WO2024040480A1 - 一种凝胶材料及其制备方法 - Google Patents

一种凝胶材料及其制备方法 Download PDF

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WO2024040480A1
WO2024040480A1 PCT/CN2022/114604 CN2022114604W WO2024040480A1 WO 2024040480 A1 WO2024040480 A1 WO 2024040480A1 CN 2022114604 W CN2022114604 W CN 2022114604W WO 2024040480 A1 WO2024040480 A1 WO 2024040480A1
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gel
hyaluronic acid
cross
spermidine
linking
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PCT/CN2022/114604
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English (en)
French (fr)
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温慧芳
李睿智
娄升凤
张堃
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爱美客技术发展股份有限公司
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Priority to PCT/CN2022/114604 priority Critical patent/WO2024040480A1/zh
Publication of WO2024040480A1 publication Critical patent/WO2024040480A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • 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, and in particular to a gel material and a preparation method thereof.
  • Polymer gel is a three-dimensional network aggregate formed by polymers through chemical bonds or physical forces.
  • the main characteristics of natural polymer-based cryogels include: hydrophilicity, high porosity, high mechanical strength, high stability and minimal non-specific interactions, and the solvent can perform mass transfer through convection, etc.
  • cryogel is usually a highly elastic material with elastic pore walls that can dry and re-expand very quickly without causing any damage to the pore structure. Therefore, these materials can be stored for long periods of time without changing their properties. Resilient even under severe deformation. When exposed to aqueous solutions, they can swell again within seconds of contact with the liquid medium.
  • the type and degree of cross-linking agent, the composition of the gel raw material solution, gel temperature and freezing rate will all affect the physical properties of the formed cryogel.
  • Hyaluronic acid (HA) or sodium hyaluronic acid (HA), also known as hyaluronic acid, is composed of ( ⁇ -1-4)D-glucuronic acid and ( ⁇ -1-3)N -Acetyl-D-glucosamine disaccharide unit is a linear polyanionic mucopolysaccharide composed of repeated connections. It is widely found in tissues such as skin, ligaments, synovial fluid, and the vitreous body of the eye. It can be used as a soft tissue filler to repair wrinkles. and some soft tissue defects and are widely used in medical fields such as surgery and plastic surgery.
  • Hyaluronic acid is a native substance in the body and has good biocompatibility and certain biological activity.
  • exogenous hyaluronic acid is easily degraded by hyaluronidase in the body and cannot be retained in the body for a long time, resulting in shortened treatment effects.
  • multiple injections are needed to achieve the therapeutic effect, so it is necessary to cross-link the hyaluronic acid molecules through chemical cross-linking agents to form a spatial network structure, and prevent the degradation of hyaluronic acid by hyaluronidase and other enzymes through a dense rigid network structure , prolong the residence time of exogenous hyaluronic acid in the body, and improve the therapeutic effect while ensuring biocompatibility.
  • Endogenous polyamines refer to polyamines that can be synthesized or produced during metabolism in the human environment.
  • the main endogenous polyamines include spermine, spermidine, and putrescine.
  • Endogenous polyamine-spermidine is a natural small molecule that is widely present in organisms and has the functions of anti-rejection, inhibiting inflammation, promoting cell autophagy and extending the life span of an individual.
  • the biological activity Molecular spermidine is used in biomedicine, tissue engineering and other fields, and may provide new solutions to the immune rejection faced by artificial tissues or organs during transplantation to achieve better physiological functions and therapeutic effects.
  • the literature (Madeo et al., Science 359, 410, 2018) reported that the endogenous polyamine-spermidine has special physiological effects, including but not limited to regulating circadian rhythm, improving hypertension, protecting cardiovascular system, and preventing Alzheimer's disease , enhance immunity, anti-cancer and even anti-aging, etc.
  • the physiological activity of spermidine is manifested in the following aspects: 1) Kidney: Reduce tension and prevent aging; 2) Heart: Lower blood pressure and prevent arteriosclerosis; 3) Brain: Prevent memory loss, resist Alzheimer's disease, and neuroprotection.
  • spermidine is a polycationic (-NH3+) fatty amine, which exists in a polyprotonated form under physiological pH conditions and has strong biological activity. It is a nucleic acid containing acidic residues.
  • phospholipids may all become spermidine binding targets. .
  • Medical cryogel material has reliable biological safety and good biocompatibility. Its high water content is very similar to the human cell matrix environment, and small molecules such as growth factors can freely enter and exit the porous structure inside the gel. Make the cryogel have good biocompatibility, degradability, cell adhesion and good combination with growth factors, so that cells will adhere to the scaffold material, differentiate and grow to form new tissue areas. It replaces the original damaged area, so it attracts more and more attention.
  • Chinese patent CN110368904A discloses freezing and thawing chitosan and cross-linking agent to obtain a solid porous gas adsorption material. However, the freezing object of this patent is chitosan, and it does not disclose that the cross-linking agent is spermidine.
  • Chinese patent CN111187432A has disclosed a method for preparing a double network hydrogel using spermidine covalent cross-linking agent.
  • This patent is based on UV curing technology and Schiff base reaction, introducing biologically active spermidine covalent Cross-linking agent to obtain a new type of hydrogel with good mechanical properties and biological properties, but the reaction conditions are not cross-linking at low temperatures, and the aldehyde groups introduced during the reaction have potential toxic risks.
  • Chinese patent CN113896915A once disclosed the active cross-linking of spermidine and hyaluronic acid, but this patent is cross-linking at room temperature and cannot form macroporous gel. During the cross-linking process, hyaluronic acid will inevitably be degraded. Resulting in a lower elastic modulus of the final gel.
  • the object of the present invention is to provide a gel material by selecting spermine or spermidine from endogenous polyamines as the cross-linking agent, and by controlling the cross-linking degree, concentration, reaction temperature, reaction time, thawing time, pH and activator type can control various physical and chemical properties of the prepared cross-linked hyaluronic acid gel.
  • the first aspect of the present invention provides a gel material, which is pre-crosslinked by endogenous polyamines and hyaluronic acid substances under the action of an activator at 20-40°C for 30-60 minutes. Then freeze cross-linking is performed, the frozen cross-linked product is thawed, and the freeze-thaw process is repeated 0 to 3 times to obtain the gel;
  • the gel material is freeze-crosslinked by endogenous polyamines and hyaluronic acid substances under the action of an activator, and the frozen-crosslinked product is not frozen after being thawed. Freeze again to produce gel directly.
  • the pore diameter of the gel material is between 50 and 660 ⁇ m, including but not limited to 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 95 ⁇ m, 100 ⁇ m, 105 ⁇ m, 110 ⁇ m, 115 ⁇ m, 120 ⁇ m, 125 ⁇ m, 130 ⁇ m, 135 ⁇ m, 140 ⁇ m, 145 ⁇ m, 150 ⁇ m, 155 ⁇ m, 160 ⁇ m, 165 ⁇ m, 170 ⁇ m, 175 ⁇ m, 180 ⁇ m, 185 ⁇ m, 190 ⁇ m, 195 ⁇ m, 200 ⁇ m, 205 ⁇ m, 210 ⁇ m, 215 ⁇ m, 220 ⁇ m, 225 ⁇ m, 230 ⁇ m, 235 ⁇ m, 240 ⁇ m, 245 ⁇ m, 250 ⁇ m, 255 ⁇ m, 260 ⁇ m, 265 ⁇ m, 270 ⁇ m, 275 ⁇ m, 280 ⁇ m
  • the porosity of the gel material is above 90%
  • Hyaluronic acid itself is considered a non-cured polysaccharide.
  • Hyaluronic acid gels are usually prepared through chemical modification or chemical cross-linking. However, there may be safety risks caused by chemical cross-linking agent residues in the gel, which is extremely serious. The application of hyaluronic acid gel is limited. Therefore, the preparation of a hyaluronic acid hydrogel with good biocompatibility, non-toxic cross-linking agent and good mechanical properties has broad application prospects.
  • the present invention uses endogenous polyamines As a cross-linking agent, the safety risk caused by the residual chemical cross-linking agent of hyaluronic acid gel is reduced, and hyaluronic acid gel is prepared by freezing cross-linking method.
  • endogenous polyamines include spermine (tetraamino compound) and/or spermidine (triamino compound);
  • the activator includes one of water-soluble carbodiimide, carbonium salt and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride. species or species;
  • the hyaluronic acid-based substances include hyaluronic acid and hyaluronic acid salts
  • the hyaluronic acid salts include potassium hyaluronate and sodium hyaluronate.
  • the cross-linking reaction efficiency of the endogenous polyamine and hyaluronic acid is higher than 85%; preferably, the cross-linking reaction efficiency is higher than 90%; more preferably, the cross-linking reaction efficiency is higher than 85%. higher than 98%;
  • the elastic modulus loss rate (G' loss rate) of the gel obtained by cross-linking is less than 12%; preferably, the elastic modulus loss rate (G' loss rate) is less than 8%; more preferably , the elastic modulus loss rate (G' loss rate) is less than 5%.
  • a second aspect of the present invention also provides a preparation method of gel material, including the following steps:
  • Adjust the pH of the mixed solution of hyaluronic acid substances and endogenous polyamines add an activator, pre-crosslink at 20 to 40°C for 30 to 60 minutes, then perform freeze crosslinking, and freeze the crosslinked product. Thaw, and then repeat the freezing-thawing process 0 to 3 times to obtain the gel material.
  • Pre-cross-linking is required before freezing to ensure that the single-site cross-linking of arginine or spermidine is completed before freezing. That is, 1 of the 4 amino groups in arginine participates in the reaction, and the reaction efficiency is about 11-25%. One of the three amino groups in ammonia participates in the reaction, and the reaction efficiency is about 11-33%.
  • one end of the cross-linked arginine or spermidine molecules on the molecular chain of the hyaluronic acid-based substance fixed by ice crystals after freezing is also fixed, bringing spermine or spermidine closer to the transparent
  • the distance between the molecular chains of hyaluronic acid substances increases the collision probability between the amino group at the other end of spermine or spermidine and the carboxyl group of the adjacent hyaluronic acid substance molecular chain, thereby improving the reaction efficiency and forming a porosity of more than 90%.
  • the gel that continuously interpenetrates the porous structure can also effectively monitor the residual amount of spermidine.
  • the endogenous polyamines include spermine and/or spermidine;
  • the activator includes water-soluble carbodiimide, carbonium salt and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) one or more of;
  • DTMM 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride
  • the hyaluronic acid substances include hyaluronic acid and hyaluronic acid salts
  • the hyaluronic acid salts include potassium hyaluronate and sodium hyaluronate
  • the solvent of the mixed solution is selected from a mixture of one or more of water, soluble alcohol, soluble ketone, DMF, DMA, and DMSO; the preferred solvent is water.
  • the soluble alcohol includes methanol, ethanol, propanol or isopropyl alcohol;
  • the soluble ketone is acetone
  • the freezing temperature is at least 5°C below the crystallization point of the solvent, more preferably at least 10 to 60°C below the crystallization point of the solvent, including but not limited to at least 10°C, 15°C, 20°C, and 25°C below the crystallization point. , 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C.
  • the molecular weight of the hyaluronic acid material is 80KDa to 3000KDa.
  • the solvent of the mixed solution is water.
  • the pH includes, but is not limited to, 3.50, 4.00, 4.10, 4.15, 4.20, 4.25, 4.30, 4.35, 4.40, 4.45, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50 . 80 , 6.85 or 6.90.
  • the pH of the mixed solution is 3.50 to 5.80.
  • Controlling the pH of the mixed solution between 3.50 and 5.80 can relatively stably regulate the pore size and porosity of the resulting gel.
  • the pore size of the resulting gel is between Between 50 and 280 ⁇ m, the porosity is between 91 and 95%, and the pH includes but is not limited to 3.50, 3.55, 3.60, 3.65, 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, 4.00, 4.05, 4.10, 4.15 . 40 , 5.45, 5.50, 5.55, 5.60, 5.65, 5.70, 5.75 or 5.80.
  • the pH of the mixed solution is 6.00-6.90;
  • controlling the pH of the mixed solution between 6.00 and 6.90 can relatively stably regulate the pore size and porosity of the gel obtained.
  • the pore size of the gel obtained is between 290 and 660 ⁇ m
  • the porosity is between 96 and 96. ⁇ 98%
  • the pH includes but is not limited to 6.00, 6.05, 6.10, 6.15, 6.20, 6.25, 6.30, 6.35, 6.40, 6.45, 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.88 Or 6.90.
  • the pH value range of the present invention is a pH condition suitable for the present invention obtained through extensive experiments.
  • the freezing temperature is between -60°C and -10°C, including but not limited to: -60°C, -59°C, -58°C, and -57°C.
  • the freezing time is 6 to 24h, including but not limited to: 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h , 19h, 20h, 21h, 22h, 23h or 24h.
  • the thawing temperature is 4 to 40°C, including but not limited to: 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C , 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C;
  • the thawing time is 4 to 10h, and the thawing time includes but is not limited to 4h, 5h, 6h, 7h, 8h, 9h or 10h;
  • the water-soluble carbodiimide activator includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-(3-dimethylaminopropyl) )-3-ethyl-carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, 1,3-bis[di(methoxymethyl)methyl]carbodiimide Imines, etc. or their salts and mixtures of one or more thereof;
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • 1-(3-dimethylaminopropyl) )-3-ethyl-carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
  • the carbonium salts include O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazole-1 -Bis(dimethylamino)carbonium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HBTU) HCTU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium tetrafluoroborate (TBTU), O-(N-succinimidyl)-bis(di Methylamino)carbonium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethylurea tetrafluoroborate
  • HATU O-(
  • the activator when the activator is a water-soluble carbodiimide activator, it needs to be used in combination with an auxiliary agent to improve the cross-linking reaction efficiency.
  • the auxiliary agent includes any one of N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (S-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt) or more;
  • the addition amount of the auxiliary agent is 10% to 50% of the mass of the carbodiimide, 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%, 30%, 31%, 32%, 33% , 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50 %.
  • the combined use of water-soluble carbodiimides and auxiliaries enables the cross-linking reaction efficiency to reach about 95%.
  • the added amount of spermine accounts for 1.0% to 62% of the molar amount of the hyaluronic acid substance, including but not limited to 1.0%, 1.5%, and 2.2%. %, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 13%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 32%, 34%, 35%, 36%, 37% ,38%,39%,40%,41%,42%,43%,44%,45%,46%,47%,48%,49%,50%,51%,52%,53%,54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61% and 62%; preferably 1.0 to 15%.
  • the added amount of spermidine accounts for 0.5% to 52% of the molar amount of the hyaluronic acid substance; including but not limited to 0.5%, 0.8%, 1.0%, 1.5%, 2%, 2.5%, 3%, 5%, 6%, 7%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% ,19%,20%,21%,22%,23%,24%,25%,26%,27%,28%,29%,30%,34%,38%,40%,41%,42 %, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51% and 52%; preferably 0.5 to 10%.
  • the mass concentration of hyaluronic acid substances in the mixed solution is 10 to 160 mg/mL, including but not limited to 10, 20, 30, 40, 50, 55, 60, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, 160mg/mL.
  • the added amount of the activator accounts for 0.5% to 150% of the molar amount of hyaluronic acid substances; preferably, it is 1% to 50%.
  • the hyaluronic acid is prepared by microbial fermentation.
  • the third aspect of the present invention also provides an application of the aforementioned gel in the preparation of medical, beauty, and health care products.
  • the medical and beauty products include: soft tissue filling materials, cartilage repair materials, tissue implant materials, coatings for biomaterial implants; tissue engineering scaffold materials, drug sustained release media, drug targeting carriers, and wound dressings.
  • the coating of the biomaterial implant includes breast fillers, catheters, cannulas, bone repairs, cartilage substitutes, micropumps and other drug delivery devices, artificial organs and blood vessels, and mesh for tissue enhancement. coating;
  • the soft tissue filling material includes medical use as filling material or joint lubricant for the face, neck, head, ears, breasts, and joints;
  • the filling material is an intradermal or subcutaneous implantable material used to improve skin quality or fill wrinkles or restore facial or body volume.
  • the wound healing drug is a wound dressing
  • the therapeutically active agent includes a chemotherapeutic agent or a biologically active factor
  • the active factors include anti-inflammatory agents, antibiotics, analgesics, anesthetics, wound healing promoters, cell growth inhibitors, immunostimulators, immunosuppressants and antiviral agents;
  • the health care products include: capsules, tablets, health drinks, etc.
  • the present invention performs a pre-crosslinking reaction before freezing to control the reaction process of the gel, by completing the single-site crosslinking of arginine or spermidine and hyaluronic acid substances before freezing (i.e., in arginine).
  • arginine One of the four amino groups participates in the reaction, and the reaction efficiency is about 11-25%. In Arginine, one of the three amino groups participates in the reaction, and the reaction efficiency is about 11-33%).
  • Single-point cross-linking can be used to freeze the One end of the cross-linked arginine or spermidine molecules on the polymer chain of the hyaluronic acid substance that is later fixed by ice crystals is also fixed, bringing the spermine or spermidine and the hyaluronic acid substance polymer chain closer together. distance, thereby increasing the collision probability between the amino group at the other end of spermine or spermidine and the carboxyl group of the adjacent hyaluronic acid molecular chain, improving the reaction efficiency, thereby forming a continuous interpenetrating porous structure with a porosity of more than 90%.
  • the overall reaction efficiency of the cryogel can be as high as over 95%, which greatly reduces the residual amount of spermine or spermidine, which has a great impact on the safety of the clinical use of the product. Very important meaning. If there is no pre-cross-linking treatment before freezing, or the reaction efficiency is lower than the above range, after freezing, the distance between the arginine or spermidine molecules and the HA molecular chain segment fixed by the ice crystal will be large, and the probability of collision will be extremely low, resulting in cross-linking. Process efficiency is reduced. If the reaction efficiency before freezing exceeds the above range, the chemical cross-linked network has basically been finalized. After freezing, ice crystals are formed inside the chemical cross-linked network, and good pore-making effects cannot be achieved. Therefore, it is difficult to obtain a gel with larger porosity if the reaction efficiency before freezing is too high or too low.
  • the present invention adjusts the pH value of the mixed solution to prepare porous gels with different pore size gradients.
  • the pH is between 3.50 and 5.80, small pore gels with pore sizes of 50 to 280 ⁇ m are obtained.
  • the pH is between 6.00 and 6.90, a macroporous gel with a pore size of 290 to 660 ⁇ m is obtained.
  • the present invention generates a scaffold with a continuous interpenetrating porous structure by cross-linking reaction between endogenous polyamines and hyaluronic acid substances at low temperature.
  • This unique structure is conducive to material exchange and promotes cell adhesion.
  • growth, and the mechanical properties of the gel scaffold are controllable, the entire process is gentle and controllable, and the yield is high. It has potential research value and broad application prospects in the fields of biomaterials and tissue engineering.
  • Figure 1 is the 1HNMR spectrum of gel A1 provided by the invention.
  • Figure 2 is a curve of room temperature cross-linking time as a function of reaction efficiency/G′ loss rate of gel A1 provided by the present invention
  • Figure 3 is an SEM test picture of the gel A3 provided by the present invention (b is a partial enlarged view of a);
  • Figure 4 is a porosity test chart of gels A1 to A6 provided by the present invention.
  • Figure 5 is a schematic diagram of the water absorption amount and water absorption rate of gels A1 to A6 provided by the present invention.
  • Figure 6 is the degradation curve of gels A1 to A6 provided by the present invention.
  • Figure 7 shows the test results of the effects of gels A1 to A3 provided by the present invention on cell proliferation
  • Figure 8 is an SEM test picture of gels B3(a) and B4(b) provided by the present invention.
  • cross-linking is the process of converting a linear structure polymer with chemical reactivity into a three-dimensional network (body) structure polymer through a chemical reaction. Often used for polymer modification.
  • cross-linking agent refers to an agent that can generate chemical bonds between linear molecules to connect linear molecules to each other to form a network structure, and is used to improve the strength of polymer materials.
  • vial refers to a borosilicate glass or soda-lime glass tube (molded) injection bottle, which is a small bottle sealed with a rubber stopper and an aluminum-plastic combination cap.
  • penicillin was mostly used to contain it, hence the name penicillin bottle.
  • mixed solution refers to a mixed solution of uncrosslinked hyaluronic acid substances dissolved in a solvent and dissolved endogenous polyamines.
  • room temperature refers to 25°C ⁇ 5°C.
  • FIG. 1 The 1HNMR spectrum of cryogel A1 is shown in Figure 1.
  • Figure 1 shows that the chemical shift around 1.8-2.0ppm is the acetyl methyl peak in hyaluronic acid, and the chemical shift around 2.5-2.8ppm is the o-aminomethylene peak on spermine. The existence of the two peaks can prove that spermine Successfully cross-linked with hyaluronic acid.
  • Example 2 HATU-catalyzed preparation of gel A2 of spermine and hyaluronic acid
  • Example 3 EDC-catalyzed preparation of gel A3 of spermidine and hyaluronic acid
  • Example 4 EDC-catalyzed preparation of gel A4 of spermidine and hyaluronic acid
  • Example 5 DMTMM catalyzed preparation of gel A5 of spermine and hyaluronic acid
  • Example 6 DMTMM catalyzed preparation of gel A6 of spermidine and hyaluronic acid
  • Test Example 1 Detection of free spermine and spermidine content
  • spermine and/or spermidine have no chromophore group and no UV and fluorescence absorption, making it difficult to measure using conventional chromatographic methods. Therefore, the determination of spermine or spermidine in samples requires derivatization and analysis; due to the Sulfonyl chloride can react with the active hydrogen on the primary or secondary amine group to remove a molecule of hydrogen chloride to generate a derivative with fluorescence and ultraviolet light. Both fluorescence and ultraviolet detectors can be used for detection, so dansyl chloride was chosen as the derivatization agent.
  • Test solution Take about 1.00g of the sample respectively, accurately weigh the mass, place it in a 100ml volumetric flask, add 0.1g of disodium hydrogen phosphate solid, then dilute to the mark with 0.1mol/L hydrochloric acid solution, shake well. Pass through a 0.45 ⁇ m filter membrane and take the remaining filtrate. Sampling needs to be carried out at different time periods during cross-linking. The sampling times are 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h and 9h respectively.
  • the chromatographic column uses octadecylsilane bonded silica gel as the filler (C18 column, 250mm ⁇ 4.6mm ⁇ 5 ⁇ m); the detection wavelength is 254nm; the column temperature is 35°C; the mobile phase A is 0.01mol/ L ammonium acetate solution, mobile phase B is acetonitrile; the flow rate is 1.0ml/min, and gradient elution is performed according to the program in the table.
  • C is the spermine or spermidine concentration of the test solution calculated after being substituted into the standard curve ( ⁇ g/ml); V is the dilution volume of the test solution (100ml); m is the mass of the sample (g).
  • the optimal freezing time for gel is when the cross-linking time is 0.5h and 1h.
  • Test Example 2 Testing of Spermine or Spermidine Reaction Efficiency
  • the actual molar ratio of spermidine or spermine to hyaluronic acid is obtained by integrating the peak area, and the ratio is calculated by the actual molar ratio and the theoretical molar ratio (calculated according to actual feeding) , to obtain reaction efficiency.
  • the characteristic H atom selected in spermidine is ⁇ -H of amino or imino, in which the number of hydrogen atoms is 8.
  • the characteristic H atom selected in spermidine is ⁇ -H of amino or imino, in which H atom There are 12 numbers in total.
  • the integral range of H atoms of spermine and spermidine is the same, 2.5 to 2.85 ppm; the characteristic H atom selected in hyaluronic acid is ⁇ -H of the carbonyl group in acetamido, in which the number of H atoms is 3. , the integration range is 1.85 ⁇ 2.05ppm; when integrating, set the integrated peak area of the carbonyl ⁇ -H in hyaluronic acid to 3, and the integral of the amino and imino ⁇ -H in spermidine or spermine can be obtained Peak area. Calculated as follows:
  • the actual cross-linking molar ratio of spermine the ⁇ -H integrated peak area of amino and imino groups in spermine/12;
  • Reaction efficiency (actual cross-linking molar ratio/theoretical cross-linking molar ratio) ⁇ 100%.
  • the reaction efficiencies of Examples 1 to 6 are all above 95%.
  • the cross-linking degrees of Example 4 and Comparative Example 1 are both 3.0%, and the reaction efficiency of the obtained gel A4 is significantly higher than that of B1 gel.
  • the cross-linking degrees of Example 1 and Comparative Example 2 are both 4.0%.
  • After freezing cross-linking The reaction efficiency of the obtained gel A1 is significantly higher than that of the B2 gel. The possible reason is the comprehensive influence of several factors such as the type of cross-linking agent, pre-cross-linking process, pH value, freezing and thawing temperature and time.
  • Comparative Example 1 The preparation process of and 2 needs to be carried out under alkaline conditions, and the cross-linked gel is unstable in a peracidic and/or overly alkaline environment and is easily degraded, which is one of the reasons for its low reaction efficiency.
  • the reaction efficiency of Comparative Example 3-4 is lower than that of Example 3-4, and the reaction efficiency of Comparative Example 3-4 is much lower than that of Example 3-4. 4. It is further explained that the reaction efficiency after the pre-cross-linking reaction in Comparative Example 4 is low (7%, not reaching single-point cross-linking), resulting in a greatly reduced efficiency of the cross-linking process after freezing.
  • the gels prepared in Examples 1 to 6 were tested for rheological properties: take 2 ml of each gel before and after sterilization, and test them using a rotational rheometer.
  • the rheometer parameters are: operating gap: 1000mm, loading Gap: 45000m, operating temperature: 37°C, deformation: 1%, frequency: 0.9Hz, operating time: 60s.
  • the rheological data of each gel are shown in Table 2; the G′ (elastic modulus) loss rate is calculated as follows:
  • G′ loss rate (G′ before sterilization – G′ after sterilization)/G′ before sterilization;
  • the G′ loss rate is an important indicator of the thermal stability of the gel.
  • the difference in elastic modulus value G' of gels A1 to A6 obtained in Examples 1 to 6 before and after sterilization is small, and the G' loss rate is within 7.75%, indicating that the elastic modulus value of gels A1 to A6 It has high thermal stability and can resist moist heat sterilization.
  • Example 1 and Comparative Example 2 the degree of cross-linking is the same, and the preparation process is freeze cross-linking.
  • the elastic modulus G′ loss rate of A1 gel before and after sterilization is 6.93%; while the loss rate of B2 gel before and after sterilization The elastic modulus G′ loss rate is 77.78%.
  • the elastic modulus G′ of A4 gel before sterilization is 6.56 times that of B1 gel, and the elastic modulus G′ of A1 gel before sterilization is 5.35 times that of B2 gel; the elasticity of A4 gel after sterilization
  • the modulus G′ is 26.8 times that of B1 gel, and the elastic modulus G′ of A1 gel after sterilization is 22.4 times that of B2 gel.
  • the thermal stability of cross-linked sodium hyaluronate gel prepared with spermine and/or spermidine as cross-linking agents is much higher than that of BDDE cross-linked sodium hyaluronate gel.
  • the possible reason is that BDDE cross-linking Hyaluronic acid gel needs to be prepared under alkaline conditions, and the amide bond of the gel is unstable and easy to break under alkaline conditions, resulting in low reaction efficiency of the resulting gel, and the non-cross-linked hyaluronic acid in the gel
  • the glycosidic bond of acid is easily broken under high temperature conditions, so its elastic modulus is significantly reduced after moist heat sterilization.
  • the elastic modulus G′ loss rate of Comparative Examples 3 to 4 is also significantly higher than that of Examples 3 to 4. This is also due to the high or low pre-crosslinking reaction efficiency of Comparative Examples 3 to 4, resulting in delays in the subsequent freezing process. The cross-linking efficiency is low, resulting in a high loss rate of elastic modulus.
  • Test Example 4 SEM of gel and measurement of pore size
  • the pore structure of the gel was observed using a scanning electron microscope.
  • the freeze-dried frozen gel sponge was cut into small squares with a blade, fixed on the electron microscope stage with conductive glue, sprayed with gold for 60 seconds, and analyzed with a field emission scanning electron microscope.
  • the scanning electron microscope results of the frozen gel sponge A3 are shown in Figure 3.
  • the scanning electron microscope results of Comparative Examples 3 to 4 are shown in Figure 8.
  • Table 3 shows the pore size distribution of gels A1 to A6. It can be seen from the figure that the pore sizes of the gels are between 50 and 660 ⁇ m, while the pore sizes obtained in Comparative Examples 3 and 4 do not exceed 10 ⁇ m. This can be further seen from Figures 3 and 8.
  • the pores of A3 have a loose porous structure with high porosity, while the gel in Figure 8 has a denser surface and a significantly smaller number of pores, which also illustrates that in Before freezing, whether the reaction efficiency is high or low will significantly affect the cross-linking efficiency in the later stage, thereby affecting the formation of the gel pore structure.
  • Example 2 405-500
  • Example 3 180-280
  • Example 4 290-395
  • Example 5 100-165
  • Example 6 510-660 Comparative example 3
  • 0.1-1 Comparative example 4 0.5-9
  • the porosity results are shown in Figure 4.
  • the porosity of the gels A1 to A6 of the present invention are all above 90%.
  • the porosity in Comparative Example 3 is only 13%, and the porosity in Comparative Example 4 is 35%. Since freezing is not carried out within the preferred reaction efficiency range, the porosity of Comparative Example 3 and Comparative Example 4 is both low.
  • Test Example 7 Measurement of Gel Water Absorption Amount and Water Absorption Rate
  • the test results of the water absorption capacity and water absorption rate of the gel are shown in Figure 5.
  • the water absorption capacity of gels A1 to A6 of the present invention within 2 minutes is more than 0.95ml, and the water absorption rate of gels A1 to A6 is more than 0.475ml/min.
  • A6 The water absorption rate can reach 1.395ml/min.
  • Figure 6 shows the degradation curve of gels A1 to A6. It can be seen from the curve that the stability of the gel is strong, and the degradation time is more than 80 minutes; and the degradation time is basically proportional to the elastic modulus of the gel, that is, the gel The greater the elastic modulus, the more difficult it is to degrade and the longer the degradation time.
  • L-929 cells were planted in cell culture medium, and 1% penicillin-streptomycin solution and 10% fetal calf serum solution were added. L-929 cells were incubated in a humid cell culture incubator containing 5% carbon dioxide at a constant temperature of 37°C for 2 days and set aside. Add 1 mL of trypsin solution containing 0.1% EDTA to the L-929 cell culture medium, and seed L929 cells in a 96-well plate at 1 ⁇ 10 4 cells/well, with 100 ⁇ L of solution per well. The gels A1-A3 obtained in Examples 1-3 were respectively transferred to 96-well plates, cured by ultraviolet lamp and sterilized. L-929 cell culture medium was then added to the wells where the gel was placed and continued to be placed in the cell culture incubator to promote cell growth. A blank cell proliferation comparison experiment was performed simultaneously.
  • the gel cell proliferation rate was measured by MTT method. After culturing for 24 hours respectively, 100 ⁇ L of MTT aqueous solution (concentration: 5 mg ⁇ mL-1) was added to each well and placed in the 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 570 nm to determine the degree of cell proliferation of L-929 caused by the gel. Cell viability is calculated according to the following formula:

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Abstract

本发明涉及一种凝胶材料，其凝胶材料由内源性多胺与透明质酸类物质在活化剂的作用下进行预交联-低温冷冻交联，之后进行解冻，再重复冷冻-解冻过程0～3次制得，内源性多胺包括精胺和/或亚精胺，活化剂包括水溶性碳二亚胺、碳鎓盐和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐(DMTMM)，本发明通过将内源性多胺与透明质酸在低温下发生交联反应而生成具有连续互穿多孔结构的支架，这种独特的结构有利于进行物质交换和促进细胞的黏附、生长。通过调控交联度及透质在反应体系中的浓度等可控制凝胶材料的孔径大小、孔隙率，还可调节凝胶支架的力学性能，整个过程温和可控，产率高，在生物材料和组织工程领域具有潜在的研究价值和广泛的应用前景。

Description

一种凝胶材料及其制备方法 技术领域
本发明涉及生物医用材料技术领域，具体涉及一种凝胶材料及其制备方法。
背景技术
高分子凝胶是高分子通过化学键或物理作用力形成的三维网络聚集体。天然高分子基冷冻凝胶主要特征包括：亲水性、高孔隙率、高机械强度、高稳定性和最小的非特异性相互作用、溶剂可以通过对流进行质量传递等。冷冻凝胶和其他大孔材料之间最重要的区别是：冷冻凝胶通常是高弹性材料，具有弹性孔壁，可以非常快速地干燥、再膨胀，但是不会对孔结构造成任何损害。因此，这些材料可以长时间储存而不改变其性能。即使在严重变形的情况下也具有弹性。当它们暴露于水溶液中时，可在与液体介质接触后几秒内再次膨胀。交联剂的类型和程度、凝胶原料溶液的组成，凝胶温度和冷冻速率等均会影响形成的冷冻凝胶的物理性质。
透明质酸(hyalourome acid，HA)或透明质酸钠(hyaluronic acid，简称HA)，又称玻尿酸，它是由(β-1-4)D-葡萄糖醛酸和(β-1-3)N-乙酰基-D-氨基葡萄糖双糖单位重复连接组成的一种线状聚阴离子粘多糖，广泛存在于皮肤、韧带、关节液和眼睛的玻璃体等组织中，可作为软组织充填剂用以修复皱纹以及一些软组织缺陷，在外科手术和整形外科等医学领域中得到广泛应用。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易被透明质酸酶降解，无法在体内长期留存，而导致治疗效果缩短，需要多次注射才能达到治疗效果，故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，通过致密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，在保证生物相容性的同时提高治疗效果。
内源性多胺是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。内源性多胺——亚精胺是一种广泛存在于生物体内的具有抗排异、抑制炎症、促进细胞自噬并可以延长个体寿命的天然小分子，借助仿生化设计思想，将生物活性分子亚精胺应用于生物医药、组织工程等领域，可能为人工组织或器官在移植过程中面临的免疫排斥反应提供新的解决方案，以实现更佳的生理功能和治疗效果。文献(Madeo等.，Science 359，410，2018)报道了内源性多胺——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。
医用冷冻凝胶材料具有可靠的生物安全性以及很好的生物相容性，其较高的含水量与人体细胞基质环境极为相似，而且生长因子等小分子可自由出入凝胶内部的多孔结构，使冷冻凝胶的生物相容性、可降解性、细胞粘附性较好以及与生长因子之间很好的结合性，这样细胞就会粘附在支架材料上分化生长形成新的组织区域来代替原来的受损区域，所以越来越受到人们的关注。中国专利CN110368904A公开了将壳聚糖、交联剂进行冷冻，解冻，得到固态多孔的气体吸附材料。但该专利的冷冻对象为壳聚糖，且没有公开交联剂为亚精胺。中国专利CN111187432A曾公开了一种利用亚精胺共价交联剂的双网络水凝胶的制备方法，该专利基于紫外光固化技术和席夫碱反应，引入具有生物活性的亚精胺共价交联剂，获得具有良好力学性能和生物性能的一种新型水凝胶，但其反应条件不是在低温下交联，且在反应过程中引入的醛基存在潜在的毒性风险。中国专利CN113896915A曾经公开了亚精胺与透明质酸的活性交联，但该专利为常温状态交联，无法形成大孔凝胶，在交联过程中也不可避免的造成透明质酸的降解而导致最终的凝胶弹性模量较低。
目前，以亚精胺等内源性多胺作为透明质酸基冷冻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冷冻交联反应条件以及冷冻凝胶性能的报道，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，通过选用了内源性多胺中的精胺或亚精胺作为交联剂，通过控制交联度、浓度、反应温度、反应时间、解冻时间、pH及活化剂类型等，可调控所制备交联透明质酸凝胶的各项理化性能。
本发明提供的技术方案如下：
本发明第一方面提供一种凝胶材料，所述凝胶材料由内源性多胺与透明质酸类物质在活化剂的作用下，在20～40℃下预交联30～60分钟，然后进行冷冻交联，将冷冻交联后的产物进行解冻，再重复冷冻-解冻过程循环0～3次得到的凝胶；
当重复冷冻-解冻过程循环0次时，所述凝胶材料由内源性多胺与透明质酸类物质在活化剂的作用下进行冷冻交联，将冷冻交联后的产物进行解冻后不再冷冻，直接制得凝胶。
进一步的，所述凝胶材料的孔径为50～660μm之间，包括不限于50μm、55μm、60μm、65μm、70μm、75μm、80μm、85μm、90μm、95μm、100μm、105μm、110μm、115μm、120μm、125μm、130μm、135μm、140μm、145μm、150μm、155μm、160μm、165μm、170μm、175μm、180μm、185μm、190μm、195μm、200μm、205μm、210μm、215μm、220μm、225μm、230μm、235μm、240μm、245μm、250μm、255μm、260μm、265μm、270μm、275μm、280μm、285μm、290μm、295μm、300μm、310μm、315μm、320μm、325μm、330μm、335μm、340μm、345μm、350μm、355μm、360μm、365μm、370μm、375μm、380μm、385μm、390μm、395μm、400μm、410μm、415μm、420μm、425μm、430μm、435μm、440μm、445μm、450μm、455μm、460μm、465μm、470μm、475μm、480μm、485μm、490μm、495μm、500μm、515μm、520μm、525μm、530μm、535μm、540μm、545μm、550μm、555μm、560μm、565μm、570μm、575μm、580μm、585μm、590μm、595μm、600μm、605μm、610μm、615μm、620μm、625μm、630μm、635μm、640μm、645μm、650μm、655μm或660μm。
和/或，所述凝胶材料的孔隙率在90％以上；
透明质酸本身被认为是非凝胶多糖，通常通过化学改性或化学交联的办法制备透明质酸凝胶，但是凝胶中可能会有化学交联剂残余引发的安全风险，这极大的限制了透明质酸凝胶的应用，因此制备一种生物相容性好、无毒性交联剂、机械性能好的透明质酸水凝胶具有广阔的应用前景，本发明采用内源性多胺作为交联剂，降低了透明质酸凝胶化学交联剂残余引发的安全风险，并通过冷冻交联的方法制备透明质酸凝胶，由现有技术可知，在透明质酸凝胶的制备过程中，降低制造温度会导致形成更小的冰晶，从而导致凝胶形成更小的孔隙，冷冻交联能够调整凝胶特定的几何形状、结构和注射后孔隙率。当冷冻速率较高，聚合物溶液的快速冷冻可能导致溶质的过度过冷，这可能导致更小和不规则的晶体形成，制造凝胶的机械性能(如柔韧性)较差，溶胀比较低，可能容易断裂。
进一步的，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)；
优选的，所述活化剂包括水溶性碳二亚胺、碳鎓盐和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐中的一种或多种；
优选的，所述透明质酸类物质包括透明质酸和透明质酸盐，所述透明质酸盐包括透明质酸钾、透明质酸钠。
进一步的，所述内源性多胺与透明质酸的交联反应效率高于85％；优选的，所述的交联反应效率高于90％；更优选的，所述的交联反应效率高于98％；
进一步的，所述的交联所得凝胶的弹性模量损失率(G’损失率)低于12％；优选的，弹性模量损失率(G’损失率)低于8％；更优选的，弹性模量损失率(G’损失率)低于5％。
本发明第二方面还提供一种凝胶材料的制备方法，包括以下步骤：
调节透明质酸类物质与内源性多胺的混合溶液的pH，加入活化剂，在20～40℃下预交联30～60分钟，再进行冷冻交联，将冷冻交联后的产物进行解冻，再重复冷冻-解冻过程0～3次，得到所述凝胶材料。
冷冻之前需要进行预交联，以保证在冷冻前完成精氨或亚精胺的单位点交联，即精氨中4个氨基有1个参与反应，反应效率在11-25％左右，亚精氨中3个氨基有1个参与反应，反应效率在11-33％左右。通过单位点交联，使冷冻后被冰晶固定的透明质酸类物质的分子链上所交联的精氨或亚精胺分子的一端也被固定，拉近了精胺或亚精胺与透明质酸类物质分子链的距离，从而提高了精胺或亚精胺另一端的氨基与邻近透明质酸类物质分子链的羧基碰撞概率，提高了反应效率，进而形成孔隙率高达90％以上的连续互穿多孔结构的凝胶，同时还可实现亚精胺残留量的有效监控。
优选的，所述内源性多胺包括精胺和/或亚精胺；
优选的，所述活化剂包括水溶性碳二亚胺、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；
优选的，所述透明质酸类物质包括透明质酸和透明质酸盐，所述透明质酸盐包括透明质酸钾、透明质酸钠；
优选的，所述混合溶液的溶剂选自：水、可溶性醇、可溶性酮、DMF、DMA、DMSO中的一种或多种的混合；优选溶剂为水。
所述可溶性醇包括甲醇、乙醇、丙醇或异丙醇；
所述可溶性酮为丙酮；
优选的，所述冷冻温度为溶剂结晶点以下的至少5℃，更优选为溶剂结晶点以下的至少10～60℃，包括不限于结晶点以下的至少10℃、15℃、20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃或60℃。
进一步的，所述透明质酸类物质的分子量为80KDa～3000KDa。
在本发明的一些实施例中，所述混合溶液的溶剂为水。
在本发明的一些实施例中，当混合溶液的溶剂为水时，所述混合溶液的pH＝3.50～6.90，
例如，所述pH包括但不限于3.50、4.00、4.10、4.15、4.20、4.25、4.30、4.35、4.40、4.45、4.50、4.60、4.70、4.80、4.90、5.00、5.10、5.20、5.30、5.40、5.50、5.60、5.70、5.80、5.90、6.00、6.10、6.20、6.30、6.32、6.35、6.38、6.40、6.42、6.45、6.50、6.55、6.58、6.60、6.62、6.65、6.68、6.70、6.73、6.75、6.80、6.85或6.90。
优选的，所述混合溶液的pH＝3.50～5.80，控制混合溶液的pH在3.50～5.80之间可相对稳 定地调控所得凝胶的孔径和孔隙率，在此pH范围内所得凝胶的孔径在50～280μm之间，孔隙率在91～95％之间，所述pH包括但不限于3.50、3.55、3.60、3.65、3.70、3.75、3.80、3.85、3.90、3.95、4.00、4.05、4.10、4.15、4.20、4.25、4.30、4.35、4.40、4.45、4.50、4.55、4.60、4.65、4.70、4.75、4.80、4.85、4.90、4.95、5.00、5.05、5.10、5.15、5.20、5.25、5.30、5.35、5.40、5.45、5.50、5.55、5.60、5.65、5.70、5.75或5.80。
更优选的，所述混合溶液的pH＝6.00～6.90；
本发明中，控制混合溶液的pH在6.00～6.90之间可相对稳定地调控所得凝胶的孔径和孔隙率，在此pH范围内所得凝胶的孔径在290～660μm之间，孔隙率在96～98％之间，所述pH包括但不限于6.00、6.05、6.10、6.15、6.20、6.25、6.30、6.35、6.40、6.45、6.50、6.55、6.60、6.65、6.70、6.75、6.80、6.85、6.88或6.90。
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的pH条件。
在本发明的一些实施例中，当混合溶液的溶剂为水时，所述冷冻温度在-60℃～-10℃，包括不限于：-60℃、-59℃、-58℃、-57℃、-56℃、-55℃、-54℃、-53℃、-52℃、-51℃、-50℃、-49℃、-48℃、-47℃、-46℃、-45℃、-44℃、-43℃、-42℃、-41℃、-40℃、-39℃、-38℃、-37℃、-36℃、-35℃、-34℃、-33℃、-32℃、-31℃、-30℃、-29℃、-28℃、-27℃、-26℃、-25℃、-24℃、-23℃、-22℃、-21℃、-20℃、-19℃、-18℃、-17℃、-16℃、-15℃、-14℃、-13℃、-12℃、-11℃或-10℃。
优选的，当混合溶液的溶剂为水时，所述冷冻时间为6～24h，包括不限于：6h、7h、8h、9h、10h、11h、12h、13h、14h、15h、16h、17h、18h、19h、20h、21h、22h、23h或24h。
优选的，当混合溶液的溶剂为水时，所述解冻温度为4～40℃，包括不限于：4℃、5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃或40℃；
优选的，当混合溶液的溶剂为水时，所述解冻时间为4～10h，所述解冻时间包括但不限于4h、5h、6h、7h、8h、9h或10h；
进一步的，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；
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。
在本发明的一些实施例中，当活化剂为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。
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(S-NHS)、叔丁醇、1-羟基苯并三唑(HOBt)中的任一种或多种；
更优选的，所述助剂的加入量为所述碳二亚胺质量的10～50％，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％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％。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95％左右。
进一步的，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类物质摩尔量的1.0～62％，包括但不限于1.0％、1.5％、2.2％、2.5％、3％、4％、5％、6％、7％、8％、9％、10％、13％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、32％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％和62％；优选为1.0～15％。
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类物质摩尔量的0.5～52％；包括但不限于0.5％、0.8％、1.0％、1.5％、2％、2.5％、3％、5％、6％、7％、8％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、34％、38％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％和52％；优选为0.5～10％。
所述混合溶液中透明质酸类物质的质量浓度为10～160mg/mL，包括但不限于10、20、30、40、50、55、60、70、75、80、90、100、110、120、130、140、150、160mg/mL。
所述活化剂加入量占透明质酸类物质摩尔量的0.5～150％；优选为1～50％。
所述透明质酸由微生物发酵法制备。
本发明第三方面还提供一种前述凝胶在制备医疗、美容、保健产品中的应用。
所述医疗、美容产品包括：软组织填充材料、软骨修复材料、组织植入材料、生物材料植入物的涂层；组织工程支架材料、药物缓释媒介、药物靶向载体、创面敷料。
优选的，所述生物材料植入物的涂层包括乳房填充物、导管、插管、骨骼修补物、软骨 替代物、微型泵和其它药物输送装置、人工脏器和血管、组织增强用的网的涂层；
优选的，所述软组织填充材料包括医疗用途的作为面部、颈部、头部、耳部、乳房、关节的填充材料或关节润滑剂；
更优选的，所述填充材料为用于改善皮肤质量或填充皱纹或恢复面部或身体体积的皮内或皮下可植入材料。
优选的，所述伤口愈合药物为伤口敷料；
优选的，所述治疗活性剂包括化学治疗剂或生物学上的活性因子；
更优选的，所述活性因子包括消炎剂，抗生素，止痛药，麻醉剂，伤口愈合促进剂，细胞生长抑制剂，免疫刺激剂，免疫抑制剂和抗病毒药；
所述保健产品包括：胶囊、片剂、保健饮品等。
本发明的有益效果：
1)本发明在冷冻处理前，进行了预交联反应以控制凝胶的反应进程，通过在冷冻前完成精氨或亚精胺与透明质酸类物质的单位点交联(即精氨中4个氨基有1个参与反应，反应效率在11-25％左右，亚精氨中3个氨基有1个参与反应，反应效率在11-33％左右)，利用单位点交联，可以使冷冻后被冰晶固定的透明质酸类物质的高分子链上所交联的精氨或亚精胺分子的一端也被固定，拉近了精胺或亚精胺与透明质酸类物质高分子链的距离，进而提高了精胺或亚精胺另一端的氨基与邻近透明质酸类物质分子链的羧基碰撞概率，提高了反应效率，从而形成孔隙率高达90％以上的连续互穿多孔结构的凝胶；另外，通过预交联反应进程的控制，可以使冷冻凝胶整体反应效率高达95％以上，极大的降低了精胺或亚精胺的残留量，对产品临床使用的安全性具有十分重要的意义。若冷冻前不进行预交联处理，或反应效率低于上述范围，在冷冻后，精氨或亚精胺分子与被冰晶固定的HA分子链段距离较大，碰撞概率极低而导致交联过程效率降低。若冷冻前反应效率超过上述范围，则化学交联网络已基本完成定型，冷冻后冰晶在化学交联网络的内部形成，无法达到良好的制孔效果。因此冷冻前的反应效率过高或过低均难以获得较大孔隙率的凝胶。
2)本发明在控制预交联反应进程的同时，调整混合溶液pH值，制备得到具有不同孔径梯度的多孔凝胶，当pH在3.50～5.80时，得到孔径为50～280μm的小孔凝胶，当pH在6.00～6.90时，得到孔径为290～660μm大孔凝胶。
3)本发明通过将内源性多胺与透明质酸类物质在低温下发生交联反应而生成具有连续互穿多孔结构的支架，这种独特的结构有利于进行物质交换和促进细胞的黏附、生长，并且凝胶支架的力学性能可控，整个过程温和可控，产率高，在生物材料和组织工程领域具有潜在的研究价值和广泛的应用前景。
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为本发明提供的凝胶A1的1HNMR图谱；
图2为本发明提供的凝胶A1的室温交联时间随反应效率/G′损失率变化曲线；
图3为本发明提供的凝胶A3的SEM测试图(b为a的局部放大图)；
图4为本发明提供的凝胶A1～A6的孔隙率测试图；
图5为本发明提供的凝胶A1～A6吸水量及吸水速率示意图；
图6为本发明提供的凝胶A1～A6的降解曲线；
图7为本发明提供的凝胶A1～A3对细胞增殖的影响测试结果；
图8为本发明提供的凝胶B3(a)与B4(b)的SEM测试图。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中，术语“交联”是将具有化学反应活性的线型结构聚合物通过化学反应变为三维网状(体型)结构聚合物的过程。常被用于聚合物改性。
本发明中，术语“交联剂”是指能在线型的分子之间产生能化学键，使线型分子相互连在一起，形成网状结构的试剂，用于提高高分子材料的强度。
本发明中，术语“西林瓶”是指：硼硅玻璃或钠钙玻璃管制(模制)注射剂瓶，是一种胶塞和铝塑组合盖封口的小瓶子。早期盘尼西林多用其盛装，故名西林瓶。
本发明中，术语“混合溶液”是指经过溶剂溶解的未交联的透明质酸类物质和溶解的内源性多胺经过混合形的溶液。
本发明中，术语“室温”是指25℃±5℃。
以下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实施例的一部分。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1HATU催化精胺与透明质酸的凝胶A1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7.9mmol)，然后加入60mL纯化水，完全溶解后，向透明质酸溶液中加入精胺0.065g(摩尔数为0.3mmol)， 此时精胺含量占透明质酸质量的1％(即占透明质酸重复结构单元摩尔数的4.0％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至3.5左右，控制温度20℃，加入0.48g HATU，继续搅拌60min(取样通过检测获得精氨的反应效率为23％，具体检测方法见试验例1，下同)，密封后放入-10℃反应6h，在室温下解冻4h后再将其放入-18℃反应6h，在室温下解冻得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次后将所得凝胶进行冻干。称取冻干后的凝胶1 g，加入浓度为25mg/mL的pH 7.0磷酸盐缓冲液共50mL，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，获得终产品冷冻凝胶A1。
该冷冻凝胶A1的1HNMR图谱如图1所示。图1中显示，化学位移1.8-2.0ppm附近为透明质酸中的乙酰甲基峰，化学位移2.5-2.8ppm附近为精胺上的邻氨基亚甲基峰，两峰的存在可以证明精胺与透明质酸成功交联。
实施例2：HATU催化精胺与透明质酸的凝胶A2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7.9mmol)，然后加入75mL纯化水，完全溶解后，向透明质酸溶液中加入精胺0.098g(摩尔数为0.47mmol)，此时精胺含量占透明质酸质量的3.3％(即占透明质酸重复结构单元摩尔数的6.0％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.9左右，控制温度40℃，加入0.72g HATU，继续搅拌30min(取样通过检测获得精氨的反应效率为19％)，密封后放入-30℃反应10h，在室温下解冻8h后再将其放入-20℃反应6h，在室温下解冻得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次，然后将所得凝胶进行冻干。称取冻干后的凝胶1g，加入浓度为25mg/mL的pH7.0磷酸盐缓冲液共50mL，待凝胶完全溶胀后，将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A2。
实施例3：EDC催化亚精胺与透明质酸的凝胶A3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7.9mmol)，然后加入100mL纯化水，完全溶解后，向透明质酸溶液中加入亚精胺0.017g(摩尔数为0.12mmol)，此时亚精胺含量占透明质酸质量的0.57％(即占透明质酸重复结构单元摩尔数的1.5％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至5.8左右，控制温度25℃，加入0.068g EDC和0.014g NHS，继续搅拌40min(取样通过检测获得亚精氨的反应效率为20％)，密封后放入-15℃冰箱反应14h，在室温下解冻6h后得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次，然后将所得凝胶进行冻干。称取冻干后的凝胶1g，加入浓度为25mg/mL的pH7.0磷酸盐缓冲液共50mL，待凝胶完全溶胀后，将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A3。
实施例4：EDC催化亚精胺与透明质酸的凝胶A4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7.9mmol)，然后加入75mL纯化水，完全溶解后，向透明质酸溶液中加入亚精胺0.034g(摩尔数为0.24mmol)，此时亚精胺含量占透明质酸质量的1.13％(即占透明质酸重复结构单元摩尔数的3.04％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.0左右，控制温度20℃，加入0.137g EDC和0.027g NHS，继续搅拌30min(取样通过检测获得亚精氨的反应效率为12％)，密封放入-60℃反应20h，在室温下解冻10h，然后在-30℃冷冻8h，在室温下解冻后得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次，然后将所得凝胶进行冻干。称取冻干后的凝胶1 g，加入浓度为25mg/mL的pH7.0磷酸盐缓冲液共50mL，待凝胶完全溶胀后，将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A4。
实施例5：DMTMM催化精胺与透明质酸的凝胶A5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7.9mmol)，然后加入50mL纯化水，完全溶解后，向透明质酸溶液中加入精胺0.04g(摩尔数为0.2mmol)，此时精胺含量占透明质酸质量的1.33％(即占透明质酸重复结构单元摩尔数的2.5％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至4.3左右，控制温度25℃，加入0.22g DMTMM，继续搅拌35min(取样通过检测获得精氨的反应效率为16％)，密封放入-18℃冰箱24h，在室温下解冻6h后得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次，然后将所得凝胶进行冻干。称取冻干后的凝胶1 g，加入浓度为25mg/mL的pH7.0磷酸盐缓冲液共50mL，待凝胶完全溶胀后，将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A5。
实施例6：DMTMM催化亚精胺与透明质酸的凝胶A6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7.9mmol)，然后加入75mL纯化水，完全溶解后，向透明质酸溶液中加入亚精胺0.057g(摩尔数为0.4mmol)，此时亚精胺含量占透明质酸质量的1.9％(透明质酸重复结构单元摩尔数的5.0％)。用6mol/L的盐酸溶液将透明质酸溶液的pH值调节至6.5左右，控制温度35℃，加入0.33g DMTMM，继续搅拌45min(取样通过检测获得亚精氨的反应效率为22％)，密封放入-20℃反应18h，在室温下解冻6h，然后在-10℃冰箱冷冻10h，在室温下解冻后得到凝胶。然后加入纯化水，用均质机粉碎凝胶后洗涤8次，然后将所得凝胶进行冻干。称取冻干后的凝胶1g，加入浓度为25mg/mL的pH7.0磷酸盐缓冲液共50mL，待凝胶完全溶胀后，将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A6。
对比例1：3％BDDE交联透明质酸冷冻凝胶制备
称取2g透明质酸钠(分子量900KDa，含透明质酸重复结构单元5.3mmol)于烧杯中，然后加入10mL的纯化水，完全溶解后，此时透明质酸的浓度为20mg/mL，向透明质酸溶液中加入BDDE(1，4-丁二醇二缩水甘油醚)0.06ml，此时BDDE含量占透明质酸质量比为3％。再加入氢氧化钠(20％)溶液1ml，搅拌30min密封后在-20℃反应16h，接着在25℃解冻24h。然后用盐酸溶液(2M)调节溶液pH值至7.0，再用pH＝7.0的磷酸盐缓冲液复配至20mg/ml，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B1。
对比例2：4％BDDE交联透明质酸冷冻凝胶制备
称取2g透明质酸钠(分子量900KDa，含透明质酸重复结构单元5.3mmol)于烧杯中，然后加入10mL的纯化水，完全溶解后，此时透明质酸的浓度为20mg/mL，向透明质酸溶液中加入BDDE(1，4-丁二醇二缩水甘油醚)0.08ml，此时BDDE与透明质酸质量比为4％。再加入氢氧化钠(20％)溶液1ml，搅拌30min密封后在-20℃反应16h，接着在25℃解冻24h。然后用盐酸溶液(2M)调节溶液pH值至7.0，再用pH＝7.0的磷酸盐缓冲液复配至20mg/ml，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B2。
对比例3
工艺步骤与实施例3相同，仅在pH值调节至5.8左右后“控制温度55℃，加入0.068g EDC和0.014g NHS，继续搅拌65min(取样通过检测获得亚精氨的反应效率为76％)”，获得终产品凝胶B3。
对比例4
工艺步骤与实施例4相同，仅在pH值调节至6.0左右后“控制温度15℃，加入0.137g EDC和0.027g NHS，继续搅拌5min(取样通过检测获得亚精氨的反应效率为7％)”，获得终产品凝胶B4。
试验例1：游离精胺和亚精胺含量检测
精胺和/或亚精胺均没有发色基团，无紫外和荧光吸收而不易采用常规色谱方法进行测定，因此对样品中精胺或亚精胺的测定需要衍生处理后进行分析；由于丹磺酰氯可以同伯胺或仲胺基上的活泼氢反应脱掉一分子的氯化氢生成具有荧光和紫外光的衍生物，可以采用荧光和紫外两种检测器进行检测，所以选择丹磺酰氯作为衍生剂。
(1)供试品溶液：分别取样品约1.00g，精密称定质量，置100ml容量瓶中，加入0.1g磷酸氢二钠固体，然后用0.1mol/L盐酸溶液稀释至刻度，摇匀，过0.45μm滤膜，取续滤液。取样时需在交联时的不同时间段进行取样，取样时间分别为0.5h、1h、2h、3h、4h、5h、6h、 7h、8h、9h。
(2)衍生法：精密量取上述供试品溶液0.5ml，置10ml具塞试管中，加入饱和碳酸氢钠溶液1.5ml和丹磺酰氯衍生剂溶液1.0ml，振荡混匀；置60℃水浴中反应30min(过程中间取出振荡两次)；取出，加饱和碳酸氢钠溶液100μl，振荡混匀，60℃水浴中保温15min；取出，加入超纯水1.0ml，置40℃水浴除去丙酮，加二氯甲烷3.0ml，振荡萃取2min，静置分层后，吸取出下层有机相；重复用二氯甲烷萃取两次(3.0ml/次)，合并二氯甲烷萃取液，50℃水浴蒸干后，用乙腈2.0ml充分振荡，使残渣溶解，混匀，过0.22μm滤膜，取续滤液；另取0.1mol/L盐酸0.5ml代替试样，同法操作，制备空白液。
(3)色谱条件：色谱柱用十八烷基硅烷键合硅胶为填充剂(C18柱，250mm×4.6mm×5μm)；检测波长为254nm；柱温为35℃；流动相A为0.01mol/L乙酸铵溶液，流动相B为乙腈；流速为1.0ml/min，按下表程序进行梯度洗脱。
Figure PCTCN2022114604-appb-000001
(4)测定：量取空白液和供试品溶液制得的衍生溶液各20μl，分别注入液相色谱仪，记录色谱图。
(5)计算方法：取系列浓度对照溶液测得的精胺或亚精胺衍生物峰面积与对应浓度进行线性回归，绘制标准曲线，相关系数(r)不得低于0.98；将供试品溶液测得的精胺或亚精胺衍生物峰面积代入标准曲线中，计算浓度C(μg/ml)，并按下表公式计算本品中游离精胺或亚精胺的含量W(％)：
Figure PCTCN2022114604-appb-000002
式中：
C为代入标准曲线后计算的供试品溶液的精胺或亚精胺浓度(μg/ml)；V为供试品溶液的稀释体积(100ml)；m为试样的质量(g)。
以冷冻凝胶A1为例，测试其反应效率随交联时间及G′损失率的变化，从而找到凝胶的最佳冷冻时间。如图2是室温交联时间随反应效率及弹性模量G′损失率之间的变化曲线。其中反应效率由游离精胺残留量结果进行计算。从图中可见，反应效率及G损失率随交联时间的延长而不断增大。在交联时间为0.5h时，反应效率是13％；在交联时间为1h时，反应效率是23％，而交联时间为2～9h这个阶段，间隔1h内反应效率在8％～11％之间，可见，在 0.5h～1h内凝胶交联较快，可能原因是初始阶段反应物的浓度较大，所以反应速率较快。另外，在交联时间分别为0.5h和1h进行冷冻的凝胶，其弹性模量G′损失率较低，分别为4.83％和6.58％。综上，在交联时间为0.5h和1h时是凝胶最佳冷冻时间。
试验例2：精胺或亚精胺反应效率检测
分别取实施例与对比例中所得透明质酸凝胶2.0g，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除去乙醇。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。将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或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，获得反应效率。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；
反应效率＝(实际交联摩尔比/理论交联摩尔比)×100％。
表1实际反应效率
Figure PCTCN2022114604-appb-000003
Figure PCTCN2022114604-appb-000004
如表1所示，实施例1～6的反应效率均在95％以上。实施例4与对比例1交联度均是3.0％，所得凝胶A4的反应效率显著高于B1凝胶；同样，实施例1与对比例2交联度均是4.0％，冷冻交联后所得凝胶A1的反应效率显著高于B2凝胶，可能原因在于交联剂的种类、预交联进程、pH值、冷冻、解冻温度与时间等几个因素的综合影响，此外，对比例1和2的制备工艺需在碱性条件下进行，而交联的凝胶在过酸和/或过碱的环境中不稳定，容易降解，这也是导致其反应效率较低的原因之一。另外，从表1中可以看出，对比例3-4与实施例3-4相比，对比例3-4的反应效率均低于实施例3-4，并且对比例4远低于实施例4，进一步说明对比例4预交联反应后的反应效率低(7％，未达到单位点交联)，导致冷冻后交联过程效率大大降低。另外，由于实施例1～6的反应效率均在95％以上，也说明精胺或亚精胺的残留量很低，实现了交联剂的充分应用，对产品的临床使用的安全性有十分重要的意义。
试验例3：凝胶流变学性能的测定
将实施例1～6制备的凝胶进行流变学性能测试：取灭菌前后的凝胶各2ml，使用旋转流变仪分别对其进行测试，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；其中G′(弹性模量)损失率的计算方式如下：
G′损失率＝(灭菌前G′-灭菌后G′)/灭菌前G′；
表2凝胶的流变学数据
Figure PCTCN2022114604-appb-000005
Figure PCTCN2022114604-appb-000006
G′损失率是凝胶热稳定性的重要表征指标，G′损失率越低表明凝胶的热稳定性越高。如表2所示，实施例1～6所得凝胶A1～A6的弹性模量值G′在灭菌前后差值较小，G′损失率均在7.75％以内，表明凝胶A1～A6的热稳定性很高，能够抵抗湿热灭菌。通过对比实施例2和6的测试数据发现，在交联度和浓度相近的情况下，精胺交联的透明质酸与亚精胺交联的透明质酸热稳定性相近，其凝胶的弹性模量值G′相近，其所得凝胶A2和A6灭菌后的G′均在3400以上。对于实施例4与对比例1，其交联度相同，且制备工艺均是冷冻交联，A4凝胶灭菌前后的弹性模量G′损失率是3.28％；而B1凝胶灭菌前后的弹性模量G′损失率是76.32％。对于实施例1与对比例2，其交联度相同，且制备工艺均是冷冻交联，A1凝胶灭菌前后的弹性模量G′损失率是6.93％；而B2凝胶灭菌前后的弹性模量G′损失率是77.78％。此外，A4凝胶灭菌前的弹性模量G′是B1凝胶的6.56倍，A1凝胶灭菌前的弹性模量G′是B2凝胶的5.35倍；A4凝胶灭菌后的弹性模量G′是B1凝胶的26.8倍，A1凝胶灭菌后的弹性模量G′是B2凝胶的22.4倍。综上所述，精胺和/或亚精胺作为交联剂制备的交联透明质酸钠凝胶其热稳定性远高于BDDE交联透明质酸钠凝胶，可能原因是BDDE交联透明质酸凝胶需在碱性条件下制备，而凝胶的酰胺键在碱性条件下不稳定，容易断裂，导致所得凝胶的反应效率较低，而凝胶中的非交联透明质酸在高温条件下其糖苷键容易断裂，所以湿热灭菌后其弹性模量显著降低。而对比例3～4的弹性模量G′损失率，也明显高于实施例3～4，这也是由于对比例3～4的预交联反应效率偏高或偏低，导致后续冷冻过程的交联效率低，从而使弹性模量的损失率偏高。
试验例4：凝胶的SEM及孔径的测定
利用扫描电子显微镜观察凝胶的孔结构。将冻干的冷冻凝胶海绵用刀片切成小方块，用导电胶固定在电镜台上，喷金60s，用场发射扫描电镜进行分析，冷冻凝胶海绵A3扫描电镜结果见图3。对比例3～4的扫描电镜结果见图8。
采用ImageJ软件和SEM图像统计分析凝胶的孔径，表3是凝胶A1～A6的孔径分布。从图中可以看出，凝胶的孔径均在50～660μm之间，而对比例3与4中获得的孔径均不超过10μm。这从图3和图8中也可以进一步看出，A3的孔为疏松的多孔结构，孔隙率高，而图8中的凝胶，表面较为致密，孔的数量明显较少，也说明了在冷冻前，反应效率的偏高或偏低，均会明显影响后期的交联效率，进而影响凝胶孔结构的形成。
表3凝胶的孔径分布
实施例 孔径(μm)
实施例1 50-98
实施例2 405-500
实施例3 180-280
实施例4 290-395
实施例5 100-165
实施例6 510-660
对比例3 0.1-1
对比例4 0.5-9
试验例5：凝胶孔隙率的测定
分别称取2g实施例1～6中制备得到的凝胶A1～A6置于500目干燥筛网中，称量并记录干燥筛网的质量M 0，并向筛网中加入NaCl溶液(0.9％)，使凝胶充分浸泡在NaCl溶液中，过夜使其充分溶胀。将筛网置于擦镜纸上擦干多余水分后称量溶胀后的凝胶及筛网的总质量并记录，记为M 1。随后把装有凝胶的筛网烘干，直至完全干燥，称量干燥的凝胶及筛网的重量并记为M 2。最后，计算凝胶的孔隙率，计算公式如下：
凝胶孔隙率(％)＝(M 1-M 2)/(M 1-M 0)。
孔隙率的结果如图4所示，本发明的凝胶A1～A6的孔隙率均在90％以上。对比例3中的孔隙率仅为13％，对比例4中的孔隙率为35％，由于未在优选的反应效率范围内进行冷冻，对比例3与对比例4的孔隙率均较低。
试验例7：凝胶吸水量及吸水速率的测定
分别称取实施例1～6中所得凝胶1g，投入到装有10ml纯化水的西林瓶中，2min后用镊子取出吸水后的凝胶，称重后记为m，计算其吸水量V(ml)及吸水速率v(ml/min)，计算公式如下：
吸水量V＝m-1；
吸水速率v＝(m-1)/2。
凝胶的吸水量和吸水速率测试结果如图5所示，本发明的凝胶A1～A6在2min内的吸水量均0.95ml以上，凝胶A1～A6的吸水速率0.475ml/min以上，A6的吸水率可达1.395ml/min。
试验例8：凝胶体外降解性能的测定
取10ml玻璃瓶，加入1.0ml的60U/ml酶溶液，不加针推入2.0ml凝胶样品后于漩涡混合器混合60s，确保凝胶样品与酶溶液均匀混合，用流变仪在37℃测定动力粘度(η，Pa·s)：装载间隙：1000μm，测试模式：flowpeak hold模式；测试时间：10800s。
如图6是A1～A6凝胶的降解曲线，从曲线中可知，凝胶的稳定性较强，降解时间均在80min以上；而且降解时间基本与凝胶的弹性模量成正比，即凝胶的弹性模量越大，越难降 解，降解时间越长。
试验例9：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2天，备用。向L-929细胞培养基中加入1mL含有0.1％EDTA的胰蛋白酶溶液，按照1×10 4个/孔将L929细胞接种于96孔板，每孔100μL溶液。将实施例1-3中所得的凝胶A1-A3分别转移至96孔板中，紫外灯固化并灭菌。然后将L-929细胞培养液加入到放置凝胶的孔中，并继续放置在细胞培养箱中以促进细胞生长。同步进行空白的细胞增殖对比实验。
凝胶细胞增殖率由MTT法测定。在分别培养了24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570nm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：
细胞增值率(％)＝(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7所示：从图中可以看出本发明的凝胶A1-A3在24小时的细胞存活率分别为126％、139％和137％，均明显高于没有加凝胶的空白对比实验的99％的细胞存活率。从细胞存活率可看出，本发明的凝胶没有细胞毒性，高孔隙率的多孔结构在一定程度上具有促进细胞增长的作用。
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 (10)

  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类物质在活化剂的作用下，在20～40℃下预交联30～60分钟，然后进行冷冻交联，将冷冻交联后的产物进行解冻，再重复冷冻-解冻过程循环0～3次得到的凝胶。
  2. 如权利要求1所述的材料，其特征在于，所述凝胶材料的孔径为50～660μm；和/或，所述凝胶材料的孔隙率在90％以上。
  3. 如权利要求1所述的材料，其特征在于，所述内源性多胺包括精胺和/或亚精胺；
    优选的，所述活化剂包括水溶性碳二亚胺、碳鎓盐和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐中的一种或多种；
    优选的，所述透明质酸类物质包括透明质酸和透明质酸盐，所述透明质酸盐包括透明质酸钾、透明质酸钠。
  4. 一种凝胶材料的制备方法，其特征在于，包括以下步骤：调节透明质酸类物质与内源性多胺的混合溶液的pH，加入活化剂，在20～40℃下预交联30～60分钟，再进行冷冻交联，将冷冻交联后的产物进行解冻，再重复冷冻-解冻过程0～3次，得到所述凝胶材料。
  5. 如权利要求4所述的制备方法，其特征在于，所述内源性多胺包括精胺和/或亚精胺；
    优选的，所述活化剂包括水溶性碳二亚胺、碳鎓盐和4-(4，6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐中的一种或多种；
    优选的，所述透明质酸类物质包括透明质酸和透明质酸盐，所述透明质酸盐包括透明质酸钾、透明质酸钠；
    优选的，所述混合溶液的溶剂选自：水、可溶性醇、可溶性酮、DMF、DMA、DMSO中的一种或多种的混合；
    优选的，所述冷冻温度为溶剂结晶点以下的至少5℃，更优选为溶剂结晶点以下的至少10～60℃。
  6. 如权利要求5所述的方法，其特征在于，所述混合溶液的pH＝3.50～6.90；所述冷冻温度在-60℃～-10℃；所述冷冻时间为6～24h；所述解冻温度为4～40℃；所述解冻时间为4～10h。
  7. 如权利要求5所述的方法，其特征在于，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、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；还包括将水溶性碳二亚胺与助剂联用；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；
    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几种的混合物。
  8. 如权利要求6所述的方法，其特征在于，控制pH在3.50～5.80，得到孔径为50～280μm的小孔凝胶，控制pH在6.00～6.90，得到孔径为290～660μm的大孔凝胶。
  9. 如权利要求4所述的方法，其特征在于，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类物质摩尔量的1.0～62％；
    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类物质摩尔量的0.5～52％；
    所述混合溶液中透明质酸类物质的质量浓度为10～160mg/mL；所述活化剂加入量占透明质酸类物质摩尔量的0.5～150％。
  10. 权利要求1～3任一项所述的凝胶或权利要求4～9任一项所述方法所得到的凝胶在制备医疗、美容、保健产品中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
CN108465128A (zh) * 2018-03-01 2018-08-31 杭州协合医疗用品有限公司 一种交联透明质酸细胞支架材料的制备方法
CN110643056A (zh) * 2019-10-12 2020-01-03 爱美客技术发展股份有限公司 高强度聚乙烯醇凝胶及其制备方法与应用
CN113896915A (zh) * 2021-11-10 2022-01-07 爱美客技术发展股份有限公司 一种凝胶材料及其制备方法与应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
CN108465128A (zh) * 2018-03-01 2018-08-31 杭州协合医疗用品有限公司 一种交联透明质酸细胞支架材料的制备方法
CN110643056A (zh) * 2019-10-12 2020-01-03 爱美客技术发展股份有限公司 高强度聚乙烯醇凝胶及其制备方法与应用
CN113896915A (zh) * 2021-11-10 2022-01-07 爱美客技术发展股份有限公司 一种凝胶材料及其制备方法与应用

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