WO2023020256A1 - 一种生物多糖水凝胶及其制备方法和应用 - Google Patents

一种生物多糖水凝胶及其制备方法和应用 Download PDF

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WO2023020256A1
WO2023020256A1 PCT/CN2022/108927 CN2022108927W WO2023020256A1 WO 2023020256 A1 WO2023020256 A1 WO 2023020256A1 CN 2022108927 W CN2022108927 W CN 2022108927W WO 2023020256 A1 WO2023020256 A1 WO 2023020256A1
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solution
polysaccharide
carboxyl
derivatives
primary amino
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PCT/CN2022/108927
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French (fr)
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韩宝芹
王硕
刘万顺
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中国海洋大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • 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
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the invention relates to an ophthalmic medical biomaterial, in particular to a biopolysaccharide hydrogel, its preparation method and its application as a vitreous substitute.
  • the vitreous body in the eyeball is a colorless transparent jelly filled between the lens and the retina, occupying more than 2/3 of the volume of the eyeball, and its main components include water, collagen, hyaluronic acid, proteoglycan, etc. 99% of the vitreous.
  • Hyaluronic acid interacts with collagen and forms a bonded state with water to stabilize the vitreous.
  • the vitreous body is highly transparent, allowing more than 90% of visible light to be transmitted.
  • the vitreous plays an important role in maintaining the shape of the eyeball, adjusting the partial pressure of oxygen in the eye, shock absorption and buffering, and maintaining the position of the retina and lens.
  • Vitrectomy is a common procedure used to treat many eye diseases such as retinopathy, retinal detachment, macular hole, etc. After vitrectomy, the vitreous cavity needs to be filled, and the vitreous substitute is filled in the vitreous cavity, pressing against the retina to keep it in the correct position and maintain the shape of the eyeball. Therefore, the vitreous filling becomes an important factor affecting the surgical effect.
  • vitreous substitutes mainly include gas, silicone oil, and perfluorocarbon liquid. These substitutes are suitable for vitreous filling in different surgical situations, but there are still many complications. For example, the filling and absorption of gas vitreous cavity is fast, the effective pressing time is short, and the patient needs to maintain a certain position after operation; perfluorocarbon liquid also has fast absorption, and the effective pressing time is about 4 weeks; It takes a long time to press, but it is not absorbed by the body tissues. There are many complications caused by the emulsification of silicone oil, such as glaucoma, cataract and other complications, so the silicone oil needs to be removed by secondary surgery. There is currently no ideal vitreous substitute.
  • the invention patent application with the publication number CN 102762647 A discloses a composition comprising a hydrogel polymer comprising oxidized hyaluronic acid and dihydrazide, wherein the dihydrazide is cross-linked with oxidized hyaluronic acid Linked to form a transparent colorless, from liquid to colloid, as a substitute for the vitreous of the eye.
  • the invention patent application with the publication number CN 110652610 A discloses a complete set of raw materials for a high cohesion double cross-linked artificial vitreous body.
  • Hyaluronic acid the second component is aminated hyaluronic acid
  • the first agent and the second agent are mixed and cross-linked to prepare a high cohesive double cross-linked artificial vitreous body.
  • the above two invention patent applications have a common feature of using oxidized ring-opened hyaluronic acid as an aldehyde group donor, which is cross-linked with dihydrazide or diamino group to form a gel.
  • the invention patent application with the publication number CN 105833344 A discloses the application of an injectable hydrogel in the preparation of intraocular fillings.
  • the injectable hydrogel consists of two doses, the first dose is a gel containing oxidized polysaccharides. liquid, the second agent is a glue solution containing chitin derivatives and/or collagen, through the cross-linking reaction between the dialdehyde groups of polysaccharides and the amino groups of chitin derivatives and/or collagen to form a viscoelastic water gel.
  • the first object of the present invention is to provide a vitreous substitute that has high light transmittance, good viscoelasticity, long degradation time, effectively improves the pressure time and has no side effects on eye tissue.
  • Biological polysaccharide hydrogel to make up for the deficiencies of the prior art.
  • Another object of the present invention is to provide a method for preparing biopolysaccharide hydrogel, and the application of the biopolysaccharide hydrogel for preparing vitreous substitutes.
  • the technical solution of the present invention while considering the excellent light transmittance, viscoelasticity, and non-toxicity of the hydrogel as a vitreous substitute, focuses on prolonging the degradation time of the hydrogel so as to prolong the pressure time on the retina .
  • the biological polysaccharide hydrogel of the present invention is composed of two agents, the first agent is a polysaccharide derivative solution with an aldehyde group in the side chain, and the second agent is a polysaccharide derivative solution with a primary amino group in the main chain or side chain, or a polysaccharide derivative solution with A solution of small molecule compounds with two or more primary amino groups.
  • the first agent of polysaccharide derivatives with aldehyde groups in side chains is a polysaccharide derivative with aldehyde groups in side chains formed by oxidation of polysaccharide derivatives grafted with ortho-dihydroxyl groups in side chains; Oxidizes the adjacent dihydroxyl structure of the polysaccharide side chain to form an aldehyde group, but does not oxidize the polysaccharide main chain, thereby ensuring that the polysaccharide molecule is not degraded by ring opening and maintaining the integrity of the polysaccharide main chain;
  • the substances reduce the specific recognition of biological enzymes to polysaccharides, thereby improving the resistance to biodegradation of polysaccharide derivatives.
  • the second agent of polysaccharide derivatives with primary amino groups in the main chain or side chains includes polysaccharide derivatives with primary amino groups in the main chain and polysaccharide derivatives with primary amino groups in the side chains; the polysaccharide derivatives with primary amino groups in the main chain
  • the polysaccharide derivative is characterized in that the main chain of the polysaccharide derivative has a primary amino group and has a branched side chain structure; the polysaccharide derivative with a primary amino group in the side chain is characterized in that the side chain of the polysaccharide derivative is branched
  • the polysaccharides of the aldehyde group donors The main chain of the derivative has no oxidative ring opening and no oxidative degradation of the molecular chain.
  • the polysaccharide derivative of the aldehyde group donor has a side chain graft structure.
  • the cross-linked structure of small molecular compounds further reduces the specific recognition of polysaccharides by biological enzymes, thereby improving the biodegradation resistance of polysaccharide derivatives.
  • the effective pressing time of the hydrogel to the retina is improved, and the purpose of being suitable as a vitreous substitute is achieved.
  • the present invention relates to a biological polysaccharide hydrogel, which is formed by mixing and reacting a first solution and a second solution, wherein the first solution is a solution of a polysaccharide derivative having an aldehyde group in a side chain, and the second solution
  • the second solution is a solution of polysaccharide derivatives with primary amino groups in the main chain or side chains, or a solution of small molecule compounds with two or more primary amino groups.
  • the first solution contains 0.5% to 15% by mass of polysaccharide derivatives with aldehyde groups in the side chain
  • the second solution contains 0.5% to 15% by mass of the main chain or side chain with primary amino groups polysaccharide derivatives, or small molecular compounds with two or more primary amino groups with a concentration of 0.2% to 30% by mass
  • the first solution and the second solution contain a mixture selected from sterile water, physiological saline, and physiologically balanced Solvent for liquid and glucose solution.
  • the polysaccharide derivative having an aldehyde group in the side chain in the first solution is an amino polysaccharide derivative having an aldehyde group in the side chain or a carboxyl polysaccharide derivative having an aldehyde group in the side chain.
  • the amino polysaccharide derivatives with side chain aldehyde groups are amino polysaccharide derivatives whose amino groups are modified to have side chains with aldehyde groups
  • the carboxyl polysaccharide derivatives with side chain aldehyde groups are those whose carboxyl groups are modified to have Carboxylated polysaccharide derivatives with aldehyde side chains.
  • Amino polysaccharide derivatives with side chain aldehyde groups or carboxyl polysaccharide derivatives with side chain aldehyde groups are amino polysaccharides with amino groups modified to have side chains with adjacent dihydroxy groups or carboxyl groups modified with adjacent dihydroxy groups Carboxylated polysaccharides in side chains are formed by oxidation.
  • the aminopolysaccharide derivatives in the first solution of the present invention include hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, carboxymethyl chitosan, or carboxyethyl chitosan;
  • Polysaccharide derivatives include chitosan derivatives with carboxyl structure, chitin derivatives with carboxyl structure, hyaluronic acid or hyaluronic acid derivatives with carboxyl structure, chondroitin sulfate, heparin, or sodium alginate.
  • the small molecule compound with two or more primary amino groups of the present invention is an aliphatic or aromatic small molecule compound with two or more primary amino groups, including ethylenediamine, propylenediamine, butylenediamine, amino Butanamide, glutamine, alanyl glutamine, lysine or other basic amino acids, small molecular compounds with dihydrazide structure, diaminobenzoic acid, diaminophenylacetic acid.
  • the small molecular compound with dihydrazide structure of the present invention includes carbohydrazide, oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, adipic acid dihydrazide or sebacic acid dihydrazide Hydrazine.
  • the polysaccharide derivatives with primary amino groups in the main chain in the second solution of the present invention are aminopolysaccharide derivatives with primary amino groups in the main chain while having a branched side chain structure; the polysaccharide derivatives with primary amino groups in the side chains have carboxyl structures in the main chain, Polysaccharide derivatives with primary amino groups in their side chains.
  • the polysaccharide derivatives with primary amino groups in the side chains are formed by condensation of carboxyl groups of carboxypolysaccharides with one amino group in small molecule compounds with two or more primary amino groups.
  • the preparation method of above-mentioned biological polysaccharide hydrogel may further comprise the steps:
  • the first solution is provided, and the first solution is a sterile solution of polysaccharide derivatives having aldehyde groups in the side chain with a mass concentration of 0.5-15%, and the pH is adjusted to be 3.5-7, wherein the side chain has aldehydes
  • the polysaccharide derivative of the base is formed by reacting an aqueous solution of a polysaccharide derivative with a grafted ortho-dihydroxy structure and an oxidizing agent;
  • the second solution is selected from any one of the following solutions:
  • step (1) Take the first solution obtained in step (1) and the second solution obtained in step (2), put them into tube A and tube B of a sterile double syringe respectively, push out the syringe to mix, and mix the first solution and the second solution Form a transparent biopolysaccharide hydrogel.
  • the polysaccharide derivatives with aldehyde groups in the side chains in the step (1) are prepared by the following method: provide the aqueous solution of the polysaccharide derivatives with a grafted ortho-dihydroxy structure with a mass concentration of 0.5% to 15%, and add the The above oxidant, wherein the molar ratio of the o-dihydroxy structure to the oxidant is 1: (0.3 ⁇ 3), room temperature or low temperature, avoid light, stir the reaction for 2min ⁇ 5h, add ethylene glycol solution to terminate the reaction for 0.5 ⁇ 2h, and dialyze the reaction product , freeze-drying, or ethanol precipitation, washing, precipitation, and drying.
  • polysaccharide derivatives with grafted ortho-dihydroxyl structures in the step (1) are prepared by the following method: respectively provide an aqueous amino polysaccharide solution with a mass concentration of 0.5 to 15% and a carboxyl-containing polysaccharide with a mass concentration of 0.2 to 30%.
  • Aqueous solution of small molecular compound with structure and ortho-dihydroxyl structure add carboxyl activating reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to small molecule
  • carboxyl activating reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to small molecule
  • adjust the pH to 3.5-7 stir and react for 0.5-3 hours
  • add the small molecule compound solution into the amino polysaccharide solution stir and adjust the pH of the reaction system to 7-9, and stir for 24 hours.
  • the small molecular compound having a carboxyl structure and an adjacent dihydroxy structure includes 2,3-dihydroxypropionic acid, 2,3,4-trihydroxybutyric acid, gluconic acid, threo Glycolic acid, arabic acid, xylonic acid, or mucic acid.
  • said step (1) has the polysaccharide derivative of grafted adjacent dihydroxyl structure also can be prepared by the following method: respectively provide the carboxypolysaccharide aqueous solution that mass concentration is 0.5 ⁇ 15% and mass concentration be 0.2 ⁇ 30%
  • carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution, adjust the pH to 3.5-7, and stir for 0.5-3 hours
  • add the small molecule compound solution to the carboxypolysaccharide solution stirring to adjust the pH of the reaction system to 7-9, stirring and reacting for 24 hours
  • dialyzing and freeze-drying the reaction product or carrying out ethanol precipitation, washing, precipitation and drying of the reaction product.
  • the small molecular compound having an amino structure and an adjacent dihydroxy structure includes aminopropylene glycol or glucosamine.
  • the polysaccharide derivatives with primary amino groups in the side chains in the step (2) are prepared by the following steps: preparing an aqueous carboxypolysaccharide solution with a mass concentration of 0.5 to 15%, and preparing a carboxypolysaccharide solution with a mass concentration of 0.2 to 30% with two or
  • For an aqueous solution of a small molecule compound with more than two primary amino groups add carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution, adjust the pH to 3.5-7, and stir for 0.5-3 hours; add the small molecule compound solution to the carboxypolysaccharide solution to adjust the pH 7 to 9, stirred and reacted for 24 hours; the reaction product was subjected to dialysis and freeze-drying, or the reaction product was subjected to ethanol precipitation, washing, precipitation and drying.
  • the oxidizing agent described therein includes sodium periodate or potassium periodate.
  • a biological polysaccharide hydrogel of the present invention is characterized in that it is formed by the mixed reaction of two agents, the first agent is a solution of polysaccharide derivatives with aldehyde groups in the side chain, and the second agent is the main A solution of polysaccharide derivatives with primary amino groups in the chain or side chain, or a solution of small molecule compounds with two or more primary amino groups.
  • the first dose of polysaccharide derivative solution having aldehyde groups in the side chain, the sugar units containing side chain aldehyde groups in the polysaccharide derivative account for 1% to 100% of the total sugar units of the polysaccharide molecule, and the polysaccharide derivative in the solution The mass percent concentration is 0.5% to 15%;
  • the second dose of polysaccharide derivative solution having primary amino groups in the main chain or side chains, and the sugar units containing primary amino groups in the main chain or primary amino groups in the polysaccharide derivatives account for the polysaccharide molecules
  • the percentage of total sugar units is 1% to 100%, and the mass percentage concentration of polysaccharide derivatives in the solution is 0.5% to 15%;
  • the second agent is a small molecule compound solution having two or more primary amino groups , the mass percent concentration of the small molecule compound in the solution is 0.2% to 30%;
  • the solvent of the first and second doses of the solution is sterile water, physiological saline, physiological balance liquid, or
  • the first dose of polysaccharide derivatives with aldehyde groups in the side chain is characterized in that it is an amino polysaccharide derivative with a side chain aldehyde group or a carboxyl polysaccharide derivative with a side chain aldehyde group; the amino polysaccharide derivative with a side chain aldehyde group
  • the derivative or the carboxyl polysaccharide derivative with a side chain aldehyde group is formed by oxidation of an amino polysaccharide derivative with a side chain grafted with a dihydroxyl group or a carboxyl polysaccharide derivative with a side chain grafted with a dihydroxyl group.
  • the aminopolysaccharide is a polysaccharide with primary amino groups, including but not limited to hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan and other chitosan derivatives;
  • the carboxyl polysaccharides include but not limited to chitosan derivatives with carboxyl structure, chitin derivatives with carboxyl structure, hyaluronic acid or hyaluronic acid derivatives with carboxyl structure , and chondroitin sulfate, heparin, sodium alginate;
  • the chitosan derivatives with carboxyl structure include carboxymethyl chitosan, carboxyethyl chitosan, succinyl chitosan and others with carboxyl structure Chitosan derivatives, said chitin derivatives with carboxyl structure include carboxymethyl chi
  • the amino polysaccharide derivative with side chain aldehyde group is characterized in that the amino polysaccharide is grafted with an ortho-dihydroxy structure on the amino group of the amino polysaccharide, thereby forming a side-chain ortho-dihydroxy structure, and the ortho-dihydroxy group is oxidized to form an aldehyde group, that is, to form Amino polysaccharide derivatives with side chain aldehyde groups;
  • the carboxyl polysaccharide derivatives with side chain aldehyde groups are characterized in that the carboxyl of the carboxyl polysaccharide is grafted with a dihydroxyl structure, thereby forming a side chain dihydroxyl structure,
  • the o-dihydroxyl group is oxidized to form an aldehyde group, that is, a carboxyl polysaccharide derivative with a side chain aldehyde group is generated.
  • the side-chain ortho-dihydroxyl structure is a structure grafted with an ortho-dihydroxyl group on the amino group of the aminopolysaccharide or on the carboxyl group of the carboxypolysaccharide.
  • Compounds that provide amino polysaccharides with an ortho-dihydroxyl structure are small molecular compounds that have both carboxyl and o-dihydroxyl groups, including but not limited to 2,3-dihydroxypropionic acid, 2,3,4-trihydroxybutyric acid, gluconic acid, Threonic acid, arabic acid, xylonic acid, mucic acid, and other compounds with carboxyl structure and adjacent dihydroxy structure; amino polysaccharides and compounds with both carboxyl structure and adjacent dihydroxy structure through the condensation reaction of amino and carboxyl groups,
  • the amino polysaccharide is grafted with a side chain adjacent to a dihydroxyl structure to form a polysaccharide derivative with an adjacent dihydroxyl side chain.
  • Compounds that provide carboxypolysaccharides with an ortho-dihydroxy structure are small molecular compounds that have both an amino structure and an ortho-dihydroxy structure, including but not limited to aminopropanediol, glucosamine, and other compounds that have both an amino structure and an ortho-dihydroxy structure; carboxyl
  • the carboxyl polysaccharide is grafted with the side chain of the ortho-dihydroxy structure through the condensation reaction of the carboxyl group and the amino group with the compound having both the amino structure and the ortho-dihydroxyl structure to form a polysaccharide derivative with an ortho-dihydroxyl side chain.
  • Polysaccharide derivatives with side chains branching adjacent dihydroxyl structures are oxidized to form polysaccharide derivatives with aldehyde groups in side chains.
  • the oxidation of the adjacent dihydroxyl structure of the side chain can obtain different aldehyde group contents by controlling the concentration, time, temperature and other conditions of the oxidizing agent, which can be 1-100% in theory.
  • the second agent is a polysaccharide derivative with primary amino groups in its main chain or side chain, or a small molecular compound with two or more primary amino groups.
  • the polysaccharide derivative with primary amino groups in its main chain is characterized in that Polysaccharide derivatives with primary amino groups in the main chain and branched side chains, including but not limited to hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, carboxymethyl chitosan, carboxyethyl chitosan Chitosan and other chitosan derivatives; the polysaccharide derivatives with primary amino groups in the side chain are characterized in that the main chain has a carboxyl structure, and the polysaccharide derivatives with primary amino groups in the side chain are carboxyl and polysaccharide derivatives of carboxyl polysaccharides.
  • One amino group in a small molecular compound with two or more primary amino groups is condensed to form a polysaccharide derivative with a primary amino group in the side chain;
  • the carboxyl polysaccharide includes but is not limited to chitosan derivatives with a carboxyl structure , chitin derivatives with carboxyl structure, hyaluronic acid and hyaluronic acid derivatives with carboxyl structure, and chondroitin sulfate, heparin, sodium alginate, the same as the carboxyl polysaccharide described in the first dose;
  • Small molecule compounds with one or more primary amino groups characterized by aliphatic or aromatic small molecule compounds with two or more primary amino groups, including but not limited to ethylenediamine, propylenediamine, butylenediamine, Aminobutyramide, glutamine, alanylglutamine, lysine and other basic amino acids, small molecular compounds with dihydrazide structure, diamino
  • the biological polysaccharide hydrogel is the first solution of polysaccharide derivatives with aldehyde groups in the side chain, and the second solution of polysaccharide derivatives with primary amino groups in the main chain or side chain, or with two or more
  • the small molecule compound solution of the primary amino group is mixed, and the aldehyde group of the first agent and the primary amino group of the second agent undergo a cross-linking reaction to form a hydrogel with high transparency, viscoelasticity and biocompatibility.
  • the preparation method of the above-mentioned biological polysaccharide hydrogel is characterized in that it includes the preparation of the first agent and the second agent and mixing the two agents.
  • the specific preparation method includes the following steps:
  • Small molecule compound solution with two or more primary amino groups prepare a sterile solution of small molecule compounds with two or more primary amino groups with a mass percent concentration of 0.2% to 30%, adjust When the pH is 7-9, determine the molar percentage of primary amino groups;
  • solution of polysaccharide derivatives with primary amino groups in the main chain prepare a sterile solution of polysaccharide derivatives with primary amino groups in the main chain at a mass percentage concentration of 0.5% to 15%, adjust the pH to 7-9, and measure the primary amino groups molar percentage of .
  • polysaccharide derivative solution with primary amino groups in the side chain prepare a carboxypolysaccharide aqueous solution with a mass concentration of 0.5 to 15%, and prepare a small molecule compound with two or more primary amino groups with a mass concentration of 0.2 to 30% Aqueous solution, add carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution, adjust the pH to 3.5-7, stir for 0.5-3h; add the small molecule compound solution to the carboxypolysaccharide solution, adjust the pH to 7-9, and stir for 24h; The reaction product is subjected to dialysis and freeze-drying, or the reaction product is subjected to ethanol precipitation, washing, precipitation and drying to obtain polysaccharide derivatives with primary amino groups in side chains. preparing a sterile solution of a polysaccharide derivative having a primary amino group in a side chain with a mass percent concentration of 0.5% to 15%, adjusting the pH to 7 to 9, and measuring the molar
  • the molar ratio of the carboxyl group in the small molecule compound with carboxyl structure and adjacent dihydroxyl structure or the carboxyl group in the carboxyl polysaccharide to the carboxyl activation reagent EDC, NHS is 1: (0.5 ⁇ 6 ): (0.5 ⁇ 6);
  • the oxidizing agent includes but not limited to sodium periodate, potassium periodate.
  • the present invention relates to the application of the above biopolysaccharide hydrogel in the preparation of intraocular vitreous substitutes.
  • the present invention also relates to the above-mentioned biopolysaccharide hydrogel, which is used as an intraocular vitreous substitute.
  • the present invention relates to an intraocular vitreous substitute, which comprises a biopolysaccharide hydrogel formed by the mixed reaction of a first solution and a second solution, wherein the first solution is a polysaccharide having an aldehyde group in a side chain Derivative solution, the second solution is a solution of polysaccharide derivatives with primary amino groups in the main chain or side chains, or a solution of small molecule compounds with two or more primary amino groups.
  • the first solution contains 0.5% to 15% by mass of polysaccharide derivatives with aldehyde groups in the side chain
  • the second solution contains 0.5% to 15% by mass of the main chain or side chain with primary amino groups polysaccharide derivatives, or small molecular compounds with two or more primary amino groups with a concentration of 0.2% to 30% by mass
  • the first solution and the second solution contain a mixture selected from sterile water, physiological saline, and physiologically balanced Solvent for liquid and glucose solution.
  • the polysaccharide derivative having an aldehyde group in the side chain in the first solution of the present invention is an amino polysaccharide derivative having an aldehyde group in the side chain or a carboxyl polysaccharide derivative having an aldehyde group in the side chain.
  • the amino polysaccharide derivatives with side chain aldehyde groups are amino polysaccharide derivatives whose amino groups are modified to have side chains with aldehyde groups
  • the carboxyl polysaccharide derivatives with side chain aldehyde groups are carboxyl modified to have aldehyde groups Carboxylated polysaccharide derivatives with side chains.
  • Amino polysaccharide derivatives with side chain aldehyde groups or carboxyl polysaccharide derivatives with side chain aldehyde groups are amino polysaccharides with amino groups modified to have side chains with adjacent dihydroxy groups or carboxyl groups modified with adjacent dihydroxy groups Carboxylated polysaccharides in side chains are formed by oxidation.
  • the aminopolysaccharide derivatives in the first solution of the present invention include hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, carboxymethyl chitosan, or carboxyethyl chitosan;
  • Carboxyl polysaccharide derivatives include chitosan derivatives with carboxyl structure, chitin derivatives with carboxyl structure, hyaluronic acid or hyaluronic acid derivatives with carboxyl structure, chondroitin sulfate, heparin, or sodium alginate.
  • the small molecule compound with two or more primary amino groups of the present invention is an aliphatic or aromatic small molecule compound with two or more primary amino groups, including ethylenediamine, propylenediamine, butylenediamine, amino Butanamide, glutamine, alanyl glutamine, lysine or other basic amino acids, small molecular compounds with dihydrazide structure, diaminobenzoic acid, diaminophenylacetic acid.
  • the small molecular compound with dihydrazide structure of the present invention includes carbohydrazide, oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, adipic acid dihydrazide or sebacic acid dihydrazide Hydrazine.
  • the polysaccharide derivatives with primary amino groups in the main chain in the second solution of the present invention are aminopolysaccharide derivatives with primary amino groups in the main chain and branched side chain structures; the polysaccharide derivatives with primary amino groups in the side chains have carboxyl structures in the main chain , A polysaccharide derivative having a primary amino group in the side chain.
  • the polysaccharide derivatives with primary amino groups in the side chains are formed by condensation of carboxyl groups of carboxypolysaccharides with one amino group in small molecule compounds with two or more primary amino groups.
  • Another aspect of the present invention also includes a biopolysaccharide hydrogel, which is used as a vitreous substitute in the eye, and the biopolysaccharide hydrogel is formed by mixing and reacting a first solution and a second solution, wherein the first The solution is a solution of polysaccharide derivatives with aldehyde groups in the side chain, and the second solution is a solution of polysaccharide derivatives with primary amino groups in the main chain or side chains, or a solution of small molecule compounds with two or more primary amino groups .
  • the first solution of the present invention contains 0.5% to 15% by mass of polysaccharide derivatives with aldehyde groups in the side chain, and the second solution contains 0.5% to 15% by mass of the main chain or side chain Polysaccharide derivatives with primary amino groups, or small molecular compounds with two or more primary amino groups with a concentration of 0.2% to 30% by mass; the first solution and the second solution contain a mixture selected from sterile water and normal saline , Physiological balance solution and solvent of glucose solution.
  • the polysaccharide derivative having an aldehyde group in the side chain in the first solution of the present invention is an amino polysaccharide derivative having an aldehyde group in the side chain or a carboxyl polysaccharide derivative having an aldehyde group in the side chain.
  • the amino polysaccharide derivatives with side chain aldehyde groups are amino polysaccharide derivatives whose amino groups are modified to have side chains with aldehyde groups
  • the carboxyl polysaccharide derivatives with side chain aldehyde groups are carboxyl modified to have aldehyde groups Carboxylated polysaccharide derivatives with side chains.
  • Amino polysaccharide derivatives with side chain aldehyde groups or carboxyl polysaccharide derivatives with side chain aldehyde groups are amino polysaccharides modified from amino groups to side chains with adjacent dihydroxy groups or carboxyl polysaccharides modified from carboxyl groups to adjacent dihydroxy groups formed by oxidation.
  • the aminopolysaccharide derivatives in the first solution of the present invention include hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxybutyl chitosan, carboxymethyl chitosan, or carboxyethyl chitosan;
  • Carboxyl polysaccharide derivatives include chitosan derivatives with carboxyl structure, chitin derivatives with carboxyl structure, hyaluronic acid or hyaluronic acid derivatives with carboxyl structure, chondroitin sulfate, heparin, or sodium alginate.
  • the small molecule compound with two or more primary amino groups of the present invention is an aliphatic or aromatic small molecule compound with two or more primary amino groups, including ethylenediamine, propylenediamine, butylenediamine, amino Butanamide, glutamine, alanyl glutamine, lysine or other basic amino acids, small molecular compounds with dihydrazide structure, diaminobenzoic acid, diaminophenylacetic acid.
  • the small molecular compound with dihydrazide structure of the present invention includes carbohydrazide, oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, adipic acid dihydrazide or sebacic acid dihydrazide Hydrazine.
  • the polysaccharide derivatives with primary amino groups in the main chain in the second solution of the present invention are aminopolysaccharide derivatives with primary amino groups in the main chain and branched side chain structures; the polysaccharide derivatives with primary amino groups in the side chains have carboxyl structures in the main chain , A polysaccharide derivative having a primary amino group in the side chain.
  • the polysaccharide derivatives with primary amino groups in the side chains are formed by condensation of carboxyl groups of carboxypolysaccharides with one amino group in small molecular compounds with two or more primary amino groups.
  • the preparation method of the biopolysaccharide hydrogel contained in the intraocular vitreous substitute of the present invention comprises the following steps:
  • the first solution is provided, and the first solution is a sterile solution of polysaccharide derivatives having aldehyde groups in the side chain with a mass concentration of 0.5-15%, and the pH is adjusted to be 3.5-7, wherein the side chain has aldehydes
  • the polysaccharide derivative of the base is formed by reacting an aqueous solution of a polysaccharide derivative with a grafted ortho-dihydroxy structure and an oxidizing agent;
  • the second solution is selected from any one of the following solutions:
  • step (1) Take the first solution obtained in step (1) and the second solution obtained in step (2), put them into tube A and tube B of a sterile double syringe respectively, push out the syringe to mix, and mix the first solution and the second solution Form a transparent biopolysaccharide hydrogel.
  • the polysaccharide derivatives with aldehyde groups in the side chain in step (1) are prepared by the following method: providing the aqueous solution of the polysaccharide derivatives with a grafted adjacent dihydroxy structure with a mass concentration of 0.5% to 15%, adding the oxidizing agent , wherein the molar ratio of the o-dihydroxy structure to the oxidizing agent is 1: (0.3 ⁇ 3), at room temperature or low temperature, avoid light, stir for 2min ⁇ 5h, add ethylene glycol solution to terminate the reaction for 0.5 ⁇ 2h, dialyze and freeze the reaction product Drying, or ethanol precipitation, washing, precipitation, and drying.
  • polysaccharide derivatives with grafted ortho-dihydroxyl structure in step (1) are prepared by the following method: respectively provide the aminopolysaccharide aqueous solution with a mass concentration of 0.5 to 15% and the aminopolysaccharide with a carboxyl structure and a mass concentration of 0.2 to 30%.
  • aqueous solution of a small molecule compound with an ortho-dihydroxy structure adding the carboxyl activation reagent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the small molecule compound solution , adjusting the pH to 3.5-7, stirring and reacting for 0.5-3 hours; adding the small molecular compound solution into the amino polysaccharide solution, stirring and adjusting the pH of the reaction system to 7-9, stirring and reacting for 24 hours.
  • the described small molecular compound with carboxyl structure and adjacent dihydroxy structure includes 2,3-dihydroxypropionic acid, 2,3,4-trihydroxybutyric acid, gluconic acid, threonic acid , arabic acid, xylonic acid, or mucic acid.
  • the polysaccharide derivative with grafted adjacent dihydroxyl structure in the step (1) can also be prepared by the following method: respectively provide the carboxypolysaccharide aqueous solution with a mass concentration of 0.5 to 15% and the carboxypolysaccharide with a mass concentration of 0.2 to 30%.
  • aqueous solution of small molecular compounds with a structure and an ortho-dihydroxyl structure For the aqueous solution of small molecular compounds with a structure and an ortho-dihydroxyl structure, add carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution, adjust the pH to 3.5-7, and stir for 0.5-3 hours; add the small molecule compound solution to the carboxypolysaccharide solution, and stir Adjusting the pH of the reaction system to 7-9, stirring and reacting for 24 hours; 3) Dialyzing and freeze-drying the reaction product, or subjecting the reaction product to ethanol precipitation, washing, precipitation and drying.
  • carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution, adjust the pH to 3.5-7, and stir for 0.5-3 hours
  • add the small molecule compound solution to the carboxypolysaccharide solution and stir Adjusting the pH of the reaction system to 7-9, stirring and reacting for 24 hours; 3) Dialyzing
  • the small molecular compound having an amino structure and an adjacent dihydroxy structure includes aminopropanediol or glucosamine.
  • the polysaccharide derivatives with primary amino groups in the side chains in step (2) are prepared by the following steps: preparing an aqueous carboxypolysaccharide solution with a mass concentration of 0.5 to 15%, and preparing a carboxypolysaccharide solution with a mass concentration of 0.2 to 30% with two or two Add the carboxyl activation reagents EDC and NHS to the carboxypolysaccharide solution for the above small molecule compound aqueous solution of primary amino groups, adjust the pH to 3.5-7, and stir for 0.5-3 hours; add the small molecule compound solution to the carboxypolysaccharide solution, and adjust the pH to 7 ⁇ 9, stirring and reacting for 24 hours; the reaction product is subjected to dialysis and freeze-drying, or the reaction product is subjected to ethanol precipitation, washing, precipitation and drying.
  • said oxidizing agent comprises sodium periodate or potassium periodate.
  • the raw material of the biological polysaccharide hydrogel of the present invention is a selected raw material with good biocompatibility, and the prepared polysaccharide hydrogel has excellent light transmittance, viscoelasticity, and tissue nontoxicity;
  • the cross-linking of small molecular compounds can reduce the specific recognition of biological enzymes to polysaccharides, thereby improving the biodegradability of cross-linked polysaccharide hydrogels, increasing the effective pressure time of hydrogels on the retina, and achieving the goal of being suitable for use as vitreous purpose of the substitute.
  • the biological polysaccharide hydrogel of the invention can keep the shape of the eyeball and press against the retina in
  • Figure 1 is the intraocular pressure detection chart of the operated eye and the normal eye after operation.
  • Figure 2 is a slit lamp observation view of the operated eye.
  • Figure 3 is a photographic observation of the fundus of the operated eye.
  • Figure 4 is a B-ultrasound examination of the operated eye.
  • Example 1 Preparation of polysaccharide derivatives grafted with aminopolysaccharides ortho-dihydroxyl structure
  • the reaction product was packed into a dialysis bag with a molecular weight cut-off of 8000-14000 for dialysis with deionized water for 48 hours, and freeze-dried to obtain polysaccharide derivatives (trihydroxybutyric acid-hydroxyethyl chitosan) grafted with aminopolysaccharides adjacent to dihydroxyl structures. sugar).
  • the graft ratio (molar ratio) of the ortho-dihydroxy structure of the polysaccharide derivative was determined to be 92%.
  • the prepared polysaccharide derivatives grafted with ortho-dihydroxyl structures have a high grafting rate of ortho-dihydroxyl groups, so as to reduce the molar ratio of primary amino groups in the aminopolysaccharides, and can be oxidized simultaneously A high proportion of aldehyde groups is obtained.
  • reaction product is precipitated with ethanol, washed, precipitated, and dried to obtain a polysaccharide derivative (aminopropylene glycol-hyaluronic acid) grafted with a carboxypolysaccharide ortho-dihydroxyl structure.
  • the rate is 88%.
  • the molar ratio of aminopropanediol and other small molecular compounds with both amino structures and ortho-dihydroxyl structures to carboxypolysaccharides can be adjusted to make the polysaccharides with carboxyl groups
  • the structure of the polysaccharide is grafted with different proportions of ortho-dihydroxyl structures to form polysaccharide derivatives with different molar ratios of side-chain ortho-dihydroxyl structures, and different proportions of ortho-dihydroxyl structures can be oxidized to obtain different proportions of aldehyde groups.
  • the reaction product was dialyzed with deionized water in a dialysis bag with a molecular weight of 8000-14000 for 48 h, and freeze-dried to obtain carboxypolysaccharide derivative 2( Oxidized aminopropylene glycol-hyaluronic acid), and the molar percentage of its aldehyde group is determined to be 46%.
  • the polysaccharide derivatives with adjacent dihydroxyl structures grafted on the side chains are oxidized by the oxidant sodium periodate to adjust the reaction temperature and control
  • the material ratio and reaction time range from a few minutes to 5 hours.
  • the oxidizing agent preferentially oxidizes the adjacent dihydroxyl structure of the polysaccharide side chain to form an aldehyde group, and does not oxidize the main chain of the polysaccharide, thereby ensuring that the main chain of the polysaccharide molecule is not degraded by ring opening and maintaining the main polysaccharide. chain integrity.
  • the oxidizing agent used includes but not limited to sodium periodate, potassium periodate;
  • the addition amount of oxidant controlling the oxidation reaction time and reaction temperature, side chain aldehyde groups with different molar ratios are obtained, while the polysaccharide main chain is not oxidized ring, the molar percentage of the side chain aldehyde unit and the sugar unit of the prepared polysaccharide derivative having an aldehyde group can be ⁇ 1%, such as 5%, 10%, 20%, 30%, 40%, 50%, 60% etc., it may be close to 100%.
  • reaction product was precipitated with ethanol, washed, precipitated, and dried to obtain a polysaccharide derivative 1 (carbohydrazide-carboxymethyl chitin) with a primary amino group in the side chain, and the grafting rate of the primary amino group was determined to be 62%.
  • the prepared polysaccharide derivatives with primary amino groups have a side chain primary amino unit and a molar percentage of sugar units can be ⁇ 1%, such as 5%, 10% , 20%, 30%, 40%, 50%, 60%, etc., can also be close to 100%.
  • the first dose Weigh 1.0 g of the aseptic sample of amino polysaccharide derivatives with aldehyde groups in the side chain prepared in Example 3, dissolve it in 10 mL of sterile normal saline, prepare a solution with a concentration of 10% by mass, and adjust the pH to 6.
  • the second dose Weigh 0.05g of a sterile sample of a small molecule compound succinic acid dihydrazide with two or more primary amino groups, dissolve it in 10mL sterile normal saline, and prepare a solution with a concentration of 0.5% by mass , adjust the pH to 7.5.
  • the first dose and the second dose of liquid are pushed out through the syringe and mixed to form a transparent polysaccharide hydrogel 1, and the gelation time is about After 2 minutes, the formed polysaccharide hydrogel is viscous and has good resilience when pressed.
  • Embodiment 8 Preparation of polysaccharide hydrogel 2
  • the first dose Weigh 0.3 g of the aseptic sample of amino polysaccharide derivatives with aldehyde groups in the side chain prepared in Example 3, dissolve it in 10 mL of sterile normal saline, prepare a solution with a concentration of 3% by mass, and adjust the pH to 5.
  • the second dose Weigh 0.2 g of the polysaccharide derivative glutamine-hyaluronic acid sterile sample with primary amino groups in the side chain prepared in Example 6, dissolve it in 10 mL sterile normal saline, and prepare the mass percentage concentration to be 2%. solution, adjusted to pH 8.
  • first dose and the second dose into tube A and tube B of a sterile double syringe respectively.
  • the first dose and the second dose are pushed out through the syringe and mixed to form a transparent polysaccharide hydrogel 2.
  • the gelation time is about After 5 minutes, the formed polysaccharide hydrogel is viscous and has good resilience when pressed.
  • Embodiment 9 Preparation of polysaccharide hydrogel 3
  • the first dose Weigh 0.3 g of the aseptic sample of carboxyl polysaccharide derivatives with aldehyde groups in the side chain prepared in Example 4, dissolve it in 10 mL of sterile deionized water, prepare a solution with a concentration of 4% by mass, and adjust the pH is 4.0.
  • the second dose Weigh 0.3 g of a sterile sample of polysaccharide derivative hydroxypropyl chitosan with primary amino groups in the main chain, dissolve it in 10 mL of sterile deionized water, prepare a solution with a concentration of 5% by mass, and adjust the pH is 8.5.
  • the first dose and the second dose are pushed out through the syringe and mixed to form a transparent polysaccharide hydrogel 3, and the gelation time is about After 6 minutes, the formed polysaccharide hydrogel is viscous and has good resilience when pressed.
  • Embodiment 10 Determination of light transmittance of polysaccharide hydrogel
  • the polysaccharide hydrogels prepared in Examples 7-9 were respectively gelled in small test tubes with a diameter of 6 mm, and the strips were taken out and cut into gel blocks with a height of 5 mm. Two pieces of each of the three different hydrogel blocks were taken, dried to a constant weight, weighed, and the solid content of the blocks before degradation was calculated.
  • the three kinds of polysaccharide hydrogels are relatively stable in the first 20 days.
  • the degradation rate in 1 day is less than 20%, and the degradation rate in 40 days is about 30%.
  • the degradation rate in 50 days and 60 days is obviously accelerated, and the degradation rate in 60 days is about 70%.
  • Experimental results show that the polysaccharide hydrogel prepared by the invention has good degradation resistance, good early stability, and the degradation time exceeds 60 days.
  • the patent application for invention with the publication number CN 102762647 A discloses that the in vitro degradation of the prepared hydrogel has a residual weight of 86.67% of the hydrogel on the 3rd day and 61.02% of the hydrogel on the 35th day.
  • the in vitro degradation time of the polysaccharide hydrogel of the present invention is longer, so the pressure time of the polysaccharide hydrogel of the present invention on the retina is longer.
  • Experimental animals were routinely anesthetized, skin disinfected, and subjected to vitrectomy.
  • the first and second doses prepared in Example 9 were injected into the vitreous cavity of the left eye of the experimental rabbit through a double syringe, forming a gel in situ, closing the Scleral incision, postoperative care of experimental rabbits, normal feeding.
  • the intraocular pressure was measured regularly after the operation, and the eye slit lamp examination, fundus photography, and B-ultrasound examination were carried out 90 days later to evaluate the filling of the hydrogel in the vitreous cavity.
  • biopolysaccharide hydrogel prepared by the present invention has no irritation in the eye, has good ocular tissue compatibility, has a good vitreous replacement effect, can press the retina and maintain the shape of the eyeball, and has good optical properties. Degraded and absorbed in the eye, the polysaccharide hydrogel has great application potential as a substitute for the vitreous humor in the eye.

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Abstract

本发明涉及一种生物多糖水凝胶及其制备方法和应用。本发明的生物多糖水凝胶是由两种溶液混合反应而成，第一溶液是侧链具有醛基的多糖衍生物溶液，第二溶液是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。本发明的制备方法，包括第一溶液和第二溶液的制备，再将第一溶液和第二溶液混合形成透明的生物多糖水凝胶。本发明的生物多糖水凝胶可以作为玻璃体替代物应用。

Description

一种生物多糖水凝胶及其制备方法和应用 技术领域
本发明涉及一种眼科医用生物材料，特别是涉及一种生物多糖水凝胶及其制备方法和作为玻璃体替代物的应用。
背景技术
眼球内的玻璃体是填充于晶状体和视网膜之间的一种无色透明胶状体，占据超过2/3的眼球体积，主要成分包括水、胶原蛋白、透明质酸、蛋白多糖等，其中水约占玻璃体的99％。透明质酸与胶原蛋白相互作用，并与水形成结合状态，使玻璃体处于稳定状态。玻璃体具有高度透明性，允许90％以上的可见光透射。玻璃体在保持眼球形状、调节眼内氧分压、减震缓冲、保持视网膜和晶状***置等方面发挥重要作用。玻璃体切除手术是用于治疗很多眼科疾病的常见手术，如视网膜病变、视网膜剥离、黄斑裂孔等。玻璃体切除后需要进行玻璃体腔填充，将玻璃体替代物填充在玻璃体腔内，顶压视网膜保持其在正确位置并保持眼球外形，因此玻璃体填充物成为影响手术效果的重要因素。
目前临床上使用的玻璃体替代物主要有气体、硅油、全氟化碳液体等。这些替代物适用于不同手术情况下的玻璃体填充，但仍存在诸多并发症。如气体玻璃体腔填充吸收较快，有效顶压时间短，并需要患者术后保持一定的***等；全氟化碳液体也存在吸收较快，有效顶压时间4周左右；常用的硅油填充顶压时间长，但不被机体组织吸收，因硅油乳化引起的并发症较多，如青光眼、白内障等并发症，因此硅油需要二次手术移除。目前还没有理想的玻璃体替代物。
作为玻璃体替代物，高度透光性、良好的粘弹性、有效的顶压时间和对眼组织无毒副作用是极为重要的。公布号为CN 102762647 A的发明专利申请公开了一种包括水凝胶聚合物的组成物，该水凝胶聚合物包含氧化透明质酸和二酰肼，其中二酰肼与氧化透明质酸交联，形成透明无色、从液态转变成胶体，作为眼球玻璃体替代物。公布号为CN 110652610 A的发明专利申请公开了一种高内聚双重交联人工玻璃体的成套原料，其第一组分为醛基化透明质酸，其醛基化透明质酸即氧化开环透明质酸，第二组分为氨基化透明质酸，第一剂与第二剂混合交联制备出高内聚双重交联人工玻璃体。上述两项发明专利申请，其共同特点是使用氧化开环的透明质酸作为醛基供体，与二酰肼或双氨基交联形成凝胶。
公布号为CN 105833344 A的发明专利申请公开了一种可注射水凝胶在制备眼内填充物中的应用，所述可注射水凝胶由两剂组成，第一剂是含氧化多糖的胶液，第二剂是含甲壳素衍生物和/或胶原蛋白的胶液，通过氧化多糖的双醛基与甲壳素衍生物和/或胶原蛋白的氨基发生交联反应，形成具有粘弹性的水凝胶。
上述现有技术都是采用氧化开环的透明质酸来形成凝胶，而氧化开环会造成透明质酸分子量大大降低，从而导致其在体内降解速度过快，作为玻璃体替代物的有效顶压时间缩短。而作为玻璃体替代物，延长体内降解时间，增加其对视网膜的有效顶压时间极为重要，以确保网膜顶压复位，避免造成网脱。
发明内容
针对上述问题，本发明的第一个目的是提供一种具有高度透光性、良好的粘弹性、降解时间长、有效提高顶压时间和对眼组织无毒副作用的适用于作为玻璃体替代物的生物多糖水凝胶，以弥补现有技术的不足。
本发明的另一个目的是提供一种生物多糖水凝胶的制备方法，以及所述生物多糖水凝胶用于制备玻璃体替代物的应用。
本发明的技术方案，在考虑作为玻璃体替代物的水凝胶的优良透光性、粘弹性、组织无毒性的同时，重点解决延长水凝胶的降解时间问题，以延长对视网膜的顶压时间。
本发明的生物多糖水凝胶是由两剂组成，第一剂是侧链具有醛基的多糖衍生物溶液，第二剂是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上伯氨基的小分子化合物溶液。
所述的第一剂侧链具有醛基的多糖衍生物，是由侧链接枝邻二羟基的多糖衍生物经氧化形成的侧链具有醛基的多糖衍生物；在控制反应条件下，氧化剂优先氧化多糖侧链的邻二羟基结构形成醛基，但不氧化多糖主链，从而保证了多糖分子不被开环降解，保持了多糖主链的完整性；同时，具有侧链接枝结构的多糖衍生物降低了生物酶对多糖的特异性识别，从而提高了多糖衍生物的抗生物降解性。
所述的第二剂主链或侧链具有伯氨基的多糖衍生物，包括主链具有伯氨基的多糖衍生物以及侧链接枝有伯氨基的多糖衍生物；所述的主链具有伯氨基的多糖衍生物，其特征是所述多糖衍生物主链具有伯氨基、同时具有侧链接枝结构；所述的侧链具有伯氨基的多糖衍生物，其特征是所述多糖衍生物的侧链接枝有伯氨基；即主链具有伯氨基的多糖衍生物以及侧链接枝有伯氨基的多糖衍生物，都具有侧链接枝结构，这些侧链接枝结构降低了生物酶对 多糖的特异性识别，从而提高了多糖衍生物的抗生物降解性。
当第一剂侧链具有醛基的多糖衍生物溶液，与第二剂主链或侧链具有伯氨基的多糖衍生物溶液混合后发生交联反应，形成水凝胶，一方面由于作为醛基供体的多糖衍生物的主链没有氧化开环，没有分子链的氧化降解，另一方面由于作为醛基供体和氨基供体的多糖衍生物都具有侧链接枝结构，这些侧链接枝结构的存在降低了生物酶对多糖的特异性识别，从而提高了多糖衍生物的抗生物降解性。当第一剂侧链具有醛基的多糖衍生物溶液，与第二剂具有两个或两个以上的伯氨基的小分子化合物溶液混合交联，形成水凝胶，由于醛基供体的多糖衍生物的主链没有氧化开环，没有分子链的氧化降解，同时醛基供体的多糖衍生物具有侧链接枝结构，当醛基供体多糖衍生物与具有伯氨基的小分子化合物发生交联，小分子化合物的交联结构进一步降低了生物酶对多糖的特异性识别，从而提高了多糖衍生物的抗生物降解性。通过提高交联多糖水凝胶的抗生物降解性，从而提高水凝胶对视网膜的有效顶压时间，达到适用于作为玻璃体替代物的目的。
本发明的具体技术方案如下：
在一个方面，本发明涉及一种生物多糖水凝胶，其是由第一溶液和第二溶液混合反应形成，其中所述第一溶液是侧链具有醛基的多糖衍生物溶液，所述第二溶液是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。
第一溶液中包含质量百分浓度为0.5％～15％的侧链具有醛基的多糖衍生物，第二溶液中包含质量百分浓度为0.5％～15％的主链或侧链具有伯氨基的多糖衍生物，或质量百分浓度为0.2％～30％的具有两个或两个以上伯氨基的小分子化合物；第一溶液和第二溶液包含选自无菌水、生理盐水、生理平衡液和葡萄糖液的溶剂。
第一溶液中的侧链具有醛基的多糖衍生物是具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物。其中具有侧链醛基的氨基多糖衍生物是其氨基被修饰成带有醛基的侧链的氨基多糖衍生物，所述具有侧链醛基的羧基多糖衍生物是其羧基被修饰成带有醛基的侧链的羧基多糖衍生物。具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物，是由氨基被修饰成带有邻二羟基的侧链的氨基多糖或羧基被修饰成带有邻二羟基的侧链的羧基多糖经氧化形成。
本发明第一溶液中的氨基多糖衍生物，包括羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、或羧乙基壳聚糖；羧基多糖衍生物，包括具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸或透明质酸衍生物、硫酸软骨素、肝素、或海藻酸钠。
本发明的具有两个或两个以上伯氨基的小分子化合物是具有两个或两个以上伯氨基的脂肪族或芳香族小分子化合物，包括乙二胺、丙二胺、丁二胺、氨基丁酰胺、谷氨酰胺、丙氨酰谷氨酰胺、赖氨酸或其它碱性氨基酸、具有二酰肼结构的小分子化合物、二氨基苯甲酸、二氨基苯乙酸。
本发明的具有二酰肼结构的小分子化合物包括碳酰肼、乙二酸二酰肼、丙二酸二酰肼、丁二酸二酰肼、己二酸二酰肼或癸二酸二酰肼。
本发明第二溶液中的主链具有伯氨基的多糖衍生物是主链具有伯氨基同时具有侧链接枝结构的氨基多糖衍生物；侧链具有伯氨基的多糖衍生物是主链具有羧基结构、侧链具有伯氨基的多糖衍生物。侧链具有伯氨基的多糖衍生物由羧基多糖的羧基与具有两个或两个以上伯氨基的小分子化合物中的一个氨基缩合形成。
上述生物多糖水凝胶的制备方法，其包括以下步骤：
(1)提供第一溶液，所述第一溶液是质量浓度为0.5～15％的侧链具有醛基的多糖衍生物无菌溶液，并调节pH为3.5～7，其中所述侧链具有醛基的多糖衍生物由具有接枝邻二羟基结构的多糖衍生物水溶液与氧化剂反应形成；
(2)提供第二溶液，所述第二溶液选自以下溶液中的任一项：
i)质量百分浓度为0.2％～30％的具有两个或两个以上的伯氨基的小分子化合物无菌溶液；
ii)质量百分浓度为0.5％～15％的主链具有伯氨基的多糖衍生物无菌溶液；
iii)质量百分浓度为0.5％～15％的侧链具有伯氨基的多糖衍生物无菌溶液；
并调节pH为7～9，测定伯氨基的摩尔百分含量；
(3)混合所述第一溶液与第二溶液：
取步骤(1)所得的第一溶液和步骤(2)所得的第二溶液，分别装入无菌双联注射器的A管和B管中，经注射器推出混合，第一溶液和第二溶液混合形成透明的生物多糖水凝胶。
其中所述步骤(1)中的侧链具有醛基的多糖衍生物通过以下方法制备得到：提供质量浓度为0.5～15％的所述具有接枝邻二羟基结构的多糖衍生物水溶液，加入所述氧化剂，其中邻二羟基结构与氧化剂的摩尔比为1﹕(0.3～3)，室温或低温，避光搅拌反应2min～5h，加入乙二醇溶液终止反应0.5～2h，将反应产物进行透析、冷冻干燥，或乙醇沉淀、洗涤、沉淀、干燥。
其中所述步骤(1)中的具有接枝邻二羟基结构的多糖衍生物通过以下方法制备得到：分别提供质量浓度为0.5～15％的氨基多糖水溶液和质量浓度为 0.2～30％的具有羧基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺加入小分子化合物溶液中，调节pH为3.5～7，搅拌反应0.5～3h；将小分子化合物溶液加入到氨基多糖溶液中，搅拌并调节反应体系pH为7～9，搅拌反应24h。
其中所述步骤(1)中，所述的具有羧基结构和邻二羟基结构的小分子化合物，包括2,3-二羟基丙酸、2,3,4-三羟基丁酸、葡萄糖酸、苏糖酸、***酸、木糖酸、或粘酸。
其中所述步骤(1)中的具有接枝邻二羟基结构的多糖衍生物还可以通过以下方法制备得到：分别提供质量浓度为0.5～15％的羧基多糖水溶液和质量浓度为0.2～30％的具有氨基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，搅拌调节反应体系pH为7～9，搅拌反应24h；3)将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
其中所述步骤(1)中，所述的具有氨基结构和邻二羟基结构的小分子化合物，包括氨基丙二醇、或氨基葡萄糖。
其中所述步骤(2)中的侧链具有伯氨基的多糖衍生物通过以下步骤制备得到：配制质量浓度为0.5～15％的羧基多糖水溶液，配制质量浓度为0.2～30％的具有两个或两个以上伯氨基的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，调节pH为7～9，搅拌反应24h；将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
其中所述羧基与羧基活化试剂EDC、NHS的摩尔比为1﹕(0.5～6)﹕(0.5～6)。
其中所述的氧化剂包括高碘酸钠或高碘酸钾。
在一个具体的实施方案中，本发明的一种生物多糖水凝胶，其特征是由两剂混合反应而成，第一剂是侧链具有醛基的多糖衍生物溶液，第二剂是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。
所述第一剂侧链具有醛基的多糖衍生物溶液，多糖衍生物中含有侧链醛基的糖单元占多糖分子总糖单元的百分数为1％～100％，多糖衍生物在溶液中的质量百分浓度为0.5％～15％；所述第二剂主链或侧链具有伯氨基的多糖衍生物溶液，多糖衍生物中含有主链伯氨基或侧链伯氨基的糖单元占多糖分子总糖单元的百分数为1％～100％，多糖衍生物在溶液中的质量百分浓度为 0.5％～15％；所述第二剂具有两个或两个以上的伯氨基的小分子化合物溶液，小分子化合物在溶液中的质量百分浓度为0.2％～30％；所述第一剂和第二剂的溶液，其溶剂是无菌水、生理盐水、生理平衡液，或本领域技术人员知道的其它含水的医用液体，如葡萄糖液等。
所述第一剂侧链具有醛基的多糖衍生物，其特征是具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物；所述具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物，是由侧链接枝邻二羟基的氨基多糖衍生物或侧链接枝邻二羟基的羧基多糖衍生物经氧化形成。所述的氨基多糖是具有伯氨基的多糖，包括但不限于羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、羧乙基壳聚糖及其它壳聚糖衍生物；所述的羧基多糖，包括但不限于具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸或透明质酸衍生物，以及硫酸软骨素、肝素、海藻酸钠；所述的具有羧基结构的壳聚糖衍生物包括羧甲基壳聚糖、羧乙基壳聚糖、琥珀酰壳聚糖及其它具有羧基结构的壳聚糖衍生物，所述的具有羧基结构的甲壳素衍生物包括羧甲基甲壳素、羧乙基甲壳素及其它具有羧基结构的甲壳素衍生物，所述的具有羧基结构的透明质酸和透明质酸衍生物包括透明质酸、羟乙基透明质酸、羟丙基透明质酸、乙酰基透明质酸、丁酰基透明质酸、氨基乙酸透明质酸、氨基丙酸透明质酸、氨基丁酸透明质酸及其它透明质酸衍生物。所述的具有侧链醛基的氨基多糖衍生物，其特征是在氨基多糖的氨基上接枝邻二羟基结构，从而形成侧链邻二羟基结构，邻二羟基经氧化形成醛基，即生成具有侧链醛基的氨基多糖衍生物；所述的具有侧链醛基的羧基多糖衍生物，其特征是在羧基多糖的羧基上接枝邻二羟基结构，从而形成侧链邻二羟基结构，邻二羟基经氧化形成醛基，即生成具有侧链醛基的羧基多糖衍生物。所述氨基多糖或羧基多糖上接枝邻二羟基结构的接枝率，可以通过控制物料比、反应条件进行调节，理论上可以在1～100％。
所述的侧链邻二羟基结构，是在氨基多糖的氨基上或羧基多糖的羧基上接枝具有邻二羟基的结构。为氨基多糖提供邻二羟基结构的化合物是同时具有羧基和邻二羟基的小分子化合物，包括但不限于2,3-二羟基丙酸、2,3,4-三羟基丁酸、葡萄糖酸、苏糖酸、***酸、木糖酸、粘酸，以及其它同时具有羧基结构和邻二羟基结构的化合物；氨基多糖与同时具有羧基结构和邻二羟基结构的化合物通过氨基与羧基的缩合反应，使氨基多糖接枝上邻二羟基结构的侧链，形成具有邻二羟基侧链的多糖衍生物。为羧基多糖提供邻二羟基结构的化合物是同时具有氨基结构和邻二羟基结构的小分子化合物，包括但不限于氨基丙二醇、氨基葡萄糖，以及其它同时具有氨基结构和邻二羟基结构的化合物；羧基多糖与同时具有氨基结构和邻二羟基结构的化合物通过 羧基与氨基的缩合反应，使羧基多糖接枝上邻二羟基结构的侧链，形成具有邻二羟基侧链的多糖衍生物。具有侧链接枝邻二羟基结构的多糖衍生物，经氧化形成了侧链具有醛基的多糖衍生物。所述侧链邻二羟基结构的氧化可以在控制氧化剂的浓度、时间、温度等条件得到不同醛基含量，理论上可以在1～100％。
所述第二剂主链或侧链具有伯氨基的多糖衍生物，或具有两个或两个以上的伯氨基的小分子化合物，所述的主链具有伯氨基的多糖衍生物，其特征是主链具有伯氨基同时具有侧链接枝结构的多糖衍生物，包括但不限于羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、羧乙基壳聚糖及其它壳聚糖衍生物；所述的侧链具有伯氨基的多糖衍生物，其特征是主链具有羧基结构、侧链具有伯氨基的多糖衍生物，是羧基多糖的羧基与具有两个或两个以上伯氨基的小分子化合物中的一个氨基缩合，形成的侧链具有伯氨基的多糖衍生物；所述的羧基多糖，包括但不限于具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸和透明质酸衍生物，以及硫酸软骨素、肝素、海藻酸钠，同第一剂所述的羧基多糖；所述的具有两个或两个以上伯氨基的小分子化合物，其特征是具有两个或两个以上伯氨基的脂肪族或芳香族小分子化合物，包括但不限于乙二胺、丙二胺、丁二胺、氨基丁酰胺、谷氨酰胺、丙氨酰谷氨酰胺、赖氨酸及其它碱性氨基酸，具有二酰肼结构的小分子化合物，二氨基苯甲酸、二氨基苯乙酸，其中具有二酰肼结构的小分子化合物包括碳酰肼、乙二酸二酰肼、丙二酸二酰肼、丁二酸二酰肼、己二酸二酰肼、癸二酸二酰肼及其它具有二酰肼结构的化合物。羧基多糖接枝伯氨基结构，通过控制物料比、反应条件，可以调节伯氨基的接枝率，理论上可以在1～100％。
所述的生物多糖水凝胶，是第一剂侧链具有醛基的多糖衍生物溶液，和第二剂主链或侧链具有伯氨基的多糖衍生物，或具有两个或两个以上的伯氨基的小分子化合物溶液，进行混合，第一剂的醛基与第二剂的伯氨基发生交联反应，形成的具有高度透明性、粘弹性、生物相容性的水凝胶。
上述生物多糖水凝胶的制备方法，其特征是包括第一剂和第二剂的制备以及将两剂进行混合，具体制备方法包括以下步骤：
第一剂的制备：
(1)具有接枝邻二羟基结构的多糖衍生物的制备：1)分别配制质量浓度为0.5～15％的氨基多糖水溶液和质量浓度为0.2～30％的具有羧基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)加入小分子化合物溶液中，调节pH为3.5～7，搅拌反应0.5～3h；将小分子化合物溶液加入 到氨基多糖溶液中，搅拌调节反应体系pH为7～9，搅拌反应24h；或2)分别配制质量浓度为0.5～15％的羧基多糖水溶液和质量浓度为0.2～30％的具有氨基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，搅拌调节反应体系pH为7～9，搅拌反应24h；3)将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥，得到具有接枝邻二羟基结构的多糖衍生物，测定多糖衍生物的邻二羟基结构的接枝率；
(2)侧链具有醛基的多糖衍生物的制备：配制质量浓度为0.5～15％的具有接枝邻二羟基结构的多糖衍生物水溶液，按邻二羟基结构与氧化剂的摩尔比为1﹕(0.3～3)加入氧化剂，室温或低温，避光搅拌反应2min～5h，加入乙二醇溶液终止反应0.5～2h，将反应产物进行透析、冷冻干燥，或乙醇沉淀、洗涤、沉淀、干燥，得侧链具有醛基的多糖衍生物，测定醛基的摩尔百分含量；
(3)侧链具有醛基的多糖衍生物溶液的制备：配制质量浓度为0.5～15％的侧链具有醛基的多糖衍生物无菌溶液，调节pH为3.5～7；
第二剂的制备：
(1)具有两个或两个以上的伯氨基的小分子化合物溶液：配制质量百分浓度为0.2％～30％的具有两个或两个以上的伯氨基的小分子化合物无菌溶液，调节pH为7～9，测定伯氨基的摩尔百分含量；
或(2)主链具有伯氨基的多糖衍生物溶液：配制质量百分浓度为0.5％～15％的主链具有伯氨基的多糖衍生物无菌溶液，调节pH为7～9，测定伯氨基的摩尔百分含量。
或(3)侧链具有伯氨基的多糖衍生物溶液：配制质量浓度为0.5～15％的羧基多糖水溶液，配制质量浓度为0.2～30％的具有两个或两个以上伯氨基的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，调节pH为7～9，搅拌反应24h；将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥，得侧链具有伯氨基的多糖衍生物。配制质量百分浓度为0.5％～15％的侧链具有伯氨基的多糖衍生物无菌溶液，调节pH为7～9，测定伯氨基的摩尔百分含量；
第一剂与第二剂混合：
取第一剂侧链具有醛基的多糖衍生物溶液，第二剂具有两个或两个以上的伯氨基的小分子化合物溶液或主链具有伯氨基的多糖衍生物溶液或侧链具有伯氨基的多糖衍生物溶液，分别装入无菌双联注射器的A管和B管中，经 注射器推出混合，第一剂和第二剂液体混合形成透明的生物多糖水凝胶。
在上述生物多糖水凝胶的制备方法中，具有羧基结构和邻二羟基结构的小分子化合物中的羧基或羧基多糖中的羧基与羧基活化试剂EDC、NHS的摩尔比为1﹕(0.5～6)﹕(0.5～6)；所述的氧化剂包括但不限于高碘酸钠、高碘酸钾。
在另一个方面，本发明涉及上述生物多糖水凝胶在制备眼内玻璃体替代物中的应用。
在另一个方面，本发明还涉及上述生物多糖水凝胶，其用作眼内玻璃体替代物。
在又一个方面，本发明涉及一种眼内玻璃体替代物，其包含由第一溶液和第二溶液混合反应形成的生物多糖水凝胶，其中所述第一溶液是侧链具有醛基的多糖衍生物溶液，所述第二溶液是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。
第一溶液中包含质量百分浓度为0.5％～15％的侧链具有醛基的多糖衍生物，第二溶液中包含质量百分浓度为0.5％～15％的主链或侧链具有伯氨基的多糖衍生物，或质量百分浓度为0.2％～30％的具有两个或两个以上伯氨基的小分子化合物；第一溶液和第二溶液包含选自无菌水、生理盐水、生理平衡液和葡萄糖液的溶剂。
本发明的第一溶液中的侧链具有醛基的多糖衍生物是具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物。其中具有侧链醛基的氨基多糖衍生物是氨基被修饰成带有醛基的侧链的氨基多糖衍生物，所述具有侧链醛基的羧基多糖衍生物是羧基被修饰成带有醛基的侧链的羧基多糖衍生物。具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物，是由氨基被修饰成带有邻二羟基的侧链的氨基多糖或羧基被修饰成带有邻二羟基的侧链的羧基多糖经氧化形成。
本发明的第一溶液中的氨基多糖衍生物，包括羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、或羧乙基壳聚糖；羧基多糖衍生物，包括具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸或透明质酸衍生物、硫酸软骨素、肝素、或海藻酸钠。
本发明的具有两个或两个以上伯氨基的小分子化合物是具有两个或两个以上伯氨基的脂肪族或芳香族小分子化合物，包括乙二胺、丙二胺、丁二胺、氨基丁酰胺、谷氨酰胺、丙氨酰谷氨酰胺、赖氨酸或其它碱性氨基酸、具有二酰肼结构的小分子化合物、二氨基苯甲酸、二氨基苯乙酸。
本发明的具有二酰肼结构的小分子化合物包括碳酰肼、乙二酸二酰肼、丙二酸二酰肼、丁二酸二酰肼、己二酸二酰肼或癸二酸二酰肼。
本发明的第二溶液中的主链具有伯氨基的多糖衍生物是主链具有伯氨基同时具有侧链接枝结构的氨基多糖衍生物；侧链具有伯氨基的多糖衍生物是主链具有羧基结构、侧链具有伯氨基的多糖衍生物。侧链具有伯氨基的多糖衍生物由羧基多糖的羧基与具有两个或两个以上伯氨基的小分子化合物中的一个氨基缩合形成。本发明的又一方面还包括一种生物多糖水凝胶，其用作眼内玻璃体替代物，所述生物多糖水凝胶是由第一溶液和第二溶液混合反应形成，其中所述第一溶液是侧链具有醛基的多糖衍生物溶液，所述第二溶液是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。
本发明的第一溶液中包含质量百分浓度为0.5％～15％的侧链具有醛基的多糖衍生物，第二溶液中包含质量百分浓度为0.5％～15％的主链或侧链具有伯氨基的多糖衍生物，或质量百分浓度为0.2％～30％的具有两个或两个以上伯氨基的小分子化合物；第一溶液和第二溶液包含选自无菌水、生理盐水、生理平衡液和葡萄糖液的溶剂。
本发明的第一溶液中的侧链具有醛基的多糖衍生物是具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物。其中具有侧链醛基的氨基多糖衍生物是氨基被修饰成带有醛基的侧链的氨基多糖衍生物，所述具有侧链醛基的羧基多糖衍生物是羧基被修饰成带有醛基的侧链的羧基多糖衍生物。具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物，是由氨基被修饰成带有邻二羟基的侧链的氨基多糖或羧基被修饰成邻二羟基的羧基多糖经氧化形成。
本发明的第一溶液中的氨基多糖衍生物，包括羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、或羧乙基壳聚糖；羧基多糖衍生物，包括具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸或透明质酸衍生物、硫酸软骨素、肝素、或海藻酸钠。
本发明的具有两个或两个以上伯氨基的小分子化合物是具有两个或两个以上伯氨基的脂肪族或芳香族小分子化合物，包括乙二胺、丙二胺、丁二胺、氨基丁酰胺、谷氨酰胺、丙氨酰谷氨酰胺、赖氨酸或其它碱性氨基酸、具有二酰肼结构的小分子化合物、二氨基苯甲酸、二氨基苯乙酸。
本发明的具有二酰肼结构的小分子化合物包括碳酰肼、乙二酸二酰肼、丙二酸二酰肼、丁二酸二酰肼、己二酸二酰肼或癸二酸二酰肼。
本发明的第二溶液中的主链具有伯氨基的多糖衍生物是主链具有伯氨基同时具有侧链接枝结构的氨基多糖衍生物；侧链具有伯氨基的多糖衍生物是主链具有羧基结构、侧链具有伯氨基的多糖衍生物。侧链具有伯氨基的多糖衍生物由羧基多糖的羧基与具有两个或两个以上伯氨基的小分子化合物中的 一个氨基缩合形成。
本发明的眼内玻璃体替代物包含的生物多糖水凝胶的制备方法包括以下步骤：
(1)提供第一溶液，所述第一溶液是质量浓度为0.5～15％的侧链具有醛基的多糖衍生物无菌溶液，并调节pH为3.5～7，其中所述侧链具有醛基的多糖衍生物由具有接枝邻二羟基结构的多糖衍生物水溶液与氧化剂反应形成；
(2)提供第二溶液，所述第二溶液选自以下溶液中的任一项：
i)质量百分浓度为0.2％～30％的具有两个或两个以上的伯氨基的小分子化合物无菌溶液；
ii)质量百分浓度为0.5％～15％的主链具有伯氨基的多糖衍生物无菌溶液；
iii)质量百分浓度为0.5％～15％的侧链具有伯氨基的多糖衍生物无菌溶液；
并调节pH为7～9，测定伯氨基的摩尔百分含量；
(3)混合所述第一溶液与第二溶液：
取步骤(1)所得的第一溶液和步骤(2)所得的第二溶液，分别装入无菌双联注射器的A管和B管中，经注射器推出混合，第一溶液和第二溶液混合形成透明的生物多糖水凝胶。
其中步骤(1)中的侧链具有醛基的多糖衍生物通过以下方法制备得到：提供质量浓度为0.5～15％的所述具有接枝邻二羟基结构的多糖衍生物水溶液，加入所述氧化剂，其中邻二羟基结构与氧化剂的摩尔比为1﹕(0.3～3)，室温或低温，避光搅拌反应2min～5h，加入乙二醇溶液终止反应0.5～2h，将反应产物进行透析、冷冻干燥，或乙醇沉淀、洗涤、沉淀、干燥。
其中步骤(1)中的具有接枝邻二羟基结构的多糖衍生物通过以下方法制备得到：分别提供质量浓度为0.5～15％的氨基多糖水溶液和质量浓度为0.2～30％的具有羧基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺加入小分子化合物溶液中，调节pH为3.5～7，搅拌反应0.5～3h；将小分子化合物溶液加入到氨基多糖溶液中，搅拌并调节反应体系pH为7～9，搅拌反应24h。
其中步骤(1)中，所述的具有羧基结构和邻二羟基结构的小分子化合物，包括2,3-二羟基丙酸、2,3,4-三羟基丁酸、葡萄糖酸、苏糖酸、***酸、木糖酸、或粘酸。
其中步骤(1)中的具有接枝邻二羟基结构的多糖衍生物还可以通过以下方法制备得到：分别提供质量浓度为0.5～15％的羧基多糖水溶液和质量浓度 为0.2～30％的具有氨基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，搅拌调节反应体系pH为7～9，搅拌反应24h；3)将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
其中步骤(1)中，所述的具有氨基结构和邻二羟基结构的小分子化合物，包括氨基丙二醇、或氨基葡萄糖。
其中步骤(2)中的侧链具有伯氨基的多糖衍生物通过以下步骤制备得到：配制质量浓度为0.5～15％的羧基多糖水溶液，配制质量浓度为0.2～30％的具有两个或两个以上伯氨基的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，调节pH为7～9，搅拌反应24h；将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
其中所述羧基与羧基活化试剂EDC、NHS的摩尔比为1﹕(0.5～6)﹕(0.5～6)。
其中所述氧化剂包括高碘酸钠或高碘酸钾。
本发明的生物多糖水凝胶的原材料是选择的生物相容性好的原材料，制备的多糖水凝胶具有优良透光性、粘弹性、组织无毒性；通过侧链接枝有醛基、保持醛基供体多糖分子结构的完整性，通过具有侧链接枝结构的氨基供体多糖分子、具有两个及以上伯氨基小分子化合物的进一步交联，从侧链接枝结构、保持多糖分子结构的完整性、小分子化合物的交联，降低生物酶对多糖的特异性识别，从而提高交联多糖水凝胶的抗生物降解性，提高水凝胶对视网膜的有效顶压时间，达到适用于作为玻璃体替代物的目的。本发明的生物多糖水凝胶作为玻璃体替代物可以保持眼球形状和顶压视网膜在原位，可以提高减震功能，具有良好的自愈合功能。
附图说明
图1是术后术眼与正常眼眼压检测图。
图2是术眼裂隙灯观察图。
图3是术眼眼底照相观察图。
图4是术眼B超检查图。
具体实施方式
下面结合附图并通过具体实施例来进一步详细说明本发明。
实施例1：氨基多糖接枝邻二羟基结构的多糖衍生物的制备
称取具有羧基结构和邻二羟基结构的小分子化合物2,3,4-三羟基丁酸5.5g溶于150mL去离子水中，加入羧基活化剂EDC 11.4g和NHS 7.0g，搅拌溶解，调节pH为4，得溶液1；称取氨基多糖羟乙基壳聚糖4.1g搅拌溶解于100mL去离子水中，得溶液2；搅拌下将溶液1滴加到溶液2中，调节pH为7.5，室温下搅拌反应24h。反应结束后，将反应产物装入截留分子量8000-14000的透析袋中去离子水透析48h，冷冻干燥得氨基多糖接枝邻二羟基结构的多糖衍生物(三羟基丁酸-羟乙基壳聚糖)。测定多糖衍生物的邻二羟基结构的接枝率(摩尔比)为92％。
在实施例1的氨基多糖接枝邻二羟基结构的多糖衍生物的制备中，可以通过增加三羟基丁酸等同时具有羧基结构和邻二羟基结构的小分子化合物的摩尔比，使具有氨基结构的多糖接枝高比例的邻二羟基结构，制备的氨基多糖接枝邻二羟基结构的多糖衍生物具有高邻二羟基接枝率，以便减少氨基多糖中伯氨基的摩尔比，同时经氧化可获得高比例的醛基。
实施例2：羧基多糖接枝邻二羟基结构的多糖衍生物的制备
称取羧基多糖透明质酸钠4g搅拌溶于200mL去离子水，加入羧基活化剂EDC 2.87g和NHS 1.73g，室温下搅拌溶解，调节pH为5，得溶液1；取具有氨基结构和邻二羟基结构的小分子化合物氨基丙二醇1.2mL(3-氨基-1,2-丙二醇)加入2mL去离子水稀释，得溶液2；将溶液2加入到溶液1中，调反应体系pH为8.5，搅拌反应24h。反应结束后，将反应产物用乙醇沉淀、洗涤、沉淀、干燥，得羧基多糖接枝邻二羟基结构的多糖衍生物(氨基丙二醇-透明质酸)，测定多糖衍生物的邻二羟基结构接枝率为88％。
在实施例2的羧基多糖接枝邻二羟基结构的多糖衍生物的制备中，可以通过调节氨基丙二醇等同时具有氨基结构和邻二羟基结构的小分子化合物与羧基多糖的摩尔比，使具有羧基结构的多糖接枝不同比例的邻二羟基结构，形成具有不同摩尔比侧链邻二羟基结构的多糖衍生物，不同比例的邻二羟基结构经氧化可获得不同比例的醛基。
实施例3：侧链具有醛基的多糖衍生物1的制备
称取实施例1制备的氨基多糖接枝邻二羟基结构的多糖衍生物3.0g，搅拌溶于100mL去离子水，得溶液1；称取氧化剂高碘酸钠2.0g搅拌溶于40mL去离子水，得溶液2；搅拌下将溶液2滴加溶液1中，室温下氧化反应10～20min。反应结束后加入终止剂乙二醇3mL终止反应，继续搅拌1h，将反应产物用乙醇沉淀、洗涤、沉淀、干燥，得侧链具有醛基的氨基多糖衍生物(氧化三羟基丁酸-羟乙基壳聚糖)，测定其醛基的摩尔百分含量为65％。
实施例4：侧链具有醛基的多糖衍生物2的制备
称取实施例2制备的羧基多糖接枝邻二羟基结构的多糖衍生物4.0g，搅拌溶于200mL去离子水，得溶液1；称取氧化剂高碘酸钠1.5g搅拌溶于40mL去离子水，得溶液2；搅拌下将溶液2滴加溶液1中，8～10℃下氧化反应40min～2h。反应结束后加入终止剂乙二醇4mL终止反应，继续搅拌1h，将反应产物用分子量8000-14000的透析袋去离子水透析48h，冷冻干燥，得侧链具有醛基的羧基多糖衍生物2(氧化氨基丙二醇-透明质酸)，测定其醛基的摩尔百分含量为46％。
在实施例3、实施例4的侧链具有醛基的多糖衍生物的制备中，侧链接枝了邻二羟基结构的多糖衍生物在氧化剂高碘酸钠的氧化作用下，调节反应温度、控制物料比和反应时间在数分钟至5小时，氧化剂优先氧化多糖侧链的邻二羟基结构形成醛基，不氧化多糖主链，从而保证了多糖分子主链不被开环降解，保持了多糖主链的完整性。使用的氧化剂包括但不限于高碘酸钠、高碘酸钾；通过控制氧化剂的加入量、控制氧化反应时间、反应温度，得到不同摩尔比的侧链醛基，而多糖主链不被氧化开环，制备的侧链具有醛基的多糖衍生物的侧链醛基单元与糖单元的摩尔百分比可以是≥1％，如5％、10％、20％、30％、40％、50％、60％等，也可以是接近100％。
实施例5：侧链具有伯氨基的多糖衍生物1的制备
称取羧基多糖羧甲基甲壳素4.5g搅拌溶解于150mL去离子水中，加入羧基活化剂EDC 3.0g和NHS 1.8g，搅拌反应1h，调节pH为6，得溶液1；称取具有两个或两个以上伯氨基的小分子化合物碳酰肼2.0g搅拌溶解于30mL去离子水，得溶液2；搅拌下将溶液2滴加至溶液中，调节pH为8，继续反应24h。反应结束后将反应产物用乙醇沉淀、洗涤、沉淀、干燥，得侧链具有伯氨基的多糖衍生物1(碳酰肼-羧甲基甲壳素)，测定伯氨基接枝率为62％。
实施例6：侧链具有伯氨基的多糖衍生物2的制备
称取羧基多糖透明质酸钠4g搅拌溶解于100mL去离子水，加入羧基活化剂EDC 1.91g和NHS 1.15g，搅拌反应1h，调节pH为6，得溶液1；称取具有两个或两个以上伯氨基的小分子化合物谷氨酰胺2.9g，搅拌溶解于20mL去离子水，得溶液2；搅拌下将溶液2滴加至溶液1中，调节pH为7，搅拌反应24h。反应结束后将反应产物用乙醇沉淀、洗涤、沉淀、干燥，得侧链具有伯氨基的多糖衍生物2，测定伯氨基接枝率为43％。
在实施例5、实施例6的侧链具有伯氨基的多糖衍生物的制备中，通过控制碳酰肼、谷氨酰胺等具有两个或两个以上伯氨基的小分子化合物的摩尔比，控制反应时间，得到不同摩尔比的侧链接枝伯氨基结构，制备的侧链具有伯氨基的多糖衍生物的侧链伯氨基单元与糖单元的摩尔百分比可以是≥1％，如5％、10％、20％、30％、40％、50％、60％等，也可以是接近100％。
实施例7：多糖水凝胶1的制备
第一剂：称取实施例3制备的侧链具有醛基的氨基多糖衍生物无菌样品1.0g，溶解于10mL无菌生理盐水，配制成质量百分浓度为10％的溶液，调节pH为6。
第二剂：称取具有两个或两个以上伯氨基的小分子化合物丁二酸二酰肼无菌样品0.05g，溶解于10mL无菌生理盐水，配制成质量百分浓度为0.5％的溶液，调节pH为7.5。
分别将第一剂和第二剂装入无菌双联注射器的A管和B管中，第一剂和第二剂液体经注射器推出混合，形成透明的多糖水凝胶1，成胶时间约2分钟，形成的多糖水凝胶按压有粘性、回弹性好。
实施例8：多糖水凝胶2的制备
第一剂：称取实施例3制备的侧链具有醛基的氨基多糖衍生物无菌样品0.3g，溶解于10mL无菌生理盐水，配制成质量百分浓度为3％的溶液，调节pH为5。
第二剂：称取实施例6制备的侧链具有伯氨基的多糖衍生物谷氨酰胺-透明质酸无菌样品0.2g，溶解于10mL无菌生理盐水，配制成质量百分浓度为2％的溶液，调节pH为8。
分别将第一剂和第二剂装入无菌双联注射器的A管和B管中，第一剂和第二剂液体经注射器推出混合，形成透明的多糖水凝胶2，成胶时间约5分钟，形成的多糖水凝胶按压有粘性、回弹性好。
实施例9：多糖水凝胶3的制备
第一剂：称取实施例4制备的侧链具有醛基的羧基多糖衍生物无菌样品0.3g，溶解于10mL无菌去离子水，配制成质量百分浓度为4％的溶液，调节pH为4.0。
第二剂：称取主链具有伯氨基的多糖衍生物羟丙基壳聚糖无菌样品0.3g，溶解于10mL无菌去离子水，配制成质量百分浓度为5％的溶液，调节pH为8.5。
分别将第一剂和第二剂装入无菌双联注射器的A管和B管中，第一剂和第二剂液体经注射器推出混合，形成透明的多糖水凝胶3，成胶时间约6分钟，形成的多糖水凝胶按压有粘性、回弹性好。
实施例10：多糖水凝胶透光率测定
分别取实施例7～9制备的多糖水凝胶适量，置于生理盐水中充分溶胀，置于玻璃比色皿中，在可见光400-800nm波长范围内测定吸光值(A)，按公示T(％)＝10 2-A计算水凝胶透光率(％)。结果见表1，水凝胶的透光率与波长有关，透光率随着波长的增大而提高，在400nm处各水凝胶的透光率均大于88％；在波长大于500nm时，各水凝胶的透光率均大于90％，表明上述实施例所制备的多糖水凝胶具有优良透光性。
表1 多糖水凝胶的透光率
波长(nm) 400 500 600 700 800
水凝胶1透光率(％) 88.5 90.8 92.4 93.7 94.8
水凝胶2透光率(％) 90.2 92.8 94.6 96.7 97.2
水凝胶3透光率(％) 89.7 91.5 93.2 95.3 96.4
实施例11：多糖水凝胶的降解性测定
分别将实施例7～9制备的多糖水凝胶在直径为6mm的小试管中成胶，取出胶条，切割成高度为5mm的胶块。分别取3种不同水凝胶胶块各2块，干燥恒重，称重，计算降解前胶块的固形物含量。配制10000单位/mL的溶菌酶磷酸缓冲液，分装于3个小三角瓶中，每个三角瓶100mL溶菌酶液，将3种多糖水凝胶各15个胶块分别放入装有溶菌酶溶液的三角瓶中，于37℃恒温箱中进行降解性测定。每10天各取2个胶块，干燥恒重，称重，计算降解胶块的固形物含量，计算降解率，结果见表2。3种多糖水凝胶在前20天相对较稳定，30天的降解率小于20％，40天的降解率在30％左右，随着降解时间的增加，降解速度加快，50天、60天的降解率明显加快，60天的降解率在70％左右。实验结果表明，本发明制备的多糖水凝胶具有较好的抗降解性，早期稳定性较好，降解时间超过60天。而公布号为CN 102762647 A的发明专利申请公开了其制备的水凝胶的体外降解在第3天的水凝胶剩余重量为86.67％，35天的水凝胶剩余重量为61.02％。与公布号为CN 102762647 A的发明专利申请比较，本发明的多糖水凝胶的体外降解时间更长，因此本发明的多糖水凝胶对视网膜的顶压时间更长。
表2 多糖水凝胶的降解率测定数据
时间(天) 10 20 30 40 50 60
水凝胶1降解率(％) 3.2％ 7.3％ 16.5％ 28.7％ 50.6％ 65.3％
水凝胶2降解率(％) 4.8％ 9.1％ 18.8％ 32.2％ 58.2％ 73.7％
水凝胶3降解率(％) 3.5％ 8.8％ 17.9％ 30.7％ 53.8％ 70.2％
实施例12：多糖水凝胶在眼内玻璃体替代物中的应用
实验动物新西兰兔3只，雌性，体重2.5～3kg，兔左眼为术眼，右眼为对照眼，试验实施例9制备的多糖水凝胶3在眼内玻璃体替代物中的应用。
实验动物按常规进行动物麻醉、皮肤消毒，进行玻璃体切除手术，将实施例9制备的第一剂和第二剂经双联注射器注入实验兔的左眼玻璃体腔内，原位形成凝胶，闭合巩膜切口，实验兔术后护理，正常饲养。术后定期测眼压，90d进行眼部裂隙灯检测，眼底照相，B超检查，评价水凝胶在玻璃体腔内的填充情况。
术后术眼眼压与正常眼眼压检测结果见图1。术后3d，术眼眼压较正常眼低，是由于手术在巩膜上做的切口还没有完全愈合，导致眼压下降。7d术眼眼压基本恢复正常，此后眼压基本稳定，与正常眼眼压没有显著性差异，表明本发明的多糖水凝胶在眼内起到了较好的维持眼压作用。与公布号为CN 105833344 A的发明专利申请比较，术眼的眼压更稳定，表明本发明的多糖水凝胶具有更好维持眼压的作用。
术后90d裂隙灯观察见图2，实验兔术眼无炎症反应，角膜正常无水肿，玻璃体保持透明，眼底清晰可见。眼底照相见图3，脉络膜上的血管清晰可见，呈网状分布，无视网膜及脉络膜出血等眼底病变。B超检查见图4，没有检测到玻璃体异物、混浊和出血、视网膜脱离等声像，与正常眼相比无明显差异。实验结果表明，本发明制备的生物多糖水凝胶眼内无刺激作用，具有良好的眼组织相容性，具有很好的玻璃体替代效果，能够顶压视网膜并保持眼球外形，光学性能好，可在眼内降解吸收，多糖水凝胶作为眼内玻璃体替代物应用具有很大的应用潜力。

Claims (23)

  1. 一种生物多糖水凝胶，其是由第一溶液和第二溶液混合反应形成，其中所述第一溶液是侧链具有醛基的多糖衍生物溶液，所述第二溶液是主链或侧链具有伯氨基的多糖衍生物溶液，或是具有两个或两个以上的伯氨基的小分子化合物溶液。
  2. 如权利要求1所述的生物多糖水凝胶，其中所述第一溶液中包含质量百分浓度为0.5％～15％的侧链具有醛基的多糖衍生物，所述第二溶液中包含质量百分浓度为0.5％～15％的主链或侧链具有伯氨基的多糖衍生物，或质量百分浓度为0.2％～30％的具有两个或两个以上伯氨基的小分子化合物；所述第一溶液和第二溶液包含选自无菌水、生理盐水、生理平衡液和葡萄糖液的溶剂。
  3. 如权利要求1所述的生物多糖水凝胶，其中所述第一溶液中的侧链具有醛基的多糖衍生物是具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物。
  4. 如权利要求3所述的生物多糖水凝胶，其中所述具有侧链醛基的氨基多糖衍生物是氨基被修饰成带有醛基的侧链的氨基多糖衍生物，所述具有侧链醛基的羧基多糖衍生物是羧基被修饰成带有醛基的侧链的羧基多糖衍生物。
  5. 如权利要求3所述的生物多糖水凝胶，其中所述具有侧链醛基的氨基多糖衍生物或具有侧链醛基的羧基多糖衍生物，是由氨基被修饰成带有邻二羟基的侧链的氨基多糖或羧基被修饰成带有邻二羟基的侧链的羧基多糖经氧化形成。
  6. 如权利要求3至5中任一项所述的生物多糖水凝胶，其中所述的氨基多糖衍生物包括羟乙基壳聚糖、羟丙基壳聚糖、羟丁基壳聚糖、羧甲基壳聚糖、或羧乙基壳聚糖；所述的羧基多糖衍生物包括具有羧基结构的壳聚糖衍生物、具有羧基结构的甲壳素衍生物、具有羧基结构的透明质酸或透明质酸衍生物、硫酸软骨素、肝素、或海藻酸钠。
  7. 如权利要求6所述的生物多糖水凝胶，其中所述的具有羧基结构的壳聚糖衍生物包括羧甲基壳聚糖、羧乙基壳聚糖、或琥珀酰壳聚糖；所述的具有羧基结构的甲壳素衍生物包括羧甲基甲壳素、或羧乙基甲壳素；所述的具有羧基结构的透明质酸或透明质酸衍生物包括透明质酸、羟乙基透明质酸、羟丙基透明质酸、乙酰基透明质酸、丁酰基透明质酸、氨基乙酸透明质酸、氨基丙酸透明质酸或氨基丁酸透明质酸。
  8. 如权利要求1所述的生物多糖水凝胶，其中所述的主链具有伯氨基的多糖衍生物是主链具有伯氨基同时具有侧链接枝结构的氨基多糖衍生物；所 述的侧链具有伯氨基的多糖衍生物是主链具有羧基结构、侧链具有伯氨基的多糖衍生物。
  9. 如权利要求8所述的生物多糖水凝胶，其中所述侧链具有伯氨基的多糖衍生物由羧基多糖的羧基与具有两个或两个以上伯氨基的小分子化合物中的一个氨基缩合形成。
  10. 如权利要求1所述的生物多糖水凝胶，其中所述的具有两个或两个以上伯氨基的小分子化合物是具有两个或两个以上伯氨基的脂肪族或芳香族小分子化合物，包括乙二胺、丙二胺、丁二胺、氨基丁酰胺、谷氨酰胺、丙氨酰谷氨酰胺、赖氨酸或其它碱性氨基酸、具有二酰肼结构的小分子化合物、二氨基苯甲酸或二氨基苯乙酸。
  11. 如权利要求10所述的生物多糖水凝胶，其中所述的具有二酰肼结构的小分子化合物包括碳酰肼、乙二酸二酰肼、丙二酸二酰肼、丁二酸二酰肼、己二酸二酰肼或癸二酸二酰肼。
  12. 一种生物多糖水凝胶的制备方法，其包括以下步骤：
    (1)提供第一溶液，所述第一溶液是质量浓度为0.5～15％的侧链具有醛基的多糖衍生物无菌溶液，并调节pH为3.5～7，其中所述侧链具有醛基的多糖衍生物由具有接枝邻二羟基结构的多糖衍生物水溶液与氧化剂反应形成；
    (2)提供第二溶液，所述第二溶液选自以下溶液中的任一项：
    i)质量百分浓度为0.2％～30％的具有两个或两个以上的伯氨基的小分子化合物无菌溶液；
    ii)质量百分浓度为0.5％～15％的主链具有伯氨基的多糖衍生物无菌溶液；
    iii)质量百分浓度为0.5％～15％的侧链具有伯氨基的多糖衍生物无菌溶液；
    并调节pH为7～9，测定伯氨基的摩尔百分含量；
    (3)混合所述第一溶液与第二溶液：取步骤(1)所得的第一溶液和步骤(2)所得的第二溶液，分别装入无菌双联注射器的A管和B管中，经注射器推出混合，第一溶液和第二溶液混合形成透明的生物多糖水凝胶。
  13. 如权利要求12所述的生物多糖水凝胶的制备方法，其中所述步骤(1)中的侧链具有醛基的多糖衍生物通过以下方法制备得到：提供质量浓度为0.5～15％的所述具有接枝邻二羟基结构的多糖衍生物水溶液，加入所述氧化剂，其中邻二羟基结构与氧化剂的摩尔比为1﹕(0.3～3)，室温或低温，避光搅拌反应2min～5h，加入乙二醇溶液终止反应0.5～2h，将反应产物进行 透析、冷冻干燥，或乙醇沉淀、洗涤、沉淀、干燥。
  14. 如权利要求12或13所述的生物多糖水凝胶的制备方法，其中所述步骤(1)中的具有接枝邻二羟基结构的多糖衍生物通过以下方法制备得到：分别提供质量浓度为0.5～15％的氨基多糖水溶液和质量浓度为0.2～30％的具有羧基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺加入小分子化合物溶液中，调节pH为3.5～7，搅拌反应0.5～3h；将小分子化合物溶液加入到氨基多糖溶液中，搅拌并调节反应体系pH为7～9，搅拌反应24h。
  15. 如权利要求14所述的生物多糖水凝胶的制备方法，其中所述步骤(1)中，所述的具有羧基结构和邻二羟基结构的小分子化合物，包括2,3-二羟基丙酸、2,3,4-三羟基丁酸、葡萄糖酸、苏糖酸、***酸、木糖酸、或粘酸。
  16. 如权利要求12或13所述的生物多糖水凝胶的制备方法，其中所述步骤(1)中的具有接枝邻二羟基结构的多糖衍生物通过以下方法制备得到：分别提供质量浓度为0.5～15％的羧基多糖水溶液和质量浓度为0.2～30％的具有氨基结构和邻二羟基结构的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，搅拌调节反应体系pH为7～9，搅拌反应24h；3)将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
  17. 如权利要求16所述的生物多糖水凝胶的制备方法，其中所述步骤(1)中，所述的具有氨基结构和邻二羟基结构的小分子化合物，包括氨基丙二醇、或氨基葡萄糖。
  18. 如权利要求12所述的生物多糖水凝胶的制备方法，其中所述步骤(2)中的侧链具有伯氨基的多糖衍生物通过以下步骤制备得到：配制质量浓度为0.5～15％的羧基多糖水溶液，配制质量浓度为0.2～30％的具有两个或两个以上伯氨基的小分子化合物水溶液，将羧基活化试剂EDC和NHS加入羧基多糖溶液中，调节pH为3.5～7，搅拌0.5～3h；将小分子化合物溶液加入到羧基多糖溶液中，调节pH为7～9，搅拌反应24h；将反应产物进行透析、冷冻干燥，或将反应产物进行乙醇沉淀、洗涤、沉淀、干燥。
  19. 如权利要求14-18中任一项所述的生物多糖水凝胶的制备方法，其中所述羧基与羧基活化试剂EDC、NHS的摩尔比为1﹕(0.5～6)﹕(0.5～6)。
  20. 如权利要求12所述的生物多糖水凝胶的制备方法，其中所述的氧化剂包括高碘酸钠或高碘酸钾。
  21. 如权利要求1至11中任一项所述的生物多糖水凝胶在制备眼内玻璃体替代物中的应用。
  22. 一种眼内玻璃体替代物，其包含如权利要求1至11中任一项所述的生物多糖水凝胶。
  23. 如权利要求1至11中任一项所述的生物多糖水凝胶，其用作眼内玻璃体替代物。
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