白、碱性明胶蛋白和酸性明胶蛋白以及它们的盐形式（如钠盐，钾盐等）和衍生物形式等，优选 透明质酸钠、硫酸软骨素、肝素钠、 碱性明胶蛋白和酸性明胶蛋白 等，特别优选透明质酸钠、硫酸软骨素和肝素钠；这些物质也可以是活性药物组份，包括类固醇、抗生素、治疗肿瘤的药物和各种多肽蛋白药物，例如皮质激素（属于类固醇），如 倍氯米松 、 倍氯美松双丙酸酯 、 布地奈德 、 地塞米松 、 泼尼松龙 、 泼尼松等；又如 各种多肽蛋白药物，如各种生长因子（碱性生长因子、酸性生长因子、血管生长因子、成骨生长因子等）、核酸（如 RNA ）等等。该活性药物组份可以固体颗粒形式分散和 / 或溶解在本发明的 双硫键交联的 生物相容高分子交联材料中 。
在医药领域的实际应用中，要求双硫键交联的 生物相容高分子交联水凝胶有合适的贮存时间，其性能需要保持稳定。然而， 双硫键交联的 高改性生物相容高分子交联水凝胶并不稳定，随着贮存时间的增加，水凝胶逐渐收缩，大量水从水凝胶中被挤出，导致动力粘度大大降低，严重影响了凝胶性能，不符合 临床医学实际应用的需要，严重制约了双硫键交联的 生物相容高分子交联水凝胶在医药领域中的应用 。例如双硫键交联的 透明质酸交联水凝胶（ 13.5% 的巯基化改性度）在室温储存 6 个月后，水凝胶的体积收缩了约 30% 。
本发明 的 双硫键交联的 生物相容高分子交联水凝胶采用本发明的 低巯基化改性度 生物相容高分子巯基化衍生物为原料，取得了意想不到的技术效果，很好地解决了上述 双硫键交联的 生物相容高分子交联水凝胶不稳定的难题。为期 6 个月的稳定性加速试验表明本发明的 双硫键交联的 生物相容高分子交联水凝胶具有很好的稳定性，将结合实施例进一步说明。
本发明的有益效果是本发明的 双硫键交联的 生物相容高分子交联材料具有很低的交联度，不仅最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时具有很高的动力粘度，有效地延缓了生物相容高分子在体内的代谢和降低了溶解性，较好地满足各种临床医学应用的要求。 本发明的有益效果还包括本发明的 双硫键交联的 生物相容高分子交联水凝胶具有很好的稳定性。
在本发明的另一方面，还提供了上述双硫键交联的 生物相容高分子交联材料 在医药领域中的应用。
本发明的双硫键交联的 生物相容高分子交联材料 在医学中的应用包括：可以用于促进伤口愈合，可用于皮肤或其它创伤的伤口敷料；也可用于防止粘连，包括外科手术（例如鼻窦炎手术）之后防止组织或器官之间的纤维性粘连；也可用作关节润滑剂用于关节炎增粘治疗等。
本发明所制备的双硫键交联的生物相容高分子交联材料在药学中的应用包括作为各种活性治疗物质的缓释载体，实现缓慢释放的性能。活性治疗物质可以是化学药物或生物学上的活性因子，包括消炎剂、抗生素、止痛药、麻醉剂、伤口愈合促进剂、细胞生长促进或抑制因子、免疫刺激剂、抗病毒药物等等。
 Advantageous Effects
本发明的有益效果是本发明的低巯基化改性度 生物相容高分子巯基化衍生物的巯基化改性度很低，最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时还具有原料用量少、反应时间短等特点，此外本发明的 低巯基化改性度 生物相容高分子巯基化衍生物可以方便地用于制备交联材料和满足多种临床医学用途的要求。另外 本发明 还克服了前述技术偏见： 即 较高的巯基化改性度对于制备后续的生物相容高分子巯基化衍生物的交联材料以及满足临床医学用途是必须的。
本发明的有益效果是本发明的双硫键交联的生物相容高分子交联材料具有很低的交联度，不仅最大限度保持了生物相容高分子的初始结构、生理功能和生物相容性，同时具有很高的动力粘度，有效地延缓了生物相容高分子在体内的代谢和降低了溶解性，较好地满足各种临床医学应用的要求。 本发明的有益效果还包括本发明的 双硫键交联的 生物相容高分子交联水凝胶具有很好的稳定性。
Description of Drawings
图 1 是本发明实施例 12 的实验结果示意图（即 左后足的重量分布图 ）。
Best Mode
Mode for Invention
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 透明质酸巯基化衍生物的制备及表征
按照 Shu 等人在 Biomacromolecules ， 3 ， 1304 ， 2002 中公开的方法制备。透明质酸 20 克 溶解于２升蒸馏水，加入 11.9 克 二硫代二丙二酰肼，搅拌溶解。然后溶液的 pH 值用 0.1 摩尔 / 升盐酸调节至 4.75 ，按照表 1 分别加入一定量的 1- 乙基 -3-(3- 二甲胺丙基 ) 碳二亚胺盐酸盐（ EDCI ） (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 。加入 1.0 摩尔／升的氢氧化钠溶液到 pH 7.0 终止反应。然后加入 100 克 二硫苏糖醇（ Diagnostic Chemical Limited ，美国 ) 和适量 1.0 摩尔 / 升的氢氧化钠溶液，搅拌。调解溶液的 pH 值为 8.5 。室温电磁搅拌反应 24 小时。此后，在上述溶液中加入１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），用大量 0.001 摩尔 / 升的盐酸和 0.2 摩尔 / 升的氯化钠溶液透析 5 天，每 8 小时换一次透析液；然后再用大量的 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，直接使用制备的溶液或冷冻干燥得到 白色絮状固体。
采用 Shu 等人在 Biomacromolecules ， 3 ， 1304 ， 2002 中报道的改进 Ellman 方法检测巯基含量并计算巯基化改性度，或者采用 氢谱核磁共振检测（1H-NMR ）（ D2O 为溶剂）测定巯基化改性度（ 以透明质酸的乙酰基的特征甲基吸收峰为内标） 。巯基化改性度是指引入巯基的数量占透明质酸侧链羧基总数量的百分比，其结果如下：
表 1. 巯基化改性度
编号 1 2 3 4 5 6 7 8 9
EDCI 投料量（ g ） 0.2 0.3 0.4 0.6 0.8 1.0 1.2 2.4 9.6
巯基化改性度（ %） 0.48 1.04 1.46 2.33 3.24 4.18 4.61 10.6 37
实施例 2 硫酸软骨素巯基化衍生物的制备及表征
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 0.6 克 二硫代二丙二酰肼， 搅拌溶解。然后溶液的 pH 值用 0.1 摩尔 / 升盐酸调节至 4.75 ，按照表 2 分别加入一定量的 1- 乙基 -3-(3- 二甲胺丙基 ) 碳二亚胺盐酸盐（ EDCI ） (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 ，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。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 ，室温电磁搅拌反应 4 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 2000 ， Sigma ，美国），用 2 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 2 升 0.001 摩尔 / 升的盐酸溶液透析 3 天，每 8 小时换透析液。最后透析管内的溶液，直接使用制备的溶液或冷冻干燥得到絮状固体。
采用 Shu 等人在 Biomacromolecules ， 3 ， 1304 ， 2002 中报道的改进 Ellman 方法检测巯基含量并计算巯基化改性度，或者采用 氢谱核磁共振检测（1H-NMR ）（ D2O 为溶剂）测定巯基化改性度（ 以硫酸软骨素的乙酰基的特征甲基吸收峰为内标） 。巯基化改性度是指引入巯基的数量占 硫酸软骨素 侧链羧基总数量的百分比，其结果如下：
表 2. 巯基化改性度
编号 1 2 3 4 5 6 7 8 9
EDCI 投料量（ g ） 0.01 0.015 0.02 0.03 0.04 0.05 0.06 0.12 0.48
巯基化改性度（ %） 0.88 1.54 1.96 3.33 4.50 5.18 6.81 15.6 42.1
实施例 3 透明质酸巯基化衍生物的制备及表征
透明质酸 10 克 溶解于 1 升 蒸馏水，按照表 3 所列数量分别加入 N- 羟基琥珀酰亚胺磺酸钠盐（ Sulfo-NHS ）、胱胺二盐酸盐（ CYS ）和 1- 乙基 -3-(3- 二甲胺丙基 ) 碳二亚胺盐酸盐（ EDCI ） ，搅拌溶解，溶液的 pH 值用 0.1 摩尔 / 升盐酸调节至 4.5~6.5 ，电磁搅拌反应一定时间。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 4.5~6.5 。加入 1.0 摩尔／升的氢氧化钠溶液到 pH 8.5 终止反应。然后加入 50 克 二硫苏糖醇（ Diagnostic Chemical Limited ，美国 ） 和适量 1.0 摩尔 / 升的氢氧化钠溶液，搅拌。调解溶液的 pH 值为 8.5 。室温电磁搅拌反应 24 小时。此后，在上述溶液中加入１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），用大量 0.001 摩尔 / 升的盐酸和 0.5 摩尔 / 升的氯化钠溶液透析 5 天，每 8 小时换一次透析液；然后再用大量的 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，直接使用制备的溶液或者冷冻干燥得到 白色絮状固体。
采用 Shu 等人在 Biomacromolecules ， 3 ， 1304 ， 2002 中报道的改进 Ellman 方法检测巯基含量并计算巯基化改性度，或者采用 氢谱核磁共振检测（1H-NMR ）（ D2O 为溶剂）测定巯基化改性度（ 以透明质酸的乙酰基的特征甲基吸收峰为内标） 。巯基化改性度是指引入巯基的数量占透明质酸侧链羧基总数量的百分比，其结果如下：
表 3. 巯基化改性度
编号 1 2 3 4 5 6 7 8 9
Sulfo-NHS （ g ） 5.43 21.62 5.43 11.62 2.72 10.86 21.62 5.43 5.43
CYS （ g ） 22.6 11.3 33.9 17 5.625 22.6 11.3 22.6 11.3
EDCI （ g ） 4.8 9.6 4.8 2.4 2.4 1.2 1.2 2.4 0.72
反应时间 ( 小时 ) 12 3 1 1 1 1 8 8 8
巯基化改性度 (%) 13.5 4.83 1.54 0.84 0.73 0.51 2.14 3.97 1.28
实施例 4 双硫键交联的透明质酸交联水凝胶的制备
实施例 1 所制备的 2 种透明质酸巯基化衍生物（巯基化改性度分别为 2.33% 和 4.18% ，表 1 中编号 4 、 6 ）分别配制成 10 、 15 和 20 mg/ml 溶液，调节溶液的 pH 值为 7.4 。上述溶液 2 毫升转移至 10 毫升玻璃瓶后密封，室温静置保存 1 周，溶液失去流动性并形成交联水凝胶，水凝胶的含水量 (g/ml) 分别为 99% 、 98.5% 和 98% 。
实施例 5 双硫键交联的硫酸软骨素交联水凝胶的制备
实施例 2 所制备的硫酸软骨素巯基化衍生物（巯基化改性度为 4.50% ，表 2 中编号 5 ）分别配制成 5 0 和 80 mg/ml 溶液，调节溶液的 pH 值为 7.4 。上述溶液 2 毫升转移至 10 毫升玻璃瓶后密封，室温静置保存 1 周，溶液失去流动性并形成交联水凝胶，水凝胶的含水量 (g/ml) 分别为 95% 和 92% 。
实施例 6 双硫键交联的透明质酸交联水凝胶的制备
实施例 3 所制备的 4 种透明质酸巯基化衍生物（巯基化改性度为 1.28% 、 1.54 % 、 2.14% 、 3.97 % ，表 3 中编号 3 、 7 、 8 、 9 ）分别配制成 5 、 7.5 和 1 0 mg/ml 溶液，调节溶液的 pH 值为 7.4 。上述溶液 2 毫升转移至 10 毫升玻璃瓶后密封，室温静置保存 10 天 ，溶液失去流动性并形成交联水凝胶，水凝胶的含水量 (g/ml) 分别为 99.5 % 、 99.25% 和 99% 。
实施例 7 双硫键交联的透明质酸交联水凝胶的制备
实施例 3 所制备的透明质酸巯基化衍生物（巯基化改性度为 2.14 % ，表 3 中编号 7 ）配制成 1 0 mg/ml 溶液，然后按照体积比 2:1 分别加入透明质酸溶液（ 5 mg/ ml ）和硫酸软骨素溶液（ 10 mg/ml ），调节溶液的 pH 值为 7.4 。上述溶液 2 毫升转移至 10 毫升玻璃瓶后密封，室温静置保存 10 天 ，溶液失去流动性并形成交联水凝胶。
实施例 8 双硫键交联的透明质酸交联水凝胶的动力粘度测定
采用旋转粘度计，在剪切速率不小于0.25Hz、25±0.1℃条件下。按照《中华人民共和国药典》（2005年版）二部附录VI G第二法测定，测定实施例6所制备的 双硫键交联的透明质酸交联水凝胶的动力粘度，其结果见表4。交联水凝胶的动力粘度比相应未改性透明质酸溶液提高了408-547倍。
表 4. 动力粘度 ( mPa·s )
透明质酸浓度
（ mg/ml ）		 巯基化改性度（ % ）
0		 巯基化改性度（ % ）
1.28		 巯基化改性度（ % ）
1.54		 巯基化改性度（ % ）
2.14		 巯基化改性度（ % ）
3.97
5 137 75000 78000 81000 88000
7.5 198 >100000 >100000 >100000 >100000
10 245 >100000 >100000 >100000 >100000
实施例 9 双硫键交联的透明质酸交联水凝胶的制备及稳定性测试
水凝胶 1 ：实施例 3 所制备的透明质酸巯基化衍生物（巯基化改性度为 13.5% ，表 3 中编号 1 ）分别配制成 10 mg/ml 溶液，调节溶液的 pH 值为 7.4 。上述溶液 2 毫升转移至 10 毫升玻璃瓶后密封，室温静置保存 10 天 ，溶液失去流动性并形成交联水凝胶。
水凝胶 2 ：实施例 6 所制备的水凝胶（采用实施例 3 所制备的 透明质酸巯基化衍生物， 透明质酸浓度 10 mg/ml ，巯基化改性度 1.54% ，表 3 中编号 3 ）。
可见，水凝胶 2 的原料的 巯基化改性度（即 1.54% ） 要 明显低于 水凝胶 1 的原料 的 巯基化改性度（即 13.5% ），即水凝胶 2 的 交联 度 就明显低于 水凝胶 1 的 交联 度 。
稳定性测试： 依据中国药典2010版第二部 ⅪⅩ C 原料药与药物制剂稳定性试验指导原则对水凝胶进行稳定性加速试验，试验条件为温度40±2℃的下放置6个月，在试验期间第0、1、2、3和6个月末，取样对水凝胶动力粘度和收缩率（%）进行测定，其结果列于表5。对于高 交联 度的水凝胶1，随着稳定性加速试验的增加，动力粘度急剧下降，并且凝胶的体积不断收缩，在1、2、3和6个月，凝胶体积的收缩率分别为10.2、35.1、39.2和41.4%，大量水从凝胶中被挤出。对于本发明的低 交联 度的水凝胶2，则保持了很好的稳定。
表5. 稳定性测试结果
时间（月）
0		 时间（月）
1		 时间（月）
2		 时间（月）
3		 时间（月）
6
水凝胶1 动力粘度 >100000 8750 <5000 <5000 <5000
水凝胶1 收缩率（%） 0 10.2 35.1 39.2 41.4
水凝胶2 动力粘度 >100000 >100000 >100000 >100000 >100000
水凝胶2 收缩率（%） 0 0 0 0 0
实施例 10 双硫键交联的透明质酸交联水凝胶 防止鼻窦炎术后鼻窦口的再狭窄化
10 只雄性巴斯德灭菌的新西兰白兔，体重 3.5-4.0 公斤，肌肉注射氯胺酮（ 35 毫克 / 公斤）及甲苯唑嗪（ 5 毫克 / 公斤）麻醉，剥离鼻外背面，碘酒消毒，然后以 3 毫升 1% 利多卡因与 1:100000 肾上腺素混合液麻醉。无菌条件下，沿中线做 2.5 毫米垂直切口，将覆盖于上颌窦的软组织及骨膜提起分离。用外科电钻打开上颌窦前壁，以 4 毫米球面切割钻将上颌窦中壁与鼻腔打通，从而形成一个直径 4 毫米边缘无黏膜的圆周形孔道。在 5 只兔子的孔道两侧中均填入 实施例 6 所制备的水凝胶（透明质酸浓度 10 mg/ml ，巯基化改性度 1.54% ）（治疗组），在 5 只兔子的孔道两侧中均不作填充（对照组）。然后将骨膜以可吸收缝线间断缝合，然后将皮肤以可吸收缝线缝合封闭上颌窦。不做其他包扎。术后给予动物正常饮食饮水。
术后两周，处死兔子。处死后将愈合的伤口切开，暴露窦腔。窦腔内的残留物用吸引器一边冲水一边轻轻吸出，用 30 度鼻内窥镜检查鼻窦内侧壁并进行摄像。每个孔径均用毫米刻度的尺子进行测量。双盲法完成对孔径的观察及测量。治疗组的鼻窦口直径 为 2.78±1.17 毫米 ，对照组的鼻窦口直径为 0.7±0.52 。
鼻窦口的再狭窄化是鼻窦炎临床手术的一个重要难题，会影响手术疗效，甚至导致鼻窦炎复发。上述结果表明 本发明的低交联度的 双硫键交联的透明质酸交联 水凝胶 可以非常显著地防止鼻窦口的狭窄化，有望在临床上得到广泛的应用。
实施例 11 双硫键交联的透明质酸交联水凝胶在术后粘连防治中的应用。
采用 Hemadeh 等（ Surgery 114: 907-10 ， 1993 ）和 Yetkin 等（ Int J Surg 7: 561-65 ， 2009 ） 报道的大鼠盲肠模型。其过程简述如下： 32 个大鼠共分为 3 组，其盲肠的浆膜用无菌纱布刮去浆膜光泽直至表面出血，然后一滴无水乙醇滴至出血表面进一步引发粘连；第一组为空白对照，不做任何治疗；第二组采用 1ml 市售透明质酸溶液（ 10 mg/ml ）进行治疗；第三组采用实施例 6 所制备的水凝胶（透明质酸浓度 10 mg/ml ，巯基化改性度 1.54% ）进行治疗；最后缝合大鼠体表伤口，两周后处死大鼠，解剖观察粘连状况。
采用 Yetkin 等的粘连评价系统进行粘连评价（ Int J Surg 2009; 7: 561-65) ，其结果如表 6 所示。空白对照组 ( 第一组 ) 产生了严重的粘连，市售透明质酸治疗组（第二组）存在一定程度粘连，本发明的双硫键交联的透明质酸交联水凝胶（第三组）具有最好的粘连防治效果。
表 6. 粘连评分
第一组 第二组 第三组
粘连评分 3.4 ±0.699 1.333 ±1.231 0.4 ±0.699
实施例 12 双硫键交联的透明质酸交联水凝胶在关节炎增粘治疗中的应用。
采用 Mihara 等（ Osteoarthritis and Cartilage 15: 543-549 ， 2007 ） 报道的兔子关节炎模型，简述如下：肌肉注射氯胺酮（ 35 毫克 / 公斤）及甲苯唑嗪（ 5 毫克 / 公斤）进行麻醉，兔子的左膝关节在膝盖骨的侧面切开 2 cm 的切口，然后切断暴露的外侧副韧带；切开 ? 肌肌腱的末端，露出外侧半月板，然后外侧半月板正中切除 3.0-4.0 mm ；缝合皮下肌肉层和皮肤层，腿部肌肉注射约 0.2 ml 氨苄西林。
半月板部分切除术后的兔子分为三组：第一组为生理盐水对照组， 分别 在术后 0 、 3 、 6 、 9 、 12 天在关节内注射 0.2 ml 生理盐水（共 5 次注射） ；第二组为透明质酸治疗组， 分别 在术后 0 、 3 、 6 、 9 、 12 天在关节内注射 0.2 ml 市售透明质酸溶液（ 10 mg/ml ）进行治疗（共 5 次注射）；第三组为本发明的双硫键交联的透明质酸交联水凝胶治疗组，在术后第 0 天关节内注射一次实施例 6 所制备的水凝胶（透明质酸浓度 10 mg/ml ，巯基化改性度 1.54% ）（共 1 次注射）。术后第 0 、 2 、 5 、 8 、 11 和 14 天对术后关节进行疼痛指数测定，疼痛指数通过测定左后足的重量分布来表征（ Mihara 等， Osteoarthritis and Cartilage 15: 543-549 ， 2007 ）；术后 15 天处死兔子，对术后关节的损伤情况进行外观和组织学评价。
对手术关节的损伤情况进行外观和组织学评价结果表明，本发明的双硫键交联的透明质酸交联水凝胶对术后关节的保护作用与透 明质酸治疗组相当，但 显著优于生理盐水对照组。 左后足的重量分布结果表明在术后 8 、 11 和 14 天, 本发明的双硫键交联的透明质酸交联水凝胶治疗组（第三组）显著优于生理盐水对照组（第一组）（ p<0.05 ）；在所有术后观察时间点， 透明质酸治疗组 ( 第二组 ) 与生理盐水对照组（第一组）的效果没有统计显著差异（ p>0.05 ），同时本发明的双硫键交联的透明质酸交联水凝胶治疗组（第三组）与 透明质酸治疗组 ( 第二组 ) 的效果没有统计显著差异（ p>0.05 ）（见图 1 ）。
上述结果表明，本发明的双硫键交联的透明质酸交联水凝胶在 关节炎增粘治疗中有显著效果，一次关节注射的疗效与未交联透明质酸五次关节注射的疗效相当。
实施例 13 含有药物的双硫键交联的透明质酸交联水凝胶的制备和表征
在实施例 6 的双硫键交联的透明质酸交联水凝胶的制备过程中 （透明质酸浓度 10 mg/ml ，巯基化改性度 3.97% ） ，分别加入 0.1~10 毫克皮质激素（例如， 倍氯米松 Beclomethasone 、 倍氯美松双丙酸酯 Beclomethasone dipropionate 、 布地奈德 Budesonide 、 地塞米松 Dexamethasone 、 泼尼松龙 Prednisolone 、 泼尼松 Prednisone 等 ），皮质激素均匀分散在所制备的交联水凝胶中。
上述含药物的 交联水凝胶 0.2 毫升放入 15 毫升塑料离心管，加入 10 毫升磷酸盐缓冲溶液，放入摇床（ 37 ℃ ， 100 转 / 分钟），间隔一定的时间测定上清液中药物的紫外吸收。测定波长如下： 倍氯米松 Beclomethasone 246 nm 、 倍氯美松双丙酸酯 Beclomethasone dipropionate 240 nm 、 布地奈德 Budesonide 248 nm 、 地塞米松 Dexamethasone 242 nm 、 泼尼松龙 Prednisolone 248 nm 、 泼尼松 Prednisone 244 nm 。
表 7 . 药物在不同时间点的累积释放百分比
时间 ( 天 ) 倍氯米松 倍氯美松双丙酸酯 布地奈德 地塞米松 泼尼松龙 泼尼松
7
14
21		 68%
87%
94%		 <1%
<1%
<1%		 26%
43%
61%		 41%
63%
75%		 95%
100%
100%		 86%
99%
100%
从上述表7的结果可以看出，双硫键交联的透明质酸交联水凝胶是一种很好的药物缓释载体，对6种皮质激素均具有较好的缓释效果。根据药物疏水性的差异，药物从水凝胶中释放的行为有很大的不同。药物疏水性越强，释放越为缓慢。例如对于相对较为亲水的泼尼松龙Prednisolone，7天内已基本释放完毕；而对于疏水性很强的倍氯美松双丙酸酯Beclomethasone dipropionate，几乎检测不到释放。
 Industrial Applicability
本发明的双硫键交联的 生物相容高分子交联材料 在医学中的应用包括：可以用于促进伤口愈合，可用于皮肤或其它创伤的伤口敷料；也可用于防止粘连，包括外科手术（例如鼻窦炎手术）之后防止组织或器官之间的纤维性粘连；也可用作关节润滑剂用于关节炎增粘治疗等。
本发明所制备的双硫键交联的生物相容高分子交联材料在药学中的应用包括作为各种活性治疗物质的缓释载体，实现缓慢释放的性能。活性治疗物质可以是化学药物或生物学上的活性因子，包括消炎剂、抗生素、止痛药、麻醉剂、伤口愈合促进剂、细胞生长促进或抑制因子、免疫刺激剂、抗病毒药物等等。
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Claims (1)

1. 一类低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，该生物相容高分子巯基化衍生物的侧链至少含有三个巯基，且巯基化改性度 ≤ 4.5% ；所述生物相容高分子巯基化衍生物是指生物相容高分子侧链基团通过化学方式引入巯基的衍生物；所述巯基化改性度是指引入巯基的数量占生物相容高分子可供改性侧链基团数量的百分比；所述 生物相容高分子 是指具有良好生物相容性的高分子，包括多糖、蛋白质以及合成高分子。
2. 按照权利要求 1 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的巯基化改性度为 0.5%~3.0% 。
3. 按照权利要求 2 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的巯基化改性度为 0.75%~2.5% 。
4. 按照权利要求 1 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述多糖是硫酸 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、壳聚糖、羧甲基壳聚糖或它们的盐形式和衍生物形式；所述合成高分子是聚丙烯酸、聚天冬氨酸、聚酒石酸、聚谷氨酸、聚富马酸或它们的盐形式和衍生物形式；所述蛋白质包括胶原蛋白、碱性明胶蛋白、酸性明胶蛋白、弹性蛋白、 核心蛋白、多糖层粘连蛋白、纤维结合蛋白或它们的盐形式 和 衍生物形式 。
5. 按照权利要求 1 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的 生物相容高分子是 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、壳聚糖、羧甲基壳聚糖、碱性明胶蛋白、酸性明胶蛋白或它们的盐形式和衍生物形式 。
6. 按照权利要求 5 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的 生物相容高分子是 硫酸 软 骨素、透明质酸或它们的盐形式和衍生物形式 。
7. 按照权利要求 1 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的 生物相容高分子 的分子量在 1000~10000000 之间。
8. 按照权利要求 7 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的 生物相容高分子 的分子量在 10000~3000000 之间。
9. 按照权利要求 8 所述的 低巯基化改性度的生物相容高分子巯基化衍生物，其特征在于，所述的 生物相容高分子 的分子量在 20000~1500000 之间。
10. 采用如权利要求 1-9 任一项所述的一种或一种以上 低巯基化改性度的生物相容高分子巯基化衍生物 制备的 双硫键交联的 生物相容高分子交联材料。
11. 按照权利要求 10 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的交联材料为包括薄膜、海绵的固态形式。
12. 按照权利要求 10 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的交联材料为水凝胶。
13. 按照权利要求 12 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的水凝胶的含水量为 95% 以上，该含水量是重量 / 体积百分比。
14. 按照权利要求 13 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的水凝胶的含水量为 98% 以上，该含水量是重量 / 体积百分比。
15. 按照权利要求 12 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的水凝胶的动力粘度大于 10000 mPa·s 。
16. 按照权利要求 15 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的水凝胶的动力粘度大于 25000 mPa·s 。
17. 按照权利要求 16 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的水凝胶的动力粘度大于 40000 mPa·s 。
18. 按照权利要求 10-17 任一项所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的交联材料还 含有一种或一种以上的多糖、蛋白质、合成高分子和活性药物组份。
19. 按照权利要求 18 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 多糖、蛋白质和合成高分子是 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、壳聚糖、羧甲基壳聚糖、胶原蛋白、碱性明胶蛋白和酸性明胶蛋白或它们的盐形式和衍生物形式 。
20. 按照权利要求 19 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 多糖、蛋白质和合成高分子是透明质酸钠、硫酸软骨素、肝素钠、 碱性明胶蛋白和酸性明胶蛋白 。
21. 按照权利要求 20 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 多糖、蛋白质和合成高分子是透明质酸钠、硫酸软骨素和肝素钠。
22. 按照权利要求 18 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 活性药物组份以固体颗粒形式分散在交联材料中，或者溶解在交联材料中。
23. 按照权利要求 18 或 22 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 活性药物组份包括类固醇、抗生素、肿瘤治疗药物以及各种多肽蛋白药物。
24. 按照权利要求 23 所述的 双硫键交联的 生物相容高分子交联材料，其特征在于，所述的 活性药物组份为皮质激素，所述皮质激素包括 倍氯米松 、 倍氯美松双丙酸酯 、 布地奈德 、 地塞米松 、 泼尼松龙 和 泼尼松。
25. 一种如权利要求 10-24 任一项所述的 双硫键交联的 生物相容高分子交联材料 在医药领域中的用途。
26. 按照 权利要求 25 所述的用途，其特征在于，所述在医药领域中的用途包括在制备术后粘连防治制剂中的用途、在制备 关节炎增粘治疗 制剂中的用途和作为 活性治疗物质 缓释载体中的用途。
27. 按照 权利要求 26 所述的用途，其特征在于，所述 活性治疗物质是化学药物或生物学上的活性因子。
28. 按照权利要求 27 所述的用途，其特征在于， 所述 活性治疗物质是消炎剂、抗生素、止痛药、麻醉剂、伤口愈合促进剂、细胞生长促进或抑制因子、免疫刺激剂或抗病毒药物 。
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