WO2023082084A1 - 一种凝胶材料及其制备方法与应用 - Google Patents

一种凝胶材料及其制备方法与应用 Download PDF

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WO2023082084A1
WO2023082084A1 PCT/CN2021/129743 CN2021129743W WO2023082084A1 WO 2023082084 A1 WO2023082084 A1 WO 2023082084A1 CN 2021129743 W CN2021129743 W CN 2021129743W WO 2023082084 A1 WO2023082084 A1 WO 2023082084A1
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cross
linking
hyaluronic acid
gel
site
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PCT/CN2021/129743
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French (fr)
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李睿智
谷诗伟
张堃
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爱美客技术发展股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/16—Nitrogen-containing compounds
    • C08K5/17—Amines; Quaternary ammonium compounds
    • 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

Definitions

  • the invention relates to the technical field of biomedical materials, in particular to a gel material and its preparation method and application.
  • Hyaluronic acid or sodium hyaluronic acid (HA for short), also known as hyaluronic acid, is composed of D-glucuronic acid through ⁇ -1,4 glycosidic bonds and N-acetylglucosamine through ⁇ -1,3 glycosidic bonds Composed of disaccharide units glycosaminoglycans. It is widely used in the field of cosmetics or eye surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects.
  • Hyaluronic acid is the original substance in the body, has good biocompatibility and has certain biological activity, but exogenous hyaluronic acid will be degraded by hyaluronidase in the body, resulting in shortened residence time in the body, resulting in treatment The effect is shortened and multiple injections are required to achieve the therapeutic effect.
  • it is necessary to cross-link hyaluronic acid molecules through chemical cross-linking agents to form a spatial network structure, and prevent hyaluronic acid from being degraded by hyaluronidase through a dense rigid network structure. Degradation effect, prolonging the residence time of exogenous hyaluronic acid in the body, ensuring biocompatibility and having good therapeutic effect at the same time.
  • the cross-linking agents of cross-linked hyaluronic acid in the market are mainly divided into two categories, one is a diepoxy cross-linking agent, mainly 1,4-butanediol diglycidyl ether (BDDE), and the other is Unsaturated sulfone crosslinking agent, mainly divinyl sulfone (DVS).
  • BDDE 1,4-butanediol diglycidyl ether
  • DVDS Unsaturated sulfone crosslinking agent
  • the mechanisms of these two types of cross-linking agents are similar, both of which use a basic catalyst as a precursor to catalyze the addition reaction between the hydroxyl group (-OH) in hyaluronic acid and the cross-linking agent to complete the cross-linking.
  • hyaluronic acid The -OH of BDDE undergoes a ring-opening addition reaction with BDDE to complete the crosslinking, and the DVS undergoes a Michael addition reaction with the -OH in hyaluronic acid to complete the crosslinking.
  • these two types of cross-linking agents have high biological toxicity, and unreacted monomers or by-products of cross-linking reactions also have the potential carcinogenicity. Since hyaluronic acid gel is a long-term implanted medical device product, there is also a lack of long-term data tracking with sufficient sample size to prove the biological safety of the product.
  • hyaluronic acid gels made of non-toxic cross-linking agents will be more sought after by more hyaluronic acid gel manufacturers all over the world.
  • non-toxic amino acid cross-linking agents have been used in hyaluronic acid gels.
  • Chinese patent CN10105713211 once disclosed the use of 4-(4,6-dimethoxytriazin-2-yl)-4-methyl Morpholine hydrochloride is a condensing agent, and lysine and its derivatives or arginine and its derivatives are used as a cross-linking agent to prepare cross-linked sodium hyaluronate gel.
  • CN106188609, CN106188584, and CN111732741 do not reflect that cross-linked hyaluronic acid can be sterilized by moist heat at 121° C. for 15 minutes.
  • CN10105713211 discloses a lysine cross-linked hyaluronic acid gel that can be sterilized by moist heat, but according to the information disclosed in this patent, the minimum degradation rate of this type of gel after moist heat sterilization has exceeded 40%, indicating that this type of gel The gel has poor resistance to heat and humidity sterilization.
  • US9907739B2 discloses the cross-linking reaction of spermine or spermidine and hyaluronic acid, but it is detected according to the method of the present invention.
  • the gel disclosed in this patent is only two-site cross-linking, and does not involve multi-site cross-linking. Linkage, that is, the crosslinking site is only the amino group in spermine or spermidine, and the imino group does not participate in the reaction, and the elastic modulus loss rate of this type of gel after moist heat sterilization is as high as 23%. It also shows that the damp heat sterilization performance of this type of gel is not good.
  • cross-linking When cross-linking occurs, it forms an amide bond with the carboxyl group in hyaluronic acid to complete Cross-linking, but due to the lack of cross-linking sites in the molecule, the formed cross-linking network structure cannot provide sufficient thermal stability protection for the main chain of hyaluronic acid, and the thermal stability of the amide bond itself is poor, so it is reflected in the gel In the middle, the degradation rate is high after moist heat sterilization, and the thermal stability is not good.
  • Endogenous polyamines mostly refer to polyamines that can be synthesized or produced during metabolism in the human body.
  • the main endogenous polyamines include spermine, spermidine, and putrescine.
  • Literature Modeo et al., Science 359,410,2018 reported that endogenous polyamines—spermidine has special physiological effects, including but not limited to regulating circadian rhythm, improving hypertension, protecting cardiovascular, preventing senile dementia, enhancing Immunity, anti-cancer and even anti-aging, etc.
  • the physiological activity of spermidine is manifested in the following aspects: 1) kidney: reduce tension, prevent aging; 2) heart: lower blood pressure, prevent arteriosclerosis; 3) brain: prevent memory loss, anti-senile dementia, neuroprotection Effect; 4) Skeleton: Prevent bone loss affected by ovariectomy; 5) Skeletal muscle: Increase the temperature of aged muscles, prevent muscle diseases; 6) Whole organism: Extend the life of the organism; 7) Immune system: Improve vaccine Immunological activity after vaccination, improve cancer-directed immunity, and prevent fatal sepsis; 8) Liver: prevent liver fibrosis and canceration, etc.
  • spermidine is a polycation (-NH3+) aliphatic amine, which exists in a multi-protonated form under physiological pH conditions, has strong biological activity, and is a nucleic acid containing acidic residues. , phospholipids, acidic proteins, pectin polysaccharides containing carboxyl groups or sulfates, and neurotransmitters and hormones with similar structures (such as dopamine, epinephrine, serotonin, thyroid hormone, etc.), all of which may become the targets of spermidine binding .
  • spermidine In terms of binding to nucleic acids, most polyamines exist in the form of polyamine-RNA complexes in cells, and the main role of spermidine is related to the structural changes and translation of RNA, such as by affecting the secondary levels of mRNA, tRNA and rRNA structure to affect the various stages of protein synthesis.spermidine can also form a stable bridge in front of the double-helix DNA strand, reduce the accessibility of ROS or other DNA damaging agents, and protect DNA from thermal denaturation and X-ray radiation. In terms of binding to proteins, spermidine can bind to a large number of negatively charged proteins and change the spatial conformation of proteins, thereby affecting their physiological functions.
  • protein kinases/phosphatases important links in various signal transduction pathways
  • enzymes involved in histone methylation and acetylation affecting gene expression by altering epigenetics
  • ion channel receptors such as AMPA, AMDA receptors
  • the object of the present invention is to provide a gel material and its preparation method and application.
  • the preparation method of the present invention can control the cross-linking sites of endogenous polyamines and hyaluronic acid, and by controlling the cross-linking reaction sites, can affect various performance parameters of the prepared cross-linked hyaluronic acid gel, and control sub-
  • the release rate of endogenous polyamines such as spermine during the degradation process of hyaluronic acid gel continues to exert the physiological activity of endogenous polyamines such as spermidine.
  • the present invention provides a gel material, which is mainly obtained by cross-linking endogenous polyamines and hyaluronic acid, and the endogenous polyamines include spermine (tetraamino compound) and /or spermidine (triamino compound); the cross-linking of the endogenous polyamine and the hyaluronic acid includes two-site cross-linking, three-site cross-linking or four-site cross-linking.
  • the present invention realizes for the first time that endogenous polyamines and hyaluronic acid are cross-linked at three or four sites to form a multi-site cross-linked active star network structure.
  • spermine or spermidine an endogenous polyamine
  • spermine or spermidine is selected as a multi-site cross-linking agent to form a dense network structure and increase the thermal stability of the thus formed amide bond cross-linked hyaluronic acid gel.
  • endogenous diamines such as putrescine
  • diamines such as putrescine have high toxicity, and the cross-linking sites are the same as those of amino acids such as lysine and arginine, so they do not have the ability to realize multi-site cross-linking. possibility of connection.
  • the present invention provides a gel material obtained by cross-linking endogenous polyamines including spermine and/or spermidine and hyaluronic acid.
  • Amine; the cross-linking of the endogenous polyamine and the hyaluronic acid comprises two-site cross-linking, three-site cross-linking or four-site cross-linking.
  • the proportion of amino residues in the gel obtained by the two-site crosslinking is lower than 20%; preferably, the proportion of amino residues in the gel obtained by crosslinking in the two-site crosslinking is less than 20%. It is less than 15%; more preferably, the proportion of amino residues in the gel obtained by cross-linking in the two sites is less than 10%.
  • the ratios of residues of amino groups and imino groups in the gel obtained by the three-position cross-linking or four-position cross-linking are both lower than 20%.
  • the proportion of amino residues in the gel obtained by cross-linking is lower than 15%; preferably, the three-position cross-linking or four-position cross-linking During the crosslinking, the proportion of amino residues in the gel obtained by crosslinking is less than 10%.
  • the proportion of imino residues in the gel obtained by cross-linking in the three-position cross-linking or four-position cross-linking is less than 15%; preferably, the three-position cross-linking or four-position In point cross-linking, the proportion of imino residues in the gel obtained by cross-linking is lower than 10%.
  • the cross-linking reaction efficiency of two-site cross-linking, three-site cross-linking or four-site cross-linking in the cross-linking reaction between endogenous polyamine and hyaluronic acid is higher than 75%; preferably, the The efficiency of the cross-linking reaction is higher than 80%; more preferably, the efficiency of the cross-linking reaction is higher than 85%.
  • the elastic modulus loss rate (G' loss rate) of the gel obtained by crosslinking is lower than 22%; preferably, the elastic modulus loss rate (G' loss rate) is lower than 15%; more preferably Yes, the elastic modulus loss rate (G' loss rate) is less than 10%.
  • the present invention provides a kind of preparation method of gel material, described method comprises the following steps:
  • the endogenous polyamines include spermine and/or spermidine.
  • spermidine and spermine There are amino groups and imino groups in spermidine and spermine; among them, spermidine contains one imino group and two amino groups, and spermine contains two amino groups and two imino groups.
  • the present invention finds that the reaction sites of spermine or spermidine and hyaluronic acid can be controlled by adjusting the pH value of the mixed solution after hyaluronic acid and endogenous polyamines are dissolved, and a double-site cross-linked hydrogel can be obtained , and three- or four-site cross-linked active star network gels.
  • the amino site reaction of spermine or spermidine can be adjusted by the pH of the solution (ie, a two-site reaction) or the amino and imino groups can react together (ie, a multi-site reaction).
  • the two-site cross-linking in the present invention means that when hyaluronic acid is cross-linked with spermine or spermidine, the amino group in spermine or spermidine is the main reaction site.
  • the two-site The proportion of amino residues in the gel obtained by point cross-linking is less than 20%;
  • the three-position or four-position cross-linking in the present invention refers to that when hyaluronic acid is cross-linked with spermine or spermidine, spermine Or the amino group and the imino group in spermidine serve as the reaction site together, in particular, the proportion of amino group and imino residue in the gel obtained by the three-position cross-linking or four-position cross-linking is both lower than 20%. .
  • the present invention finds that controlling the pH value of the solution between 5.00 and 5.49 can improve the reactivity of the imino group and relatively stably realize the three-site or four-site reaction; the pH value of the solution is controlled between 4.50-4.99 or 5.50-6.50 , can provide imino reactivity, realize multi-site co-participation reaction relatively stably, and prepare active star-shaped network gel. Therefore, the present invention obtains hyaluronic acid hydrogel with imino as the cross-linking site for the first time, and finds that hydrogels with different cross-linking sites can obtain different gel properties under the same cross-linking degree.
  • the three-site or four-site reaction can be realized relatively stably by controlling the pH of the mixed solution between 5.00 and 5.49, the pH including but not limited to 5.00, 5.10, 5.20, 5.30, 5.40 or 5.49.
  • Controlling the pH of the mixed solution between 4.50-4.99 or 5.50-6.50 can achieve relatively stable two-site crosslinking reaction, the pH includes but not limited to 4.50, 4.60, 4.70, 4.80, 4.90, 4.99, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, or 6.50.
  • the experimental results are not ideal, and the pH range of the present invention is the pH condition suitable for the method of the present invention obtained through a large number of experiments.
  • the method of the present invention is an endogenous polyamine (spermine and spermidine) multi-site active reaction technology (Spermidine/Spermine Multisite Active Reaction Technology, referred to as SMART).
  • the method of the invention can control the cross-linking sites of endogenous polyamines and hyaluronic acid, and further affect various performance parameters of the cross-linked hyaluronic acid gel by controlling the cross-linking reaction sites.
  • it can also control the release rate of endogenous polyamines such as spermidine during the degradation process of hyaluronic acid gel, and continuously exert the physiological activity of endogenous polyamines such as spermidine.
  • an activator is added during the two-site crosslinking reaction, three-site crosslinking reaction or four-site crosslinking reaction;
  • the activator includes water-soluble carbodiimide, phosphonium bromide salt formed from triphenylphosphine and bromide, carbonium salt and 4-(4,6-dimethoxytriazin-2-yl )-4-methylmorpholine hydrochloride (DMTMM) in one or more.
  • water-soluble carbodiimide phosphonium bromide salt formed from triphenylphosphine and bromide
  • carbonium salt and 4-(4,6-dimethoxytriazin-2-yl )-4-methylmorpholine hydrochloride (DMTMM) in one or more.
  • DTMM 4-(4,6-dimethoxytriazin-2-yl )-4-methylmorpholine hydrochloride
  • the water-soluble carbodiimide activators include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-(3-dimethyl Aminopropyl)-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[bis(methoxymethyl)methyl ] Carbodiimide, etc. or their salts and one or more mixtures thereof.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • 1-(3-dimethyl Aminopropyl)-3-ethyl-carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
  • 1,3-bis[bis(methoxymethyl)methyl ] Carbodiimide etc. or their salts and one or more mixtures thereof.
  • the phosphonium bromide salt formed from triphenylphosphine and bromide includes phosphonium salt formed from triphenylphosphine and carbon tetrabromide, triphenylphosphine and N-bromosuccinimide phosphonium salts etc.
  • the phosphonium bromide salt The desired phosphonium salt is obtained by known methods from triphenylphosphine and bromide in dichloromethane.
  • the carbenium salts include O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate (HATU), O-(benzene Triazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbon Onium hexafluorophosphate (HCTU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbenium tetrafluoroborate (TBTU), O-(N-succinimide base)-bis(dimethylamino)carbenium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetrafluoroborate One or more mixtures of methylurea
  • the auxiliary agent includes N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, 1-hydroxybenzotriazole (HOBt) Any one or more; more preferably, the additive is added in an amount of 10-30% of the carbodiimide mass, including but not limited to 10%, 11%, 12%, 13%, 14% , 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, and 30%.
  • the combined use of water-soluble carbodiimide and additives makes the cross-linking reaction efficiency reach about 70%.
  • the cross-linking efficiency of carbonium salt is the highest, which is 80-95%.
  • the cross-linking efficiency is as high as 95%.
  • the cross-linking efficiency of phosphonium bromide salt and DMTMM is second, and the cross-linking efficiency is 70%. ⁇ 85% range.
  • the carbonium salt of HATU is used as the reaction activator.
  • the molecular formula structure of HATU is as follows (1):
  • the amount of the activator added is the amount of the endogenous polyamine substance 200-280%; including but not limited to 210%, 220%, 230%, 240%, 250%, 260%, 270% and 280%; when carrying out three-site and/or four-site cross-linking (imino sites also participate in the reaction), the amount of the activator added is 300% to 550% of the amount of the endogenous polyamine substance; including but not limited to 320%, 350%, 370%, 390%, 400%, 420%, 450%, 470%, 500%, 520%, and 550%
  • the amount of the activator added is 400-550% of the amount of the spermine substance; when the endogenous polyamine is spermidine , the added amount of the activator is 300-400% of the amount of the spermidine substance.
  • the amount of the activator added is related to the amino crosslinking site of the cross-linking agent spermine or spermidine.
  • each molecule of the activator can activate a carboxyl group and perform a reaction with an amino group (or imino group). Amide coupling reaction.
  • the amount of spermine added accounts for 0.3-35% of the mass of hyaluronic acid, including but not limited to 0.4%, 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% , 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% and 35%; when the endogenous polyamine is spermidine , the amount of spermidine added accounts for 0.5% to 40% of the mass of hyaluronic acid; including but not limited to 0.5%, 0.8%, 1%, 3%, 5%, 8%, 10%, 11%, 12% %, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%,
  • the mass concentration of hyaluronic acid in the aqueous solution of the reaction system is 10-150 mg/mL, including but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150 mg/mL.
  • the mass concentration of hyaluronic acid is 1-50 mg/mL, including but not limited to 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50mg/mL.
  • the molecular weight of the hyaluronic acid is 100KDa (kilodalton) to 3000KDa; preferably, the hyaluronic acid is hyaluronic acid prepared by microbial fermentation.
  • the temperature of the crosslinking reaction is 10 ⁇ 60°C, the cross-linking reaction time is 14 ⁇ 24h;
  • the activator includes water-soluble carbodiimide and/or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride
  • the crosslinking reaction The temperature is 40-60°C, and the cross-linking reaction time is 14-24h.
  • the two-site cross-linked gel and the three-site or four-site cross-linked active star-shaped network gel can be controlled by different reaction temperatures and the ratio of the activator to spermine or spermidine . Because the imino group is affected by the steric hindrance effect, the reactivity of the imino group is lower than that of the primary amino group. Therefore, only a lower temperature and a lower amount of activator are required for the two-site reaction in which only the primary amino group participates.
  • the crosslinking reaction can be completed; however, when the imino group is required to participate in the reaction, in order to increase the reactivity of the imino group, it is necessary to increase the reaction temperature and prolong the reaction time to improve the crosslinking reaction efficiency of the imino group.
  • cross-linking reaction between spermidine and hyaluronic acid, spermine and hyaluronic acid is shown below, wherein (3) is the cross-linking reaction between hyaluronic acid and spermine (4-site star network). (4) The cross-linking reaction between hyaluronic acid and spermidine (3-site star network).
  • the hyaluronic acid gel prepared by the SMART multi-site active reaction technology in the present invention fully utilizes the different activities of the amino group and the imino group in the spermine or spermidine molecule, and controls the reaction conditions, such as pH, temperature, active agent
  • One or more of the types and amounts can be used to obtain double-site cross-linked gels, and active star-shaped network gels with three-site or four-site cross-linked gels.
  • the gel material is further eluted with an eluent, pulverized, and dried;
  • the eluent is an organic solvent; more preferably, the organic solvent is a soluble alcohol or a soluble ketone; more preferably, the organic solvent is ethanol or acetone;
  • volume ratio of the gel to the eluent in the reaction system during pulverization is 1:1-5;
  • the particle size of the gel after pulverization is 10-500 ⁇ m.
  • the preparation method further includes reconstitution and moist heat sterilization of the dried gel material
  • the reconstituted solution is a phosphate buffer solution; more preferably, the mass concentration of phosphate buffer salt in the phosphate buffer solution is 5-40 mg/mL;
  • the temperature of the moist heat sterilization is 120-130° C.
  • the time of the moist heat sterilization is preferably 15-45 minutes.
  • the gel is eluted with an organic solvent and the gel particles are pulverized, and the residual activator can be removed by repeated washing.
  • the drying can be vacuum drying, and the organic solvent can be removed by drying. Pulverizing the gel into a lower particle size can ensure the dissolution efficiency of the residual activator during multiple washings.
  • the vacuum-dried gel particles are reconstituted with a phosphate buffer solution, filled in a pre-filled syringe and subjected to moist heat sterilization to obtain the final product gel.
  • the pH range of the phosphate buffer solution is 6.8-7.6.
  • the concentration of the final hyaluronic acid gel is 1-35 mg/mL.
  • the method comprises the steps of:
  • the loss rate of the elastic modulus is low, and the lowest can be controlled within 10%, which can effectively maintain the rheological properties of the gel before sterilization, to a large extent It improves the thermal stability of hyaluronic acid gel.
  • polyamines such as spermidine are designed as hyaluronic acid cross-linking agents, and the whole hyaluronic acid gel is used as a slow-release gel of polyamines such as spermidine as a whole, that is, the gel is in vivo During the natural degradation process, polyamines such as spermidine can be released continuously.
  • the three-site or four-site active star-shaped network gel of the present invention can stably release spermidine monomer under the enzymatic trigger of hyaluronidase, and continuously exert the biological activity of spermidine.
  • the present invention also provides the gel material obtained by the above preparation method.
  • the present invention also provides the use of the gel material or the gel material prepared by the preparation method in preparing tissue filling and repairing materials or drug carriers.
  • the gel material or the gel material prepared by the preparation method is used in the preparation of medicines, medical cosmetology and cosmetic products, such as soft tissue filling, soft tissue repair, or cosmetic injections for eliminating facial skin wrinkles.
  • the gel material provided by the present invention realizes multi-site crosslinking between endogenous polyamines and hyaluronic acid to form a denser network structure
  • the preparation method of the present invention can realize the control of the number of cross-linking reaction sites, which in turn can affect the performance of the prepared cross-linked hyaluronic acid gel;
  • the present invention can control the release rate of endogenous polyamines such as spermidine during the degradation process of hyaluronic acid gel, and continuously exert the physiological activity of endogenous polyamines such as spermidine;
  • the present invention improves the ease of use of the gel in the fields of soft tissue filling, soft tissue repair, medical cosmetology, etc., and is suitable for popularization and application.
  • Fig. 1 is the 1 H NMR spectrum of spermidine cross-linked hyaluronic acid provided by the present invention
  • Fig. 2 is a schematic diagram of cell proliferation of spermidine-crosslinked hyaluronic acid provided by the present invention
  • Fig. 3 is the release curve of spermidine in cross-linked hyaluronic acid gel with cross-linked hyaluronic acid under thermal degradation conditions provided by the present invention
  • Fig. 4 is the release curve of spermidine in spermidine cross-linked hyaluronic acid gel with cross-linked hyaluronic acid under enzymatic conditions provided by the present invention.
  • Example 1 Preparation of double-site cross-linked hydrogel of spermine and hyaluronic acid catalyzed by HATU
  • Example 2 Preparation of three-site cross-linked hydrogel of spermidine and hyaluronic acid catalyzed by HATU
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and dried in vacuum at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 143 ⁇ m
  • D0.9 is 201 ⁇ m.
  • Example 3 Preparation of four-site cross-linked hydrogel of spermine and hyaluronic acid catalyzed by HATU
  • Example 4 Preparation of double-site cross-linked hydrogel of spermidine and hyaluronic acid catalyzed by triphenylphosphonium bromide succinimide
  • Example 5 Preparation of three-site cross-linked hydrogel of spermidine and hyaluronic acid catalyzed by triphenylphosphonium bromide succinimide
  • Example 6 Preparation of three-site cross-linked hydrogel of spermidine and hyaluronic acid catalyzed by DMTMM
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and dried in vacuum at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 144 ⁇ m
  • D0.9 is 206 ⁇ m.
  • Example 7 Preparation of four-site cross-linked hydrogel of spermine and hyaluronic acid catalyzed by EDC
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and dried in vacuum at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 125 ⁇ m
  • D0.9 is 193 ⁇ m.
  • Example 8 HATU catalyzed the combination of spermidine and spermine to participate in the multi-site cross-linking of hyaluronic acid Hydrogel preparation
  • Example 9 EDC catalyzes the three-site crosslinking of spermidine and hyaluronic acid
  • Example 10 Preparation of four-site cross-linked hydrogel of spermine and hyaluronic acid catalyzed by HATU
  • Example 11 Preparation of three-site cross-linked hydrogel of spermidine and hyaluronic acid catalyzed by HATU
  • Example 12 EDC catalyzes the four-site crosslinking of spermine and hyaluronic acid
  • Example 13 Preparation of double-site cross-linked hydrogels catalyzed by HATU under acidic conditions of spermidine and hyaluronic acid
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and dried in vacuum at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 138 ⁇ m
  • D0.9 is 210 ⁇ m.
  • Comparative Example 1 Preparation of HATU-catalyzed cross-linked hydrogel between spermidine and hyaluronic acid under strong acid conditions Weigh 3.0 g of sodium hyaluronate (molecular weight 900 KDa, containing 7.4 mmol of hyaluronic acid repeating structural units), and then add 98 mL of purified Water, after completely dissolving, the concentration of hyaluronic acid is 30mg/mL at this moment, adds spermidine 0.022g (mole number is 0.15mmol) to hyaluronic acid solution, now spermidine content accounts for 1% of hyaluronic acid quality.
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and then vacuum-dried at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 143 ⁇ m
  • D0.9 is 201 ⁇ m.
  • Comparative example 2 EDC catalyzes the cross-linking reaction of spermine and hyaluronic acid
  • the gel was prepared according to the method disclosed in US9907739B2.
  • the precipitate was separated, washed continuously with 200 mL of absolute ethanol for 5 times, then placed in a vacuum oven, and dried in vacuum at 40° C. for 24 h at a vacuum degree of -0.09 MPa. After complete drying, take 1.0 g of dry gel, add 50 mL of pH 7.0 phosphate buffer with a concentration of 10 mg/mL in total, and prepare a hydrogel with a hyaluronic acid concentration of 20 mg/mL. After the gel is completely swollen, The gel was filled into a pre-filled syringe, and subjected to moist heat sterilization at 121°C for 15 minutes to obtain the final product gel.
  • the gel particle diameter D0.5 is 155 ⁇ m
  • D0.9 is 221 ⁇ m.
  • the actual molar ratio of spermidine to hyaluronic acid is obtained by integrating the peak area, and the cross ratio is obtained by comparing the actual molar ratio with the theoretical molar ratio (calculated according to actual feeding). connection efficiency.
  • the characteristic H atom selected in spermidine is ⁇ -H of amino or imino group, wherein the number of hydrogen atoms is 8, and the characteristic H atom selected in spermidine is ⁇ -H of amino group or imino group, wherein the H atom
  • the total number is 12, and the integral range of the H atoms of spermine and spermidine is the same, which is 2.5-2.85ppm;
  • the characteristic H atom selected in hyaluronic acid is the ⁇ -H of the carbonyl group in the acetamido group, and the total number of H atoms is 3 1, the integration range is 1.85-2.05ppm; when integrating, set the integration peak area of the carbonyl ⁇ -H in hyaluronic acid to 3, you can get the integration of amino and imino ⁇ -H in spermidine or spermine Peak area. Calculated as follows:
  • Cross-linking efficiency (actual cross-linking molar ratio/theoretical cross-linking molar ratio) ⁇ 100%.
  • Example Theoretical crosslinking molar ratio Actual crosslinking molar ratio Cross-linking efficiency Example 1 2.0% 1.7% 85%
  • Example 2 2.0% 1.9% 95%
  • Example 3 2.0% 1.9% 95%
  • Example 4 2.0% 1.5% 75%
  • Example 5 2.0% 1.7% 85%
  • Example 6 2.0% 1.6% 80%
  • Example 7 2.0% 1.5% 75%
  • Example 8 4.0% 3.9% 97% Comparative example 1 2.0% 0.9% 45%
  • Comparative example 2 2.0% 1.0% 50%
  • the activation efficiency of HATU can be as high as nearly 95%, while the activation efficiency of EDC+NHS is only 75%.
  • the cross-linking efficiency of spermine and spermidine is close, and the efficiency of spermine is slightly lower, which may be due to the higher content of imino in spermine. Because of the large steric hindrance of imino, the reactivity is not as good as that of spermine. primary amino group.
  • Example 15 Effect of spermidine cross-linked hyaluronic acid gel on cell proliferation
  • the L-929 cells were planted in the cell culture medium, and 1% penicillin-streptomycin solution and 10% fetal bovine serum solution were added. L-929 cells were incubated in a humidified cell culture incubator containing 5% carbon dioxide at 37°C for 3 days. Then, the spermidine three-site cross-linked hyaluronic acid gel obtained in Example 2 was transferred into a 96-well plate, cured by ultraviolet light and sterilized. Again, add the L-929 cell culture solution to the wells where the spermidine cross-linked hyaluronic acid gel was placed, and add 1 mL of trypsin solution containing 0.1% EDTA, the number of L-929 cells in each well was 1 ⁇ 10 5 pcs. And continue to be placed in the cell culture incubator to promote cell growth.
  • the cell proliferation rate of hyaluronic acid gel cross-linked with spermidine was determined by MTT method. After culturing for 24, 48 and 72 hours respectively, 100 ⁇ L of MTT aqueous solution (concentration: 5 mg ⁇ mL-1) was added to each well and placed in an incubator to continue culturing for 4 hours. Then remove the MTT solution, add 150 ⁇ L of dimethyl sulfoxide to dissolve the formazan crystals, and use a microplate reader to detect the absorbance of the solution at a wavelength of 490 nm to determine the degree of cell proliferation of the gel on L-929. Cell viability was calculated according to the following formula:
  • Example 16 Detection of rheological properties of spermine or spermidine cross-linked hyaluronic acid gel
  • G' loss rate (G' before sterilization - G' after sterilization)/G' before sterilization;
  • the parameters of the rheometer are: operating gap: 1000mm, loading gap: 45000m, operating temperature: 37°C, deformation: 1%, frequency: 0.9Hz, running time: 60s.
  • the rheological data of each gel are shown in Table 2:
  • Example G'(Pa) before sterilization After sterilization G'(Pa) G' loss rate
  • Example 1 466 411 11.8%
  • Example 2 533 494 7.3%
  • Example 3 632 588 7.0%
  • Example 4 448 388 13.4%
  • Example 5 505 459 9.1%
  • Example 6 485 433 10.7% Example 7 581 512 11.9% Example 8 1005 958 4.7% Example 9 843 725 14.0% Example 10 0.82 0.70 14.6% Example 11 1.54 1.31 14.9% Example 12 985 878 10.9% Example 13 455 403 11.4% Comparative example 2 405 311 23.2%
  • the G' loss rate can be considered as an indicator of the thermal stability of the gel, and the lower the G' loss rate, the higher the thermal stability of the gel.
  • the G' loss rate has a certain relationship with the cross-linking efficiency in Example 14, wherein, in the case of similar cross-linking degree and cross-linking efficiency Below (Examples 2 and 3), the thermal stability of hyaluronic acid cross-linked with spermidine is similar to that of hyaluronic acid cross-linked with spermine, but the elastic modulus of spermine is higher and the thermal stability is higher.
  • the possible reason The cross-linked network structure formed by the 4-site cross-linking of spermine is more dense, and the same theory can also be used for the three-site cross-linked spermidine gel and the two-site cross-linked spermidine gel, that is, double
  • the thermal stability of gels with one-site crosslinking is worse than that of three-site crosslinking.
  • the order of thermal stability of the gel is: HATU>triphenylphosphonium salt>DMTMM>EDC+auxiliary, the order is the same as in Example 14.
  • the hyaluronic acid concentration in the cross-linked hyaluronic acid hydrogel also has a great influence on the thermal stability of the gel.
  • Examples 10 and 11 adopted the same cross-linking sites as in Examples 2 and 3, but due to the implementation
  • the hyaluronic acid concentrations in the cross-linked hyaluronic acid hydrogels in Examples 10 and 11 were 1 mg/mL and 2 mg/mL respectively, while the corresponding concentrations in Examples 2 and 3 were 20 mg/mL, thus resulting in a relatively large difference in G' Large, and the result of increased G' drop value.
  • the amount of activator added also has a greater impact on the performance of the hydrogel.
  • Example 13 Under the same conditions as in Example 1 and Example 13, the amount of HATU activator added in Example 13 was reduced by 28.6% compared to Example 1 ( That is, 164.8 ⁇ mol), resulting in the result that G' in Example 13 is significantly lower than that in Example 1, and the ratio of G' reduction is also significantly higher than that in Example 1.
  • Example 17 Detection of the release of spermidine in the gel during the degradation of hyaluronic acid under thermal degradation conditions
  • Example 2 Take the finished gel in Example 2, 2 mL of each sample, seal it in a vial, place it in a blast drying oven at 125°C for accelerated hydrolysis, take out 3 parallel samples every 15 minutes, and cool to about 25°C Finally, the pH was adjusted to 12 with sodium hydroxide, and 20 mL of chromatographic grade acetone was added respectively, and after filtration with a 0.2 ⁇ m filter membrane, the acetone filtrate was injected into the Agilent 7890B gas chromatograph, and the SGE H 2 capillary column was used to (30m ⁇ 0.53mm ⁇ 1.0 ⁇ m), the chromatographic conditions are: inlet temperature 220°C, pressure 40°C, separation flow rate 30°C, split ratio 10:1, separation time 0.8, column temperature: 100°C for 0.5min, 20°C /min increased to 180°C and maintained for 1min, detector (FID): temperature 250°C, air flow rate 350mL/min, H2 flow rate 35mL/min, make-up N2 flow
  • the total degradation time was 90 minutes, and a total of 6 detection points were set up, and the detection results are shown in Figure 3.
  • the purpose of this experiment is to simulate the release of spermidine monomer due to the hydrolysis of the cross-linking site during the thermal degradation of hyaluronic acid.
  • the degradation process of cross-linked hyaluronic acid takes a long time, ranging from a few months to more than a year, so this experiment uses hyaluronic acid gel cross-linked with spermidine.
  • the test of the release of spermidine during the accelerated hydrolysis process at a high temperature of 100°C can observe the degradation process of hyaluronic acid and the release process of spermidine in a relatively short period of time.
  • the spermidine monomer can be continuously released in the original form, and it can also be proved that the gel is in vivo Continuously release spermidine, and exert the specific physiological activity of spermidine.
  • Example 18 Detection of the release of spermidine in the gel during the degradation of hyaluronic acid under enzymatic conditions
  • the total degradation time was 180min, and a total of 6 detection points were set up, and the detection results are shown in Figure 4.
  • the purpose of this experiment is to simulate the process of releasing spermidine monomer due to gel degradation during the enzymatic degradation of hyaluronic acid.
  • the release of spermidine is slowly released along with the degradation process of hyaluronic acid.
  • the release rate of spermidine is almost unchanged, indicating that the use of spermidine to crosslink
  • the obtained hyaluronic acid gel can release spermidine uniformly and stably during enzymatic degradation.
  • This experiment can also prove that the cross-linking technology in the present invention is an active reaction technology.
  • the spermidine monomer can be continuously released in the original form, and it can also be proved that the gel is in vivo Continuously release spermidine, and exert the specific physiological activity of spermidine.
  • ninhydrin add 100ml of purified water to dissolve, mix well; store in the dark at 2-8°C for later use.
  • Take 54.6g of sodium acetate add 20ml of 1mol/L acetic acid solution to dissolve, add water to dilute to 500ml, and set aside.
  • Residual amount ratio (A 0 -A i )/A 0 ⁇ 100%
  • A0 is the absorbance value measured by the control solution
  • Ai is the absorbance value measured by the sample.
  • one spermidine molecule contains one imino group and two amino groups
  • one spermine molecule contains two imino groups and two amino groups
  • Example Amino residues imino residues Example 1 10.34% 86.61%
  • Example 2 7.31% 8.02%
  • Example 3 8.53% 9.45%
  • Example 4 18.13% 89.45%
  • Example 5 10.16% 12.31%
  • Example 6 13.35% 13.43%
  • Example 7 17.41% 14.31%
  • Example 8 6.32% 7.88% Comparative example 1 39.25% 97.13% Comparative example 2 40.35% 98.31%
  • Examples 1 and 4 are two-site crosslinking, and its main reactive group is amino, and the reaction efficiency is as high as 89.66% (100%-amino residues, the same below), and the lowest amino reaction efficiency exceeds 80%, the imino reaction efficiency does not exceed 15%, therefore, under this condition, the two-site cross-linking reaction mainly involves the amino group.
  • the reaction efficiency between the amino group and the imino group of spermidine or spermine all reached more than 90%, and in Examples 5-7, the reaction efficiency of the imino group also exceeded 85%.

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Abstract

一种凝胶材料及其制备方法与应用。该凝胶材料由内源性多胺与透明质酸交联得到，所述内源性多胺包括精胺和/或亚精胺；内源性多胺与透明质酸的交联包含双位点交联、三位点交联或四位点交联。通过控制反应交联位点影响凝胶的各项性能以及多胺的降解释放速度。所述凝胶经湿热灭菌后，弹性模量损失率低，可有效保持凝胶在灭菌前的流变学性能，很大程度上提高了透明质酸凝胶的热稳定性，也提高了凝胶在软组织填充、软组织修复、医疗美容等领域方面的易用性。

Description

一种凝胶材料及其制备方法与应用 技术领域
本发明涉及生物医用材料技术领域，尤其是涉及一种凝胶材料及其制备方法与应用。
背景技术
透明质酸或透明质酸钠(hyaluronic acid，简称HA)，又称玻尿酸，是由D-葡萄糖醛酸通过β-1,4糖苷键与N-乙酰葡糖胺通过β-1,3糖苷键组成的双糖单位糖胺聚糖。广泛用于化妆品领域或眼科手术中，也可作为软组织充填剂用以修复皱纹以及一些软组织缺陷。透明质酸为体内原生物质，具有良好的生物相容性并具有一定的生物活性，但外源性透明质酸在体内会受到透明质酸酶的降解作用而导致体内停留时间缩短，而导致治疗效果缩短，需要多次注射才能达到治疗效果。为避免透明质酸受到透明质酸酶的降解，需要通过化学交联剂将透明质酸分子进行交联，形成空间网络结构，通过密集的刚性网络结构阻止透明质酸酶等对透明质酸的降解作用，延长外源性透明质酸在体内的停留时间，保证生物相容性的同时具有良好的治疗效果。
目前市场中的交联透明质酸的交联剂主要分为两类，其一为双环氧类交联剂，主要为1,4-丁二醇二缩水甘油醚(BDDE)，其二为不饱和砜交联剂，主要为二乙烯基砜(DVS)。此两类交联剂的机理类似，均是以碱性催化剂为先导条件，催化透明质酸中的羟基(-OH)与交联剂发生加成反应而完成交联，其中，透明质酸中的-OH与BDDE发生开环加成反应完成交联，DVS则与透明质酸中的-OH发生迈克尔加成反应完成交联。然而，这 两类交联剂均具有较高的生物毒性，同时未反应的单体或交联反应副产物也存在这潜在的致癌性。由于透明质酸凝胶为长期植入的医疗器械产品，临床方面也缺乏足够样本量的长期数据追踪以证明该产品的生物安全性。在当前市场需求方面，延长透明质酸凝胶在体内的停留时间以及增加透明质酸凝胶的粘弹性质均为目前行业的主要趋势。目前能有效延长透明质酸凝胶体内停留时间的方法为增大交联度，同时交联度的增加也能够带来粘弹性的增加，但在使用传统的BDDE或DVS交联剂的情况下，增加交联剂的加入量，则会增加人们对该类产品安全性方面的担忧。
在此前提下，在全世界范围内，选择无毒交联剂制成透明质酸凝胶的也将更加受到更多透明质酸凝胶厂商的追捧。目前，无毒的氨基酸交联剂已经被使用在透明质酸凝胶中，中国专利CN10105713211曾经公开了以4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐为缩合剂，赖氨酸及其衍生物或精氨酸及其衍生物为交联剂制备的交联透明质酸钠凝胶。中国专利CN106188609、CN106188584、CN111732741曾公开了以1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐类碳二亚胺为缩合剂，N-羟基琥珀酰亚胺类琥珀酰亚胺为催化剂，精氨酸及其衍生物、赖氨酸及其衍生物(含聚赖氨酸)为交联剂制备的交联透明质酸钠凝胶。以上专利均公开了以赖氨酸或精氨酸等双氨基氨基酸为交联剂制备的透明质酸钠凝胶。以赖氨酸或精氨酸交联剂为例，该交联透明质酸水凝胶在湿热灭菌时表现的性能差异较大。其中，CN106188609、CN106188584、CN111732741均未体现交联透明质酸能够进行121℃15分钟的湿热灭菌。CN10105713211中公开了能够湿热灭菌的赖氨酸交联透明质酸凝胶，但根据该专利公开的信息显示，该类凝胶在湿热灭菌后的最低降解率已超过40％，说明此类凝胶的耐湿热灭菌性能不佳。US9907739B2中公开了精胺或亚精胺与透明质酸的交联反应，但根据本发明的方法进行检测，该专利中公开的凝胶仅为双位点交联，并未涉及多位点交联，即交联位点仅为精胺或亚精胺中的氨基基团，亚氨基基团并 未参与反应，且该类凝胶在湿热灭菌后的弹性模量损失率高达23％，亦说明此类凝胶的耐湿热灭菌性能不佳。此外，Xiang Mei Yan等人发表的文章(Journal of Biomaterials Applications,2011,27(2):179-186)中公开了采用己二胺交联的透明质酸水凝胶，但该凝胶在灭菌后弹性模量下降值也超过20％。主要原因在于赖氨酸、精氨酸以及己二胺等此类二元氨基分子中仅存在两个氨基交联位点，在发生交联时，与透明质酸中的羧基形成酰胺键而完成交联，但由于分子中交联位点较少，形成的交联网络结构无法为透明质酸主链提供足够的热稳定性保护，加之本身酰胺键热稳定性较差，因此体现在凝胶中表现为湿热灭菌后降解率较高，热稳定性不佳。
内源性多胺多指在人体环境中能够合成或代谢过程中产生的多胺，主要的内源性多胺包括精胺、亚精胺与腐胺等。文献(Madeo等.,Science 359,410,2018)报道了内源的多胺——亚精胺具有特殊的生理作用，包括但不限于调节昼夜节律、改善高血压、保护心血管、预防老年痴呆、增强免疫力、抗癌甚至抗衰老等。亚精胺的生理活性作用表现在以下几个方面：1)肾脏：降低紧张度，防止衰老；2)心脏：降血压，防止动脉硬化；3)大脑：防记忆衰退，抗老年痴呆，神经保护作用；4)骨骼：防止由卵巢切除影响的骨流失；5)骨骼肌：提高年老肌肉的温度，预防肌肉疾病；6)全生物体：延长生物体的寿命；7)免疫系统：提高疫苗接种后的免疫活性，提高癌症定向免疫力，预防致命性脓毒症；8)肝脏：预防肝纤维化与癌变等。亚精胺产生生理活性的主要机理表现为：亚精胺为多阳离子(-NH3+)脂肪胺，在生理pH条件下以多质子化形式存在，具有很强的生物活性，含有酸性残基的核酸、磷脂、酸性蛋白质、含羧基或硫酸盐的果胶多糖以及具有相似结构的神经递质和激素(如多巴胺、肾上腺素、血清素、甲状腺激素等)，都有可能成为亚精胺结合的目标。在与核酸结合方面，大多数多胺在细胞内以多胺-RNA复合物的形式存在，亚精胺的主要作用与RNA的结构变化与翻译有关，如通过影响mRNA、tRNA和rRNA的二级结构来 影响蛋白质合成的各个阶段。亚精胺还能在双螺旋DNA链之前形成稳定的桥，降低ROS或其他DNA损伤剂的可及性，保护DNA免受热变性与X射线辐射。在与蛋白结合方面，亚精胺能够与大量负电荷蛋白结合，改变蛋白质空间构象，从而影响其生理功能。例如蛋白激酶/磷酸酶(多种信号传导途径的重要环节)、参与组蛋白甲基化和乙酰化的酶(通过改变表观遗传影响基因的表达)、离子通道受体(如AMPA、AMDA受体)等等。
目前，以亚精胺等内源性多胺作为透明质酸凝胶交联剂的报道较少，也几乎没有关于内源性多胺与透明质酸的交联反应条件以及交联水凝胶的性能的报道，鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种凝胶材料及其制备方法与应用。本发明的制备方法可控制内源性多胺与透明质酸的交联位点，通过控制交联反应位点，影响制得的交联透明质酸凝胶的各项性能参数，并控制亚精胺等内源性多胺在透明质酸凝胶降解过程中的释放速度，持续发挥亚精胺等内源性多胺的生理活性作用。
本发明提供的技术方案如下：
在一个方面，本发明提供了一种凝胶材料，所述凝胶材料主要由内源性多胺与透明质酸交联得到，所述内源性多胺包括精胺(四氨基化合物)和/或亚精胺(三氨基化合物)；所述内源性多胺与所述透明质酸的交联包含双位点交联、三位点交联或四位点交联。特别地，本发明首次实现了将内源性多胺与透明质酸以三位点或四位点进行交联形成多位点交联的活性星形网络结构。
本发明选择了内源性多胺的精胺或亚精胺作为多位点交联剂，形成致密的网络结构，增加了如此形成的酰胺键交联透明质酸凝胶的热稳定性。本发明没有选择使用腐胺等内源二胺的原因在于，腐胺等二胺具有较高毒 性，且交联位点与赖氨酸、精氨酸等氨基酸相同，不具备实现多位点交联的可能性。
在一个实施方案中，本发明提供了一种凝胶材料，所述凝胶材料由内源性多胺与透明质酸交联得到，所述内源性多胺包括精胺和/或亚精胺；所述内源性多胺与所述透明质酸的交联包含双位点交联、三位点交联或四位点交联。
进一步的，所述的双位点交联得到的凝胶中氨基残留量占比低于20％；优选的，所述的双位点交联中交联得到的凝胶中氨基残留量占比低于15％；更优选的，所述的双位点中交联得到的凝胶中氨基残留量占比低于10％。
进一步的，所述的三位点交联或四位点交联得到的凝胶中氨基和亚氨基残留量占比均低于20％。
进一步的，所述的三位点交联或四位点交联中交联得到的凝胶中氨基残留量占比低于15％；优选的，所述的三位点交联或四位点交联中交联得到的凝胶中氨基残留量占比低于10％。
进一步的，所述的三位点交联或四位点交联中交联得到的凝胶中亚氨基残留量占比低于15％；优选的，所述的三位点交联或四位点交联中交联得到的凝胶中亚氨基残留量占比低于10％。
进一步的，所述内源性多胺与透明质酸交联反应中双位点交联、三位点交联或四位点交联的交联反应效率高于75％；优选的，所述的交联反应效率高于80％；更优选的，所述的交联反应效率高于85％。
进一步的，所述的交联得到的凝胶的弹性模量损失率(G’损失率)低于22％；优选的，弹性模量损失率(G’损失率)低于15％；更优选的，弹性模量损失率(G’损失率)低于10％。
在另一个方面，本发明提供了一种凝胶材料的制备方法，所述方法包括以下步骤：
调节透明质酸与内源性多胺的混合溶液的pH＝4.50～6.50，加入活化剂，使所述透明质酸和所述内源性多胺发生双位点、三位点或四位点交联反应，得到所述凝胶材料；
其中，所述内源性多胺包括精胺和/或亚精胺。
亚精胺与精胺中存在氨基基团与亚氨基基团；其中，亚精胺含有一个亚氨基与两个氨基，精胺含有两个氨基与两个亚氨基。本发明发现可通过调节透明质酸与内源性多胺溶解后的混合溶液的pH值控制精胺或亚精胺与透明质酸的反应位点，可以获得双位点交联的水凝胶，以及三位点或四位点交联的活性星形网络凝胶。在反应过程中，可通过溶液pH调节精胺或亚精胺的氨基位点反应(即双位点反应)或者使得氨基、亚氨基共同反应(即多位点反应)。本发明所述的双位点交联是指透明质酸与精胺或亚精胺交联时，精胺或亚精胺中氨基基团为主要反应位点，特别的，所述的双位点交联得到的凝胶中氨基残留量占比低于20％；本发明所述的三位点或四位点交联是指透明质酸与精胺或亚精胺交联时，精胺或亚精胺中氨基和亚氨基共同作为反应位点，特别的，所述的三位点交联或四位点交联得到的凝胶中氨基和亚氨基残留量占比均低于20％。
本发明发现将溶液pH值控制在5.00～5.49之间可以提高亚氨基的反应活性，相对稳定地实现三位点或四位点反应；将溶液pH值控制在4.50～4.99或5.50～6.50之间，可以提供亚氨基反应活性，相对稳定地实现多位点共同参与反应，制备活性星形网络凝胶。因此本发明首次获得了以亚氨基为交联位点的透明质酸水凝胶，并且发现不同交联位点的水凝胶在相同交联度的情况下可获得不同的凝胶性能。
在本发明中，控制混合溶液的pH在5.00～5.49之间可相对稳定地实现三位点或四位点反应，所述pH包括但不限于5.00、5.10、5.20、5.30、5.40或5.49。控制混合溶液的pH在4.50～4.99或5.50～6.50之间可相对稳定地实现双位点交联反应，所述pH包括但不限于4.50、4.60、4.70、4.80、4.90、 4.99、5.50、5.60、5.70、5.80、5.90、6.00、6.10、6.20、6.30、6.40或6.50。在本发明限定的pH之外，实验结果并不理想，本发明的pH值范围为经过大量实验摸索得出的适合本发明方法的pH条件。
本发明方法为内源性多胺(精胺与亚精胺)多位点活性反应技术(Spermidine/Spermine Multisite Active Reaction Technology，简称SMART)。本发明方法可控制内源性多胺与透明质酸的交联位点，通过控制交联反应位点，进而影响交联透明质酸凝胶的各项性能参数。此外，还可以控制亚精胺等内源性多胺在透明质酸凝胶降解过程中的释放速度，持续发挥亚精胺等内源性多胺的生理活性作用。
在一个实施方案中，在所述双位点交联反应、三位点交联反应或四位点交联反应的过程中加入活化剂；
优选地，所述活化剂包括水溶性碳二亚胺、三苯基膦与溴化物形成的溴化鏻盐、碳鎓盐和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)中的一种或多种。
在一个实施方案中，所述水溶性碳二亚胺活化剂包括1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)、1-(3-二甲基氨基丙基)-3-乙基-碳二亚胺、1-环己基-3-(2-吗啉乙基)碳二亚胺、1,3-二[二(甲氧甲基)甲基]碳二亚胺等或其盐以及其中一种或几种的混合物。
在一个实施方案中，所述三苯基膦与溴化物形成的溴化鏻盐包括三苯基膦与四溴化碳形成的鏻盐、三苯基膦与N-溴代琥珀酰亚胺形成的鏻盐等。所述溴化鏻盐通过已知方法，由三苯基膦与溴化物在二氯甲烷中获得所需鏻盐。
在一个实施方案中，所述碳鎓盐包括O-(7-氮杂苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐(HATU)、O-(苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐(HBTU)、O-(5-氯苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐(HCTU)、O-(苯并三氮唑-1-基)-二(二甲氨基)碳鎓四氟硼酸盐(TBTU)、 O-(N-丁二酰亚胺基)-二(二甲氨基)碳鎓四氟硼酸盐(TSTU)、2-(5-降冰片烯-2,3-二甲酰亚胺基)-1,1,3,3-四甲基脲四氟硼酸盐(TNTU)中的一种或几种的混合物。
在所述活化剂中，当使用水溶性碳二亚胺活化剂时，需要同时与助剂联合使用以提高交联反应效率。优选地，所述助剂包括N-羟基琥珀酰亚胺(NHS)、磺酸化N-羟基琥珀酰亚胺(Sulfo-NHS)、叔丁醇、1-羟基苯并三唑(HOBt)中的任一种或多种；更优选地，所述助剂的加入量为所述碳二亚胺质量的10～30％，包括但不限于10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％和30％。水溶性碳二亚胺与助剂联合使用使得交联反应效率可达到70％左右。
在三苯基膦与溴化物形成的溴化鏻盐、碳鎓盐和4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐这三类活化剂中，碳鎓盐的交联效率最高，为80～95％，其中采用HATU催化剂时，交联效率高达95％；溴化鏻盐与DMTMM的交联效率次之，交联效率在70～85％范围内。
在一个优选的方案中，使用碳鎓盐的HATU作为反应活化剂。HATU的分子式结构如下(1)所示：
Figure PCTCN2021129743-appb-000001
HATU参与酰胺缩合反应的机理如下所示：
Figure PCTCN2021129743-appb-000002
所述交联反应中，当进行双位点交联时(精胺或亚精胺的主要反应位点为氨基)，所述活化剂的加入量为所述内源性多胺物质的量的200～280％；包括但不限于210％、220％、230％、240％、250％、260％、270％和280％；当进行三位点和/或四位点交联时(亚氨基位点也参与反应)，所述活化剂的加入量为所述内源性多胺物质的量的300～550％；包括但不限于320％、350％、370％、390％、400％、420％、450％、470％、500％、520％和550％
优选地，当所述内源性多胺为精胺时，所述活化剂的加入量为所述精胺物质的量的400～550％，当所述内源性多胺为亚精胺时，所述活化剂的加入量为所述亚精胺物质的量的300～400％。
活化剂的加入量与交联剂精胺或亚精胺的氨基交联位点相关，以上活化剂使用时，每一分子的活化剂可以活化一个羧基，并与一个氨基(或亚氨基)进行酰胺偶联反应。
在一个实施方案中，所述交联反应中，当所述内源性多胺为精胺时，所述精胺的加入量占所述透明质酸质量的0.3～35％，包括但不限于0.4％、0.5％、0.8％、1％、3％、5％、8％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％和35％；当所述内源性多胺为亚精胺时，所述亚精胺加入量占所述透明质酸质量的0.5～40％；包括但不限于0.5％、0.8％、1％、3％、 5％、8％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、34％、38％和40％。
进一步地，所述反应体系水溶液中透明质酸的质量浓度为10～150mg/mL，包括例如但不限于10、20、30、40、50、60、70、80、90、100、110、120、130、140、150mg/mL。
进一步地，在最终获得的交联透明质酸水凝胶中，透明质酸的质量浓度为1-50mg/mL，包括但不限于1、3、5、10、15、20、25、30、35、40、45、50mg/mL。
在一个实施方案中，所述透明质酸的分子量为100KDa(千道尔顿)至3000KDa；优选地，所述透明质酸为由微生物发酵法制备的透明质酸。
在一个实施方案中，所述交联反应中，当所述活化剂包括碳鎓盐和/或三苯基膦与溴化物形成的溴化鏻盐时，所述交联反应的温度为10～60℃，交联反应的时间为14～24h；
当所述活化剂包括水溶性碳二亚胺和/或4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐时，所述交联反应的温度为40～60℃，交联反应的时间为14～24h。
在反应过程中，可通过不同的反应温度以及活化剂与精胺或亚精胺的配比以控制双位点交联凝胶以及三位点或四位点交联的活性星形网络凝胶。由于亚氨基受空间位阻效应的影响，亚氨基的反应活性低于伯氨基，因此在仅有伯氨基参与的双位点反应时，仅需要较低的温度和较低的活化剂加入量即可完成交联反应；然而需要亚氨基参与反应时，为增加亚氨基的反应活性，需要提高反应温度，并延长反应时间以提高亚氨基的交联反应效率。
亚精胺与透明质酸、精胺与透明质酸的交联反应如下所示，其中(3)为透明质酸与精胺(Spermine)交联反应(4位点星形网络)。(4)为透明质酸与亚精胺(Spermidine)交联反应(3位点星形网络)。
Figure PCTCN2021129743-appb-000003
本发明采用SMART多位点活性反应技术制得的透明质酸凝胶，充分利用精胺或亚精胺分子内氨基与亚氨基的不同活性，通过控制反应条件，例如pH、温度、活性剂的类型与添加量中的一种或多种，可分别获得双位点交联凝胶，以及三位点或四位点交联的活性星形网络凝胶。
在一个实施方案中，所述凝胶材料还经过洗脱剂洗脱、粉碎、干燥处理；
优选地，所述洗脱剂为有机溶剂；更优选地，所述有机溶剂为可溶性醇或可溶性酮；更优选地，所述有机溶剂为乙醇或丙酮；
进一步地，粉碎时反应体系中凝胶与洗脱剂的体积比为1：1～5；
进一步地，粉碎后所述凝胶的粒径为10～500μm。
在一个实施方案中，所述制备方法方法还包括对干燥后的凝胶材料进行复溶、湿热灭菌；
优选地，所述复溶的溶液为磷酸盐缓冲溶液；更优选地，所述磷酸盐缓冲溶液中磷酸缓冲盐的质量浓度为5～40mg/mL；
进一步地，所述湿热灭菌的温度为120～130℃，湿热灭菌的时间优选为15～45min。
在一个具体的实施方案中，采用有机溶剂对所述凝胶进行洗脱并粉碎凝胶颗粒，可反复多次洗涤去除残留的活化剂。所述干燥可采用真空干燥，干燥可去除有机溶剂。将所述凝胶粉碎成较低的粒径可保证多次清洗过程中残留活化剂的溶出效率。
在一个具体的实施方案中，将真空干燥后的凝胶颗粒用磷酸盐缓冲溶液复溶，灌装在预灌封注射器中并进行湿热灭菌，获得终产品凝胶。所述磷酸盐缓冲溶液的pH值范围为6.8～7.6。终产品透明质酸凝胶的浓度为1～35mg/mL。
在一个具体的实施方案中，所述方法包括以下步骤：
(a)直接将透明质酸与内源性多胺溶解水中，调节溶液pH值；
(b)加入活化剂完成亚精胺和/或精胺与透明质酸的交联反应；
(c)加入有机洗脱剂并粉碎凝胶颗粒，多次洗涤去除残留活化剂，并通过真空干燥去除有机溶剂；
(d)将真空干燥后的凝胶颗粒用磷酸盐缓冲溶液复溶，灌装在预灌封注射器中并进行湿热灭菌，获得终产品凝胶。
本发明方法制备的透明质酸凝胶经湿热灭菌后，弹性模量损失率较低，最低可控制在10％以内，可有效保持凝胶在灭菌前的流变学性能，很大程度上提高了透明质酸凝胶的热稳定性。
本发明在将亚精胺等多胺设计为透明质酸交联剂的同时，又将整个透明质酸凝胶作为整体的亚精胺等多胺的缓释凝胶，即该凝胶在体内自然降解过程中，可以持续原型释放亚精胺等多胺物质。本发明三位点或四位点的活性星形网络凝胶在透明质酸酶的酶促引发下，可稳定释放亚精胺单体，持续发挥亚精胺的生物活性作用。
本发明还提供了上述制备方法获得的凝胶材料。
在另一个方面，本发明还提供了所述凝胶材料或所述制备方法制得的凝胶材料在制备组织填充与修复材料或药物载体方面的用途。例如用于制备药物、医疗美容和化妆品用途的产品中，如软组织填充、软组织修复方面，或者消除面部皮肤皱纹的美容注射剂等。
有益效果：
(1)本发明提供的凝胶材料实现了内源性多胺与透明质酸进行多位点交联，形成更加致密的网络结构；
(2)本发明获得的透明质酸凝胶经湿热灭菌后，弹性模量损失率低(最低可控制在10％以内)，可有效保持凝胶在灭菌前的流变学性能，很大程度上提高了透明质酸凝胶的热稳定性；
(3)本发明的制备方法可以实现控制交联反应位点的数目，进而可影响制备的交联透明质酸凝胶的性能；
(4)本发明可控制亚精胺等内源性多胺在透明质酸凝胶降解过程中的释放速度，持续发挥亚精胺等内源性多胺的生理活性作用；
(5)本发明提高了凝胶在软组织填充、软组织修复、医疗美容等领域方面的易用性，适于推广应用。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍， 显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明提供的亚精胺交联透明质酸的 1H NMR图谱；
图2为本发明提供的亚精胺交联透明质酸的细胞增殖示意图；
图3为本发明提供的热降解条件下亚精胺交联透明质酸凝胶中亚精胺随交联透明质酸释放量曲线；
图4为本发明提供的酶促条件下亚精胺交联透明质酸凝胶中亚精胺随交联透明质酸释放量曲线。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1：HATU催化精胺与透明质酸的双位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入精胺0.03g(摩尔数为0.15mmol)，此时精胺含量占透明质酸质量的1％(透明质酸重复结构单元摩尔数的2％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.20左右，随后加入搅匀，加入0.32mmol HATU，精胺摩尔数的213％，继续搅拌均匀，密封放入25℃恒温箱反应24h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa 的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为158μm，D0.9为196μm。
实施例2：HATU催化亚精胺与透明质酸的三位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的2％)。加入0.48mmol HATU，即亚精胺摩尔数的320％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.40左右，随后加入搅匀，密封放入50℃恒温箱反应14h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为143μm，D0.9为201μm。
实施例3：HATU催化精胺与透明质酸的四位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入精胺0.03g(摩尔数为0.15mmol)，此时精胺含量占透明质酸质量的1％(透明质酸重复结构单元摩尔数的2％)。 加入0.65mmol HATU，精胺摩尔数的433％，搅匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.30左右，继续搅拌均匀，密封放入60℃恒温箱反应14h。反应结束后加入40mL丙酮，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入丙酮200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL丙酮连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为139μm，D0.9为186μm。
实施例4：溴化琥珀酰亚胺三苯基鏻盐催化亚精胺与透明质酸的双位点交联水凝胶制备
将0.2mol三苯基膦与0.2mol N-溴代琥珀酰亚胺于1000mL二氯甲烷中溶解，20-25℃搅拌24h，反应完毕用使用旋转蒸发仪将二氯甲烷去除，得到溴化琥珀酰亚胺三苯基鏻盐，密封后备用。
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的2％)。加入0.32mmol上述步骤获得的溴化琥珀酰亚胺三苯基鏻盐，即亚精胺摩尔数的213％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.00左右，随后加入搅匀，密封放入25℃恒温箱反应24h。反应结束后加入40mL丙酮，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入丙酮200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL丙酮连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全 干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为141μm，D0.9为191μm。
实施例5：溴化琥珀酰亚胺三苯基鏻盐催化亚精胺与透明质酸的三位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的2％)。加入按照实施例4中的方法制备得到的溴化琥珀酰亚胺三苯基鏻盐0.48mmol，即亚精胺摩尔数的320％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.20左右，随后加入搅匀，密封放入40℃恒温箱反应24h。反应结束后加入40mL丙酮，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入丙酮200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL丙酮连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为133μm，D0.9为178μm。
实施例6：DMTMM催化亚精胺与透明质酸的三位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的 2％)。加入DMTMM 0.48mmol，即亚精胺摩尔数的320％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.10左右，随后加入搅匀，密封放入40℃恒温箱反应24h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为144μm，D0.9为206μm。
实施例7：EDC催化精胺与透明质酸的四位点交联水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入精胺0.03g(摩尔数为0.15mmol)，此时精胺含量占透明质酸质量的1％(透明质酸重复结构单元摩尔数的2％)。加入0.65mmol EDC，精胺摩尔数的433％，并加入EDC质量20％NHS，搅匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.45左右，继续搅拌均匀，密封放入60℃恒温箱反应14h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为125μm，D0.9为193μm。
实施例8：HATU催化亚精胺同精胺联用参与透明质酸的多位点交联 水凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中分别加入亚精胺0.022g(摩尔数为0.15mmol)，精胺0.03g(摩尔数为0.15mmol)，此时亚精胺或精胺的含量各占透明质酸重复结构单元摩尔数的2％。加入0.48mmol HATU，即亚精胺摩尔数的320％，再加入0.65mmol HATU，即精胺摩尔数的433％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.00左右，随后加入搅匀，密封放入50℃恒温箱反应14h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为134μm，D0.9为236μm。
实施例9：EDC催化亚精胺与透明质酸的三位点交联
称取6g透明质酸钠(分子量120KDa)，然后加入35mL的纯化水，完全溶解后，此时透明质酸的浓度为150mg/mL，向透明质酸溶液中加入亚精胺2.4g(摩尔数为16.6mmol)，此时亚精胺含量占透明质酸质量的40％。在三位点交联的前提下，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.2左右，随后加入搅匀，加入66.4mmol EDC，即亚精胺摩尔数的400％，同时加入Sulfo-NHS 2.4g(EDC质量的20％)继续搅拌均匀，密封放入60℃鼓风干燥箱反应24h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟， 持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共20mL，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为145μm，D0.9为213μm。
实施例10：HATU催化精胺与透明质酸的四位点交联水凝胶制备
称取0.5g透明质酸钠(分子量2800KDa)，然后加入38.5mL的纯化水，完全溶解后，此时透明质酸的浓度为13.3mg/mL，向透明质酸溶液中加入精胺1.5mg(摩尔数为7.4μmol)，此时精胺含量占透明质酸质量的0.3％。在四位点交联的前提下，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.2左右，随后加入搅匀，加入29.6μmol HATU，即精胺摩尔数的400％，继续搅拌均匀，密封放入30℃鼓风干燥箱反应24h。反应结束后加入40mL丙酮，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入丙酮200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL丙酮连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶0.4g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共400mL，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为156μm，D0.9为231μm。
实施例11：HATU催化亚精胺与透明质酸的三位点交联水凝胶制备
称取0.5g透明质酸钠(分子量2800KDa)，然后加入38.5mL的纯化水，完全溶解后，此时透明质酸的浓度为13.3mg/mL，向透明质酸溶液中加入亚精胺2.5mg(摩尔数为17.2μmol)，此时亚精胺含量占透明质酸质量的0.5％。在三位点交联的前提下，用6mol/L的盐酸溶液将透明质酸溶 液的pH值调节至5.2左右，随后加入搅匀，加入51.6μmol HATU，即亚精胺摩尔数的300％，继续搅拌均匀，密封放入30℃鼓风干燥箱反应24h。反应结束后加入40mL丙酮，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入丙酮200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL丙酮连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶0.4g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共200mL，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为125μm，D0.9为201μm。
实施例12：EDC催化精胺与透明质酸的四位点交联
称取6g透明质酸钠(分子量120KDa)，然后加入35mL的纯化水，完全溶解后，此时透明质酸的浓度为150mg/mL，向透明质酸溶液中加入精胺2.1g(摩尔数为10.4mmol)，此时精胺含量占透明质酸质量的35％。在四位点交联的前提下，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.2左右，随后加入搅匀，加入57.2mmol EDC，即精胺摩尔数的550％，同时加入HOBt 2.0g(EDC质量的20％)继续搅拌均匀，密封放入60℃鼓风干燥箱反应24h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共20mL，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为148μm，D0.9为197μm。
实施例13：HATU催化亚精胺与透明质酸偏酸条件下的双位点交联水 凝胶制备
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的2％)。加入0.48mmol HATU，即亚精胺摩尔数的320％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至4.60左右，随后加入搅匀，密封放入50℃恒温箱反应14h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为138μm，D0.9为210μm。
对比例1：强酸条件下HATU催化亚精胺与透明质酸交联水凝胶制备称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入亚精胺0.022g(摩尔数为0.15mmol)，此时亚精胺含量占透明质酸质量的0.7％(透明质酸重复结构单元摩尔数的2％)。加入0.48mmol HATU，即亚精胺摩尔数的320％，搅拌均匀，用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至3.20左右，随后加入搅匀，密封放入50℃恒温箱反应14h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀， 将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为143μm，D0.9为201μm。
对比例2：EDC催化精胺与透明质酸进行交联反应
本对比例依照US9907739B2中公开的方法进行凝胶制备。
称取3.0g透明质酸钠(分子量900KDa，含透明质酸重复结构单元7.4mmol)，然后加入98mL的纯化水，完全溶解后，此时透明质酸的浓度为30mg/mL，向透明质酸溶液中加入精胺0.03g(摩尔数为0.15mmol)，此时精胺含量占透明质酸质量的1％(透明质酸重复结构单元摩尔数的2％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.2左右，搅匀，加入7.4mmol EDC，即透明质酸结构单元摩尔数的100％，精胺摩尔数的49倍，同时加入EDC质量20％的HOBt，继续搅拌均匀，密封放入25℃恒温箱反应24h。反应结束后加入40mL无水乙醇，采用IKA T25高剪切分散机对凝胶颗粒进行粉碎，粉碎转速为10000转/分钟，持续5min。粉碎完成后继续加入无水乙醇200mL使凝胶完全脱水。分离沉淀，将沉淀用200mL无水乙醇连续清洗5次，再将沉淀放入真空烘箱后，以-0.09MPa的真空度40℃真空干燥24h。完全干燥后，取干凝胶1.0g，加入浓度为10mg/mL的pH7.0磷酸盐缓冲液共50mL，配得透明质酸浓度为20mg/mL的水凝胶，待凝胶完全溶胀后，将凝胶灌装至预灌封注射器中，以121℃、15min的条件进行湿热灭菌，获得终产品凝胶。凝胶粒径D0.5为155μm，D0.9为221μm。
实施例14：精胺或亚精胺交联效率检测
将实施例1-8与对比例1-2中获得的透明质酸凝胶中各取出2.0ml，每 份凝胶分别加无水乙醇50ml，搅拌30min至凝胶变为白色沉淀。将沉淀分离，-0.09MPa的真空度40℃真空干燥24h去除乙醇。取出凝胶，分别加入0.5mol/L的硫酸溶液10mL，于90℃处理2h后，分别用6mol/L氢氧化钠溶液中和至pH＝7。溶液放入真空干燥箱中，-0.09MPa的真空度60℃真空干燥24h去除多余水分。以10mg/mL的浓度溶解于氘代水中，采用400M核磁共振谱仪进行1H谱扫描。
通过对 1H NMR图谱中的关键特征峰进行积分，通过积分峰面积获得亚精胺与透明质酸的实际摩尔比，通过实际摩尔比与理论摩尔比(按照实际投料计算)进行比值，获得交联效率。其中，亚精胺中选择的特征H原子为氨基或亚氨基的α-H，其中氢原子数共8个，精胺中选择的特征H原子为氨基或亚氨基的α-H，其中H原子数共12个，精胺与亚精胺的H原子的积分范围相同，为2.5-2.85ppm；透明质酸中选择的特征H原子为乙酰氨基中羰基的α-H，其中H原子数共3个，积分范围为1.85-2.05ppm；积分时，设定透明质酸中的羰基α-H的积分峰面积为3，即可得到亚精胺或精胺中氨基与亚氨基α-H的积分峰面积。计算公式如下：
亚精胺交联实际摩尔比＝亚精胺中氨基与亚氨基α-H积分峰面积/8；
精胺实际交联摩尔比＝精胺中氨基与亚氨基α-H积分峰面积/12；
交联效率＝(实际交联摩尔比/理论交联摩尔比)×100％。
实施例1-8的交联效率如表1所示，核磁谱图如图1所示：
表1.实际交联效率
实施例 理论交联摩尔比 实际交联摩尔比 交联效率
实施例1 2.0％ 1.7％ 85％
实施例2 2.0％ 1.9％ 95％
实施例3 2.0％ 1.9％ 95％
实施例4 2.0％ 1.5％ 75％
实施例5 2.0％ 1.7％ 85％
实施例6 2.0％ 1.6％ 80％
实施例7 2.0％ 1.5％ 75％
实施例8 4.0％ 3.9％ 97％
对比例1 2.0％ 0.9％ 45％
对比例2 2.0％ 1.0％ 50％
由表1的数据中进行分析，发现当双位点交联时，交联效率低于多位点交联。进行双位点交联的实施例1、4与实施例2、3、5对比时发现，双位点交联的效率普遍低于多位点交联，可能原因在于活化剂用量、pH值与温度三个条件的综合影响导致了双位点交联时交联效率较低；在同样进行多位点交联时，各活化剂进行活化的效率大小顺序为：HATU>三苯基鏻盐>DMTMM>EDC+助剂。其中，HATU活化效率可高达近95％，而EDC+NHS的活化效率仅75％。当相同条件下，精胺与亚精胺的交联效率接近，精胺的效率略低，可能由于精胺中的亚氨基含量较高所致，因亚氨基空间位阻较大，反应活性不及伯氨基。另外，通过对对比例的分析，在多位点pH值范围以外时，氨基与亚氨基的反应活性被抑制，因此交联效率较低；低于多位点交联的试验温度时，即使按照多位点交联的条件进行反应，最终得到的交联效率也与双位点交联的相近，且反应效率更低。
实施例15：亚精胺交联透明质酸凝胶对细胞增殖的影响
将L-929细胞种植在细胞培养基中，加入1％的盘尼西林-链霉素溶液以及10％的胎牛血清溶液。L-929细胞在潮湿的含有5％二氧化碳的细胞培养箱中，37℃下恒温孵化3天。然后，再将实施例2中所得的亚精胺三位点交联透明质酸凝胶转移进入96孔板中，紫外灯固化并灭菌。再次，将L-929细胞培养液加入到放置了亚精胺交联透明质酸凝胶的孔中，并加入1mL含有0.1％EDTA的胰蛋白酶溶液，每孔中的L-929细胞数量为1×10 5个。并继续放置在细胞培养箱中以促进细胞生长。
亚精胺交联的透明质酸凝胶细胞增殖率由MTT法测定。在分别培养了24、48以及72小时之后，向每个孔中加入100μL的MTT水溶液(浓度为 5mg·mL-1)并置于培养箱中继续培养4小时。然后移除MTT溶液，加入150μL的二甲亚砜以溶解甲瓒结晶，采用酶标仪在490nm波长处检测溶液的吸光度以确定该凝胶对L-929的细胞增殖程度。细胞存活率根据下式计算：
细胞存活率(％)＝(As/Ac)×100％；
其中，As为样品溶液在570nm处的吸光度，Ac为空白对照在570nm处的吸光度。
细胞增值结果如图2所示：从图中可以看出亚精胺交联透明质酸凝胶在24、48以及72小时的细胞存活率分别为112％、123％和138％。从这两类凝胶的细胞存活率可以看出，亚精胺交联透明质酸水凝胶没有细胞毒性，其细胞存活率随时间增长而升高的现象表明该水凝胶在一定程度上会起到促进细胞增长的作用。
实施例16：精胺或亚精胺交联透明质酸凝胶的流变学性能检测
将实施例1-13与对比例2中获得的透明质酸凝胶中，分为灭菌前与灭菌后两个类型各取出2.0ml，采用TA DHR-2型平板流变仪检测凝胶的弹性模量(G’)，并计算弹性模量损失率。其中，G’损失率由下式进行计算：
G’损失率＝(灭菌前G’-灭菌后G’)/灭菌前G’；
流变仪参数为，操作间隙：1000mm，装载间隙：45000m，运行温度：37℃，形变量：1％，频率：0.9Hz，运行时间：60s。各凝胶流变学数据如表2所示：
表2.凝胶的流变学数据
实施例 灭菌前G’(Pa) 灭菌后G’(Pa) G’损失率
实施例1 466 411 11.8％
实施例2 533 494 7.3％
实施例3 632 588 7.0％
实施例4 448 388 13.4％
实施例5 505 459 9.1％
实施例6 485 433 10.7％
实施例7 581 512 11.9％
实施例8 1005 958 4.7％
实施例9 843 725 14.0％
实施例10 0.82 0.70 14.6％
实施例11 1.54 1.31 14.9％
实施例12 985 878 10.9％
实施例13 455 403 11.4％
对比例2 405 311 23.2％
G’损失率可认为是凝胶热稳定性的表征指标，G’损失率越低则凝胶的热稳定性越高。由表2中的数据可以看出，以实施例1-7为例，G’损失率与实施例14中的交联效率有一定的关系，其中，在相近交联度与交联效率的情况下(实施例2与3)，亚精胺交联的透明质酸与精胺交联的透明质酸热稳定性相近，但精胺的弹性模量更高且热稳定性更高，可能原因在于精胺的4位点交联形成的交联网络结构更加致密，同样的理论也可用于三位点交联的亚精胺凝胶与双位点交联的亚精胺凝胶，即双位点交联的凝胶热稳定性较三位点交联的更差。四种活化剂对比，凝胶的热稳定性顺序为：HATU>三苯基鏻盐>DMTMM>EDC+助剂，顺序同实施例14。此外，交联透明质酸水凝胶中的透明质酸浓度对凝胶热稳定性也有较大影响，实施例10与11采用了与实施例2与3相同的交联位点，但由于实施例10与11中交联透明质酸水凝胶中透明质酸浓度分别为1mg/mL与2mg/mL，而实施例2与3中相应的浓度为20mg/mL，因此造成了G’相差较大，且G’下降值增大的结果。另外，活化剂加入量也对水凝胶性能影响较大，实施例1与实施例13在其他条件相同的情况下，实施例13中HATU活化剂的加入量较实施例1中减少28.6％(即164.8μmol)，造成实施例13中的G’较实施例1有明显下降且G’下降比例也较实施例1有明显提高的结果。
实施例17：热降解条件下凝胶中亚精胺随透明质酸降解过程中的释放检测
取实施例2中的成品凝胶，每个样品2mL，于西林瓶中密封后，置于鼓风干燥箱125℃条件下进行加速水解，每隔15min取出3个平行样品，冷却至25℃左右后，分别用氢氧化钠将pH调节至12后，并分别加入色谱级丙酮20mL，采用0.2μm滤膜过滤后，将丙酮滤液进样至安捷伦7890B型气相色谱仪中，采用SGE H 2毛细管柱(30m×0.53mm×1.0μm)，色谱条件为：进样口温度220℃，压力40，分离流速30，分流比10:1，分离时间0.8，柱温：100℃保持0.5min，以20℃/min升到180℃保持1min，检测器(FID)：温度250℃，空气流速350mL/min，H2流速35mL/min，尾吹N2流速30mL/min。
总降解时间90min，共设6个检测点，检测结果如图3所示。
本实验的目的在于模拟透明质酸在热降解过程中，由于交联位点的水解而释放亚精胺单体地过程。然而，由于正常生理环境下，交联透明质酸地降解过程时间较长，短则数月，长则超过1年，因此本实验采用了亚精胺交联的透明质酸凝胶在高于100℃的高温条件下加速水解过程中亚精胺的释放量检测实验，可以在较短地时间内观测到透明质酸地降解过程与亚精胺的释放过程。如图3所示，亚精胺的释放随着透明质酸的降解过程缓慢释放，初期释放速度较慢，后期随着交联透明质酸网络的瓦解，亚精胺的释放速度显著加快，累积释放量也有显著提高，说明该凝胶中亚精胺的释放呈现一个先慢后快的过程。本实验同时可以证明本发明中的交联技术为活性反应技术，在交联透明质酸的持续降解过程中可以持续地以原型释放亚精胺单体，也由此可以证明该凝胶在体内可持续释放亚精胺，并发挥亚精胺特定的生理活性作用。
实施例18：酶促条件下凝胶中亚精胺随透明质酸降解过程中的释放检测
取实施例2中的成品凝胶，每个样品2mL，于西林瓶中密封后，每个样品中加入1000u的透明质酸酶进行加速水解，每隔30min取出3个平行样品，冷却至25℃左右后，取出0.5mL上清液，分别用氢氧化钠将pH调节至12后，并分别加入色谱级丙酮20mL，采用0.2μm滤膜过滤后，将丙酮滤液进样至安捷伦7890B型气相色谱仪中，采用SGE H 2毛细管柱(30m×0.53mm×1.0μm)，色谱条件为：进样口温度220℃，压力40，分离流速30，分流比10:1，分离时间0.8，柱温：100℃保持0.5min，以20℃/min升到180℃保持1min，检测器(FID)：温度250℃，空气流速350mL/min，H2流速35mL/min，尾吹N2流速30mL/min。
总降解时间180min，共设6个检测点，检测结果如图4所示。
本实验的目的在于模拟透明质酸在酶促降解过程中，由于凝胶降解而释放亚精胺单体地过程。如图4所示，亚精胺的释放随着透明质酸的降解过程缓慢释放，整个透明质酸凝胶的降解过程中，亚精胺的释放速度几乎不变，说明使用亚精胺交联得到的透明质酸凝胶在酶促降解时，可均匀稳定地释放亚精胺。本实验同时可以证明本发明中的交联技术为活性反应技术，在交联透明质酸的持续降解过程中可以持续的以原型释放亚精胺单体，也由此可以证明该凝胶在体内可持续释放亚精胺，并发挥亚精胺特定的生理活性作用。
实施例19：双位点与多位点交联凝胶中剩余活性位点的检测
取茚三酮约2g，加纯化水100ml使溶解，混匀；避光2-8℃条件保存备用。取醋酸钠54.6g，加1mol/L醋酸溶液20ml溶解后，加水稀释至500ml，备用。分别取亚精胺标准品与精胺标准品，置10ml量瓶中，用纯化水稀释至刻度，摇匀，配制成亚精胺标准使用液500μg/ml，精胺标准使用液500ug/ml；使用时定量稀释至10、50、100、200μg/ml，并使用原500μg/ml标准使用液作为标准曲线。
取实施例1-8，对比例1、2中湿热灭菌前的水凝胶与上述精胺与亚精胺 系列标准溶液，各取1mL，依次加入上述醋酸-醋酸钠缓冲液2.0ml和茚三酮溶液2.0ml，加塞，充分混匀后置70℃水浴加热30min，取出迅速冷却至室温，加纯化水稀释至25ml，混匀；取混合液在565nm波长处长处测定氨基基团衍生物的吸光度，同时在400nm波长处检测亚氨基基团衍生物的吸光度；同法用纯化水进行空白校正。
按下式计算氨基或亚氨基残余量占比(％)：
残余量占比＝(A 0-A i)/A 0×100％
式中，A0为对照液测得的吸光度值，Ai为样品测得的吸光度值。
其中，一个亚精胺分子中含有一个亚氨基基团与两个氨基基团，一个精胺分子中含有两个亚氨基基团与两个氨基基团。
实际检测结果如表3所示：
表3.凝胶中氨基或亚氨基残留量占比
实施例 氨基残留量 亚氨基残留量
实施例1 10.34％ 86.61％
实施例2 7.31％ 8.02％
实施例3 8.53％ 9.45％
实施例4 18.13％ 89.45％
实施例5 10.16％ 12.31％
实施例6 13.35％ 13.43％
实施例7 17.41％ 14.31％
实施例8 6.32％ 7.88％
对比例1 39.25％ 97.13％
对比例2 40.35％ 98.31％
从表3结果可知，实施例1、4为双位点交联，其主要反应基团为氨基，反应效率高达89.66％(100％-氨基残留量，下同)，最低的氨基反应效率也超过80％，亚氨基反应效率不超过15％，因此，该条件下主要为氨基基 团参与的双位点交联反应。实施例2、3、8中，亚精胺或精胺的氨基与亚氨基的反应效率均达到90％以上，实施例5-7中，亚氨基的反应效率也超过85％。说明在该条件下，大部分地亚精胺发生了三位点交联，精胺发生了四位点交联。进一步证实了本发明中三位点与四位点交联的真实性。通过对对比例的检测，发现在本发明中限定的条件之外时，亚精胺或精胺中的亚氨基反应效率降低至不超过10％，且氨基基团的反应效率也有所降低。其中，虽然对比例2总活化剂的加入量为交联剂的49倍，但由于反应条件如pH，反应温度等的不合适，精胺中的亚氨基均未能参与反应，且氨基的反应效率不超过60％。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (14)

  1. 一种凝胶材料，其特征在于，所述凝胶材料由内源性多胺与透明质酸交联得到，所述内源性多胺包括精胺和/或亚精胺；所述内源性多胺与所述透明质酸的交联包含双位点交联、三位点交联或四位点交联；
    所述的双位点交联得到的凝胶中氨基残留量占比低于20％；
    所述的三位点交联或四位点交联得到的凝胶中氨基和亚氨基残留量占比均低于20％。
  2. 权利要求1所述的凝胶材料，其特征在于，所述的双位点交联、三位点交联或四位点交联中交联得到的凝胶中氨基残留量占比低于15％；更进一步的，所述的双位点交联、三位点交联或四位点交联中交联得到的凝胶中氨基残留量占比低于10％。
  3. 权利要求1或2所述的凝胶材料，其特征在于，所述的三位点交联或四位点交联中交联得到的凝胶中亚氨基残留量占比低于15％；更进一步的，所述的三位点交联或四位点交联中交联得到的凝胶中亚氨基残留量占比低于10％。
  4. 权利要求1或2所述的凝胶材料，其特征在于，所述内源性多胺与透明质酸交联反应的交联效率高于75％；
    进一步的，所述内源性多胺与透明质酸交联的交联反应效率高于80％；更进一步的，所述内源性多胺与透明质酸交联的交联反应效率高于85％。
  5. 一种凝胶材料的制备方法，其特征在于，包括以下步骤：
    调节透明质酸与内源性多胺的混合溶液的pH＝4.5～6.5，加入活化剂，使所述透明质酸和所述内源性多胺发生双位点、三位点或四位点交联反应，得到所述凝胶材料；
    所述内源性多胺包括精胺和/或亚精胺；
    其中，当所述活化剂包括碳鎓盐和/或三苯基膦与溴化物形成的溴化鏻盐时，所述交联反应的温度为10～60℃，交联反应的时间为14～24h；
    当所述活化剂包括水溶性碳二亚胺和/或4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐时，所述交联反应的温度为40～60℃，交联反应的时间为14～24h。
  6. 根据权利要求5所述的一种凝胶材料的制备方法，其特征在于，调节透明质酸与内源性多胺的混合溶液的pH＝4.50～4.99或5.50～6.50，加入活化剂碳鎓盐和/或三苯基膦与溴化物形成的溴化鏻盐，交联反应的温度为10～60℃，交联反应的时间为14～24h，使所述透明质酸和所述内源性多胺发生双位点交联反应，得到所述凝胶材料。
  7. 根据权利要求5所述的一种凝胶材料的制备方法，其特征在于，调节透明质酸与内源性多胺的混合溶液的pH＝5.00～5.49，加入活化剂碳鎓盐和/或三苯基膦与溴化物形成的溴化鏻盐，交联反应的温度为10～60℃，交联反应的时间为14～24h，使所述透明质酸和所述内源性多胺发生三位点或四位点交联反应，得到所述凝胶材料；
    或者，调节透明质酸与内源性多胺的混合溶液的pH＝5.00～5.49，加入活化剂水溶性碳二亚胺和/或4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐，交联反应的温度为40～60℃，交联反应的时间为14～24h，使所述透明质酸和所述内源性多胺发生三位点或四位点交联反应，得到所述凝胶材料。
  8. 根据权利要求5-7任意一项所述的制备方法，其特征在于，
    所述水溶性碳二亚胺包括1-乙基-3-(3-二甲基氨基丙基)碳二亚胺、1-(3-二甲基氨基丙基)-3-乙基-碳二亚胺、1-环己基-3-(2-吗啉乙基)碳二亚胺、1,3-二[二(甲氧甲基)甲基]碳二亚胺或其盐中的一种或多种；所述三苯基膦与溴化物形成的溴化鏻盐包括三苯基膦与四溴化碳形成的鏻盐、三苯基膦与N-溴代琥珀酰亚胺形成的鏻盐中的一种或多种；所述碳鎓盐包括O-(7-氮杂苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐、O-(苯并三氮唑-1-基)-二(二 甲氨基)碳鎓六氟磷酸盐、O-(5-氯苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐、O-(苯并三氮唑-1-基)-二(二甲氨基)碳鎓四氟硼酸盐、O-(N-丁二酰亚胺基)-二(二甲氨基)碳鎓四氟硼酸盐、2-(5-降冰片烯-2,3-二甲酰亚胺基)-1,1,3,3-四甲基脲四氟硼酸盐中的一种或多种。
  9. 根据权利要求5-7任意一项所述的制备方法，其特征在于，当使用水溶性碳二亚胺活化剂时，还包括与助剂联用；优选地，所述助剂包括N-羟基琥珀酰亚胺、磺酸化N-羟基琥珀酰亚胺、叔丁醇、1-羟基苯并三唑中的任一种或多种。
  10. 根据权利要求5-7任意一项所述的制备方法，其特征在于，所述交联反应中，当进行双位点交联时，所述活化剂的加入量为所述内源性多胺物质的量的200～280％；
    当进行三位点和/或四位点交联时，所述活化剂的加入量为所述内源性多胺物质的量的300～550％；优选地，当所述内源性多胺为精胺时，所述活化剂的加入量为所述精胺物质的量的400～550％，当所述内源性多胺为亚精胺时，所述活化剂的加入量为所述亚精胺物质的量的300～400％。
  11. 根据权利要求5-7任意一项所述的制备方法，其特征在于，所述交联反应中，当所述内源性多胺为精胺时，所述精胺的加入量占所述透明质酸质量的0.3～35％；当所述内源性多胺为亚精胺时，所述亚精胺加入量占所述透明质酸质量的0.5～40％；
    进一步地，所述反应体系水溶液中透明质酸的质量浓度为10～150mg/mL；
    进一步地，在最终获得的交联透明质酸水凝胶中，透明质酸的质量浓度为1～50mg/mL。
  12. 根据权利要求5-7任意一项所述的制备方法，其特征在于，所述凝胶材料还经过洗脱剂洗脱、粉碎、干燥处理；
    优选地，所述洗脱剂为有机溶剂；更优选地，所述有机溶剂为可溶性醇或可溶性酮；更优选地，所述有机溶剂为乙醇或丙酮；
    进一步地，粉碎时反应体系中凝胶与洗脱剂的体积比为1：1～5；
    进一步地，粉碎后所述凝胶的粒径为10～500μm。
  13. 根据权利要求12所述的制备方法，其特征在于，所述方法还包括对干燥后的凝胶材料进行复溶、湿热灭菌；
    优选地，所述复溶的溶液为磷酸盐缓冲溶液；更优选地，所述磷酸盐缓冲溶液中磷酸缓冲盐的质量浓度为5～40mg/mL；
    进一步地，所述湿热灭菌的温度为120～130℃，湿热灭菌的时间优选为15～45min。
  14. 根据权利要求1-4任意一项所述的凝胶材料或权利要求5-13任意一项所述制备方法制得的凝胶材料在制备组织填充与修复材料或药物载体方面的用途。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103189078A (zh) * 2010-10-29 2013-07-03 汇美迪斯有限公司 含有透明质酸和l-精氨酸的抗粘连剂
WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
WO2014064633A1 (en) * 2012-10-24 2014-05-01 Teoxane Process for preparing a crosslinked gel

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103189078A (zh) * 2010-10-29 2013-07-03 汇美迪斯有限公司 含有透明质酸和l-精氨酸的抗粘连剂
WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
WO2014064633A1 (en) * 2012-10-24 2014-05-01 Teoxane Process for preparing a crosslinked gel

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