WO2023221082A1 - 一种接枝聚合物材料及其制备方法与应用 - Google Patents

一种接枝聚合物材料及其制备方法与应用 Download PDF

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WO2023221082A1
WO2023221082A1 PCT/CN2022/094065 CN2022094065W WO2023221082A1 WO 2023221082 A1 WO2023221082 A1 WO 2023221082A1 CN 2022094065 W CN2022094065 W CN 2022094065W WO 2023221082 A1 WO2023221082 A1 WO 2023221082A1
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plla
solution
salt
solvent
active ester
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PCT/CN2022/094065
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French (fr)
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李睿智
张堃
谷诗伟
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爱美客技术发展股份有限公司
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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
    • 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
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
    • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
    • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids
    • C08G63/08—Lactones or lactides

Definitions

  • the invention relates to the technical field of biomedical materials, and in particular to a graft polymer material and its preparation method and application.
  • Hyaluronic acid or sodium hyaluronate, also known as hyaluronic acid, is composed of D-glucuronic acid through a ⁇ -1,4 glycosidic bond and N-acetylglucosamine through a ⁇ -1,3 glycosidic bond Glycosaminoglycans are composed of disaccharide units.
  • HA is widely used in the field of cosmetics or ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects.
  • Hyaluronic acid is a native substance in the body and has good biocompatibility and certain biological activity.
  • hyaluronic acid can be modified in two ways to achieve cross-linking.
  • One is to cross-link hyaluronic acid molecules with chemical cross-linking agents to form
  • the spatial network structure prevents the degradation of hyaluronic acid by hyaluronidase and others through the dense rigid network structure, prolongs the residence time of exogenous hyaluronic acid in the body, ensures biocompatibility and has good therapeutic effects.
  • the second is to hydrophobically modify the side chain of hyaluronic acid to reduce the affinity of hyaluronidase to hyaluronic acid, which can effectively increase the contact probability of hyaluronidase to hyaluronic acid, thereby improving the absorption of hyaluronic acid in the body. residence time.
  • Chinese patent document CN107286608A discloses a method of coupling PLGA and HA through a melting method, in which PLGA and HA are coupled through the coupling agent cystamine.
  • Chinese patent CN104162169B discloses a cystamine-derived PLGA coupled with HA in a mixed solvent of N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and water.
  • DMF N,N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • Acta Biomaterialia , 2009, 5 (9): 3394-3403. disclosed a method of coupling HA and PLGA using adipic acid dihydrazide as a coupling agent. This method also uses adipic acid dihydrazide to couple Hyaluronic acid is ammoniated and then coupled to the active ester of hydroxysuccinimide (NHS) of PLGA in a single solution of DMSO.
  • NHS hydroxysuccinimide
  • graft copolymer of hyaluronic acid and polylactic acid (PLA, racemic). Since racemic PLA is an amorphous polymer, it has good solubility and can be dissolved in organic solvents such as tetrahydrofuran, acetone, and DMSO. Dissolved, a mixed solvent of water and organic solvent can also be used when grafting with HA. A homogeneous coupling reaction between HA and PLA occurs, the grafting rate is higher, and the reaction conditions are easier to control.
  • Chinese patent CN104056275B discloses that the terminal aldehyde group of HA is ammoniated and coupled with PLA in a mixed solvent of DMSO and water to obtain a HA-PLA graft copolymer.
  • Fabio Salvatore Palumbo et al (Palumbo F S, Pitarresi G, Mandracchia D, et al. New graft copolymers of hyaluronic acid and polylactic acid:Synthesis and characterization[J].
  • Carbohydrate Polymers, 2006, 66(3):379-385. ) disclosed that HA is first quaternized, and then the coupling reaction between PLA and HA is carried out in a composite solution of DMSO and methylene chloride.
  • the quaternary ammonium salt is removed through a cation exchange resin.
  • Giovanna Pitarresi et al (Pitarresi G, Palumbo F S, Fiorica C, et al.Injectable in situ forming microgels of hyaluronic acid-g-polylactic acid for methylprednisolone release[J].European Polymer Journal, 2013,49(3):718 -725.) disclosed that quaternized HA was coupled with PLA in a mixed solution of DMSO and methylene chloride to obtain a HA-PLA graft copolymer.
  • the preparation of graft polymers of HA and polyester currently reported includes two methods. One is to couple hyaluronic acid with an NHS-activated ester of polyester after ammoniation, and the other is to use hyaluronic acid.
  • the hydroxyl groups are coupled directly to the NHS activated ester of the polyester.
  • the second method has lower reaction efficiency due to the lower reactivity of hydroxyl groups.
  • the first method uses exogenous polyamines, and exogenous polyamines have certain biological toxicity.
  • the graft polymer prepared by this method has certain safety risks, and the NHS activation of polyester Ester is prone to hydrolysis in water, and its coupling reaction with high molecular weight hyaluronic acid (molecular weight greater than 100KDa) that can only be dissolved in water results in low coupling reaction efficiency.
  • Poly-L-lactic acid is a polymer of L-lactic acid, which can be degraded into L-lactic acid in the body and is one of the metabolites of human polysaccharides. Therefore, PLLA has good tissue compatibility and is widely used in bone nails and bone. boards, as well as medical beauty and other fields. Due to its excellent crystallization properties, PLLA has poor solubility and can only be dissolved in limited organic solvents such as halogenated organic compounds (such as chloroform, dichloromethane, hexafluoroisopropanol, etc.).
  • organic solvents such as halogenated organic compounds (such as chloroform, dichloromethane, hexafluoroisopropanol, etc.).
  • the present invention provides a grafted polymer material and its preparation method and application.
  • NDHC Nano Disperse Heterogeneous Coupling
  • a graft polymer which is mainly obtained by coupling PLLA and HA or its salt through an ammoniating reagent.
  • the ammoniating reagent includes spermine (SM) and/or spermidine ( SPD).
  • the amination reagent is spermine (SM).
  • the amination reagent is spermidine (SPD).
  • the molecular weight of PLLA is 3-200KDa (for example, 3, 5, 10, 20, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200KD).
  • the specific optical rotation range of PLLA is -155° to -160°.
  • one end of PLLA is carboxyl-terminated, and the other end may be hydrogen or alkyl (eg, methyl)-terminated.
  • the molecular weight of HA or its salt is 100-3000KDa (for example, 100, 200, 400, 500, 600, 800, 900, 1000, 1500, 2000, 2500, 3000KDa).
  • the salt of HA can be its sodium salt, potassium salt, calcium salt, especially the sodium salt, that is, sodium hyaluronate.
  • the molecular weight of the graft polymer is 103-5000KDa (for example, 103, 200, 400, 500, 600, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000KDa).
  • the graft polymer has the following structure:
  • R is
  • R' is H or C 1-6 alkyl
  • n is an integer from 250 to 8500
  • n is an integer from 40 to 3000.
  • the grafted polymer is in the state of nanomicelle particles in the aqueous solution, and the particle size range of the nanoparticles is 10-900nm (such as 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900nm).
  • R' is H or methyl
  • a method for preparing a graft polymer which includes the following steps:
  • PLLA reacts with hydroxyl compounds in the presence of an activator to obtain PLLA-active ester
  • step (1) React the PLLA-active ester obtained in step (1) with an ammoniating reagent to obtain ammoniated PLLA as shown below;
  • step (3) React the ammoniated PLLA obtained in step (2) with HA or its salt to obtain a graft polymer, as shown in Formula I.
  • the hydroxy compound described in step (1) is selected from: N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, 1-hydroxybenzo Any one or more of triazole (HOBt).
  • the PLLA-active ester prepared in this way is one or more of PLLA-NHS active ester, PLLA-Sulfo-NHS active ester, PLLA-HOBt active ester or PLLA-tert-butanol active ester.
  • the PLLA active ester described in step (1) can be PLLA-NHS active ester, and its structure can be as follows:
  • R' is H or C 1-6 alkyl, especially H or methyl.
  • step (1) includes:
  • step (1) also includes a purification step (1-2): adding the reaction product obtained in step (1-1) to a precipitant, separating the precipitate, and removing the solvent.
  • a purification step (1-2) adding the reaction product obtained in step (1-1) to a precipitant, separating the precipitate, and removing the solvent.
  • step (1-1) is performed under the protection of an inert gas; more specifically, the inert gas is, for example, nitrogen or helium.
  • the inert gas is, for example, nitrogen or helium.
  • the molar ratio of PLLA to the hydroxy compound is 1:1-20 (for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:15, 1:20), especially 1:1-5, where the molar amount of PLLA is calculated from the ratio of the input mass to its weight average molecular weight.
  • the molar ratio of the hydroxy compound to the activator is 1:1-20 (for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 , 1:8, 1:10, 1:15, 1:20), especially 1:1-5.
  • a solvent needs to be used to dissolve PLLA and perform a homogeneous reaction.
  • the solvent used is a halogenated organic compound, such as dichloromethane, chloroform, dichloroethane or hexafluoroisopropanol. wait.
  • the concentration of PLLA in halogenated organic matter is 50-200 mg/ml (mass volume concentration, such as 50, 60, 80, 90, 100, 110, 120, 140, 160, 180, 200 mg/ml).
  • reaction temperature range in step (1-1) is 15-70°C (such as 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70°C), especially 30 -50°C.
  • reaction time in step (1-1) is 10-24 hours (for example, 10, 12, 14, 16, 18, 20, 22, 24 hours).
  • the activator described in step (1-1) is selected from: phosphonium bromide salt formed from water-soluble carbodiimide, triphenylphosphine and bromide, carbonium salt and 4-(4,6-di One or more of methoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM).
  • DTMM methoxytriazin-2-yl-4-methylmorpholine hydrochloride
  • the water-soluble carbodiimide can be selected from: 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 Amines, 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 phosphonium bromide salt formed by triphenylphosphine and bromide can be selected from: the phosphonium salt formed by triphenylphosphine and carbon tetrabromide, the phosphonium salt formed by triphenylphosphine and N-bromosuccinimide Salt etc.
  • the phosphonium bromide salt can be obtained by reacting triphenylphosphine and bromide in a solvent such as dichloromethane by a known method.
  • the carbonium salt can be selected from: O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-1-yl) Azol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate Fluorophosphate (HCTU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium tetrafluoroborate (TBTU), O-(N-succinimidyl) -Bis(dimethylamino)carbonium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyl Urea
  • the precipitating agent described in step (1-2) is an ether, especially a C 2-10 ether, such as diethyl ether, tert-butyl methyl ether, methyl ethyl ether, etc.
  • the volume ratio of the precipitant to the reaction product described in step (1-2) is 1-10:1 (for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 7:1, 8:1, 9:1, 10:1).
  • step (1-1) the method of adding the reaction product obtained in step (1-1) to the precipitating agent as described in step (1-2) is dropwise addition.
  • any suitable method can be used to separate the precipitate in step (1-2), such as suction filtration.
  • the method of removing the solvent in step (1-2) is to vacuum dry the separated precipitate; specifically, the drying temperature can be 35-45°C (for example, 40°C); specifically, The drying time can be 6-48 hours, and the vacuum degree ranges from -0.06 to -0.1MPa (for example -0.08MPa).
  • reaction formula of PLLA-NHS active ester obtained by reacting PLLA with NHS is as follows:
  • R' is H or C 1-6 alkyl, especially H or methyl.
  • the PLLA-active ester described in step (2) is PLLA-NHS active ester, and its structure can be as follows,
  • step (2) includes:
  • step (2) also includes a purification step: (2-2) Add the reaction product obtained in step (2-1) to a precipitant, separate the precipitate, and remove the solvent.
  • step (2-1) is performed under the protection of an inert gas; more specifically, the inert gas is, for example, nitrogen or helium.
  • the inert gas is, for example, nitrogen or helium.
  • the solvent A of the PLLA-active ester solution in step (2-1) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, chloroform, dichloroethane, hexafluoroisopropanol, etc. .
  • the solvent B of the ammoniating reagent solution in step (2-1) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, chloroform, dichloroethane, hexafluoroisopropanol, etc.
  • the mass percentage concentration of the PLLA-active ester solution described in step (2-1) can be 0.1-50% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 20%, 30%, 40%, 50%).
  • the concentration of the ammoniating reagent solution in step (2-1) can be 1-100 mg/ml (mass volume concentration, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 60, 70, 80, 90, 100mg/ml), such as 5-50mg/ml.
  • the mass ratio of PLLA-active ester and amination reagent (SM or SPD) described in step (2-1) is 1:0.01-15 (for example, 1:0.01, 1:0.02, 1:0.04, 1: 0.05, 1:0.06, 1:0.075, 1:0.08, 1:0.1, 1:0.2, 1:0.4, 1:0.5, 1:0.6, 1:0.8, 1:1,, 1:2, 1:4 , 1:5, 1:6, 1:8, 1:10, 1:12, 1:15), especially 1:0.01-1.
  • step (2-1) the method of adding the PLLA-active ester solution to the ammoniation reagent solution as described in step (2-1) is dropwise addition.
  • the temperature of the reaction described in step (2-1) is 10-70°C (for example, 10, 20, 30, 40, 50, 60, 70°C), especially 40-70°C.
  • reaction time in step (2-1) is 6-48 hours (for example, 6, 12, 18, 24, 30, 36, 42, 48 hours), especially 18-30 hours.
  • the precipitating agent in step (2-2) is an ether or an alcohol, especially a C 2-10 ether or a C 1-6 alcohol, such as diethyl ether, tert-butyl methyl ether, methyl ethyl ether, or methanol, ethanol. , isopropyl alcohol, etc.
  • the volume ratio of the precipitant to the reaction product described in step (2-2) is 1-10:1 (for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 7:1, 8:1, 9:1, 10:1).
  • step (2-1) the method of adding the reaction product obtained in step (2-1) to the precipitating agent as described in step (2-2) is dropwise addition.
  • the method of separating the precipitate in step (2-2) can be any suitable method, such as suction filtration.
  • the method of removing the solvent in step (2-2) is to vacuum dry the separated precipitate; specifically, the drying temperature can be 35-45°C; specifically, the drying time can be 6 -48 hours, vacuum degree range is -0.06 to -0.1MPa.
  • step (3) includes:
  • step (3-2) Add an activator (and auxiliary agent, if necessary) to the system obtained in step (3-2), and react;
  • step (3) also includes a purification step: (3-4) removing small molecule reactants from the reaction product obtained in step (3-3) (for example, through dialysis or ultrafiltration), and freeze-drying;
  • step (3) further includes a purification step: (3-5) Immerse the product obtained in step (3-4) into solvent E, remove solvent E (can be repeated multiple times), and dry under vacuum.
  • the solvent C of the solution of HA or its salt in step (3-1) is water, that is, the solution of HA or its salt is an aqueous solution of HA or its salt.
  • the mass percentage concentration of the solution of HA or its salt in step (3-1) is 0.1-50% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 20%, 30%, 40%, 50%).
  • the solution of HA or its salt described in step (3-1) also includes a polymer emulsifier, such as polyethylene glycol and polyvinyl alcohol; more specifically, the molecular weight of polyethylene glycol can be 1000-50000Da (for example, 1000, 2000, 4000, 6000, 8000, 10000, 15000, 20000, 30000, 40000, 50000Da), especially 1000-20000Da; more specifically, the polyvinyl alcohol molecular weight can be 5000-500000Da (for example, 5000, 10000, 20000Da) , 40000, 50000, 60000, 80000, 100000, 200000, 300000, 400000, 500000Da), especially 5000-50000Da, the alcoholysis degree can be 60-100% (such as 78%, 88%, 98%), especially 87 -89%.
  • a polymer emulsifier such as polyethylene glycol and polyvinyl alcohol
  • the molecular weight of polyethylene glycol can be 1000-50000Da ( For example
  • the polymer emulsifier accounts for 0.1-30% of the total mass of HA or its salt solution (for example, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 20%, 30%), especially 0.5-10%.
  • the pH of the solution of HA or its salt in step (3-1) is 5.0-6.5 (for example, 5.0, 5.5, 6.0, 6.5), and the pH can be adjusted by, for example, hydrochloric acid.
  • the solvent D of the ammoniated PLLA solution in step (3-1) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, chloroform, dichloroethane, hexafluoroisopropanol, etc.
  • solvent A, solvent B and solvent D may be the same or different.
  • the mass percentage concentration of the ammoniated PLLA solution in step (3-1) is 0.1-50% (for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1% , 2%, 3%, 4%, 5%, 6%, 8%, 10%, 20%, 30%, 40%, 50%).
  • the mass ratio of HA or its salt and ammoniated PLLA in step (3-1) is 1:0.01-2 (for example, 1:0.01, 1:0.05, 1:0.1, 1:0.2, 1:0.3, 1 :0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.5, 1:2), especially 1:0.1-1.
  • the ultrasonic treatment time in step (3-2) can be 5-60 minutes (for example, 5, 10, 20, 30, 40, 50, 60 minutes).
  • the power of the ultrasonic treatment in step (3-2) can be 1000-5000W (for example, 1000, 2000, 3000, 4000, 5000W).
  • the ammoniated PLLA solution is dispersed into nano-droplets in the hyaluronic acid aqueous solution, and the particle size range of the nano-droplets is 10-900 nm (for example, 10, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900nm).
  • the amount of activator added in step (3-3) is 0.01-100% of the mass of ammoniated PLLA (for example, 0.01%, 0.1%, 5%, 10%, 20%, 30%, 40%, 50 %, 60%, 70%, 80%, 90%, 100%).
  • the activator described in step (3-3) is selected from: phosphonium bromide salt formed from water-soluble carbodiimide, triphenylphosphine and bromide, carbonium salt and 4-(4,6-di One or more of methoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM).
  • DTMM methoxytriazin-2-yl-4-methylmorpholine hydrochloride
  • the water-soluble carbodiimide can be selected from: 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 Amines, 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 auxiliary agent is selected from: N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, 1-hydroxybenzotriazole (HOBt) any one or more of them.
  • NHS N-hydroxysuccinimide
  • Sulfo-NHS sulfonated N-hydroxysuccinimide
  • HOBt 1-hydroxybenzotriazole
  • step (3-3) is: adding an activator and an auxiliary agent to the system obtained in step (3-2) and reacting.
  • the activator is EDC and the auxiliary agent is NHS.
  • the phosphonium bromide salt formed by triphenylphosphine and bromide can be selected from: the phosphonium salt formed by triphenylphosphine and carbon tetrabromide, the phosphonium salt formed by triphenylphosphine and N-bromosuccinimide Salts, etc.; in one embodiment of the present invention, the phosphonium bromide salt is a phosphonium salt formed by triphenylphosphine and N-bromosuccinimide.
  • the phosphonium bromide salt can be obtained by reacting triphenylphosphine and bromide in a solvent such as dichloromethane by a known method.
  • the carbonium salt can be selected from: O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HATU), O-(benzotriazol-1-yl) Azol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate Fluorophosphate (HCTU), O-(benzotriazol-1-yl)-bis(dimethylamino)carbonium tetrafluoroborate (TBTU), O-(N-succinimidyl) -Bis(dimethylamino)carbonium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyl Urea
  • the temperature of the reaction described in step (3-3) is 10-70°C (for example, 10, 20, 30, 40, 50, 60, 70°C), especially 30-40°C.
  • reaction time in step (3-3) is 6-48 hours (for example, 6, 12, 18, 24, 30, 36, 42, 48 hours), especially 18-30 hours.
  • the method of removing small molecule reactants described in step (3-4) can be dialysis or ultrafiltration, and the time of dialysis or ultrafiltration can be 6-48 hours.
  • the solvent E described in step (3-5) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, chloroform, dichloroethane, hexafluoroisopropanol, etc.; in one aspect of the present invention
  • solvent E is methylene chloride.
  • the preparation method of graft polymer includes the following reaction route:
  • an ammoniated PLLA which has the following structure and can be used as an intermediate for preparing graft polymers:
  • R is
  • R' is H or C 1-6 alkyl
  • n is an integer from 40 to 3000.
  • R' is H or methyl
  • the molecular weight of aminated PLLA is 3-200KDa (for example, 3, 5, 10, 20, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200KD).
  • the specific optical rotation range of PLLA is -155° to -160°.
  • a method for preparing ammoniated PLLA described in the third aspect which includes steps (1) and (2) described in the second aspect of the present invention.
  • a fifth aspect of the present invention there is provided the use of the graft polymer described in the first aspect and the graft polymer prepared by the method described in the second aspect in the preparation of drugs.
  • the drug contains active ingredients and the graft polymer as a drug carrier to achieve sustained release, controlled release, targeted drug delivery and other purposes.
  • the drug is an anti-tumor drug
  • its active ingredient can be, for example, ixabepilone, mitomycin, plicamycin, bleomycin, pianthraquinone, amrucin Bicin, Varrubicin, Pirarubicin, Mitoxantrone, Idarubicin, Zorubicin, Arubicin, Epirubicin, Daunorubicin, Doxorubicin, Doxycycline Elements; trabectedin, cabazitaxel, paclitaxel, docetaxel, demecomycin, teniposide, etoposide, vinorelin, vinflunine, vinorelbine, vindesine, vinorelbine Neosine, vinblastine; Fluridine, Trifluridine, Tegafur, Fluorouracil, Decitabine, Azacitidine, Capecitabine, Gemcitabine, Carmofur, Cytarabine, Nelarabine ,
  • a pharmaceutical composition which contains an active ingredient and the graft polymer described in one aspect, and the graft polymer prepared by the method described in the second aspect as a pharmaceutical carrier.
  • the seventh aspect of the present invention there is provided the use of the graft polymer described in the first aspect and the graft polymer prepared by the method described in the second aspect in the preparation of tissue engineering materials.
  • the tissue engineering materials can be bone tissue engineering materials, cartilage tissue engineering materials, cornea tissue engineering materials, cardiovascular tissue engineering materials, liver tissue engineering materials, etc.
  • an eighth aspect of the present invention there is provided the use of the graft polymer described in the first aspect and the graft polymer prepared by the method described in the second aspect in the preparation of soft tissue filler.
  • the soft tissue filler can be used to eliminate wrinkles (such as wrinkles around the eyes, forehead lines, glabella lines, perioral lines, nasolabial folds, tear troughs, nasolabial folds, neck lines, hand wrinkles, stretch marks, etc.), Anti-aging, scar and wound repair, etc.
  • wrinkles such as wrinkles around the eyes, forehead lines, glabella lines, perioral lines, nasolabial folds, tear troughs, nasolabial folds, neck lines, hand wrinkles, stretch marks, etc.
  • the present invention has the following outstanding features:
  • endogenous polyamines serve as a non-toxic, harmless and safer ammoniation reagent.
  • PLLA is ammoniated through such ammoniation reagents. Due to the ammoniation The final PLLA is not easily hydrolyzed, making it easier to undergo a coupling reaction with hyaluronic acid or its salts in a water environment, which can effectively promote the coupling of PLLA and HA or its salts; at the same time, it can also exert various functions of spermidine.
  • Biological activity ensures that all components can be degraded and metabolized, and the degradation products are endogenous substances in the human body, thereby improving the ease of use of the graft polymer in the field of preparing drugs, tissue engineering materials or soft tissue fillers, and is suitable for Promote applications.
  • the present invention can disperse liquid beads into nanoparticles through a two-liquid phase grafting system, and more It is conducive to internal grafting and improves grafting efficiency.
  • the HA-b-PLLA graft copolymer prepared by the present invention has high grafting efficiency and uniform particle size distribution, with a particle size of 50-900 nm.
  • the graft polymer has good stability and is amphiphilic. , can be used in fields such as drug carriers, tissue engineering materials, and soft tissue repair.
  • Figure 1 shows the 1 H NMR spectrum of the PLLA-NHS activated ester prepared by the present invention.
  • Figure 2 shows the 1 H NMR spectrum of ammoniated PLLA (SPD ammoniated) prepared in the present invention.
  • Figure 3 shows the 1 H NMR spectrum of HA-b-PLLA prepared in the present invention.
  • Figure 4 shows the FT-IR spectrum of HA-b-PLLA prepared in the present invention.
  • Figure 5 shows the particle size distribution diagram of HA-b-PLLA prepared in the present invention in aqueous solution.
  • the main raw materials such as hyaluronic acid or its salt, poly-L-lactic acid, NHS, and activator used in the following examples are all commercially available products.
  • the obtained ammoniated PLLA was dried and stored. The yield was about 75 %.
  • the 1 H NMR spectrum of ammoniated PLLA is shown in Figure 2. SPD ammoniated PLLA with spermidine forms amminated PLLA with two ammoniated connection methods, and the peak positions of the two 1 H NMR spectra are basically the same.
  • Example 4 Coupling reaction of ammoniated PLLA (SPD ammonia) and HA (HATU activation)
  • the median particle size of the hexafluoroisopropanol solution of ammoniated PLLA is 100nm.
  • Add 50mg HATU, 35 The reaction was stirred for 24 hours at °C, and the resulting solution was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 30KDa. The water was removed by freeze-drying. The sponge-like product was soaked in 40 ml of methylene chloride for 1 hour. The solvent was poured out, and the soaking was repeated three times, and then the resulting product was HA-b-PLLA can be obtained by vacuum drying to remove methylene chloride.
  • the product is labeled HA-b-PLLA1.
  • the 1 H NMR spectrum of HA-b-PLLA1 is shown in Figure 3 (in Figure 2, two kinds of HA-b-PLLA formed from two aminated PLLA and HA, both 1 H
  • the peak positions of the NMR spectra are basically the same, so one of them is selected as an illustration in Figure 3).
  • Figure 3 shows that the chemical shift around 1.8-2.0ppm is the methyl peak on the acetamido group in hyaluronic acid, and the chemical shift around 4.9-5.1ppm is the methine peak on L-polylactic acid.
  • the product is a snowflake-like loose powder, and its micelle particle size distribution in aqueous solution is shown in Figure 5. It can be obtained from the frequency curve in Figure 5 that the median particle size of HA-b-PLLA1 is approximately 520nm, and It can be seen from the figure that the frequency curve is a sharp single peak, which further indicates that the particle size of the prepared graft polymer is very uniform.
  • Example 5 Coupling reaction of ammoniated PLLA (SPD ammonia) and HA (EDC activation)
  • the product is labeled HA-b-PLLA2.
  • HA-b-PLLA2 By integrating the peaks with chemical shifts around 1.8-2.0ppm and 4.9-5.1ppm in the 1 H NMR spectrum, the grafting of PLLA and HA in HA-b-PLLA2 was calculated. The efficiency is around 25%.
  • the median micelle size in aqueous solution is approximately 180 nm.
  • the product is labeled HA-b-PLLA3.
  • HA-b-PLLA3 By integrating the peaks with chemical shifts around 1.8-2.0ppm and 4.9-5.1ppm in the 1 H NMR spectrum, the grafting of PLLA and HA in HA-b-PLLA3 was calculated. The efficiency is around 56%.
  • the FT-IR spectrum of HA-b-PLLA3 is shown in Figure 4.
  • Characteristic stretching vibration peak which indicates that PLLA is successfully grafted to HA molecules. Its median micelle size in aqueous solution is approximately 450 nm.
  • Example 7 Coupling reaction of ammoniated PLLA (SM ammonia) and HA (DMTMM activation)
  • the median particle size of the ammoniated PLLA chloroform solution is 250 nm.
  • HA-b-PLLA can be obtained by drying and removing methylene chloride.
  • the grafting efficiency of PLLA and HA was calculated to be about 42%.
  • the median micelle size in aqueous solution is approximately 240 nm.
  • Example 8 Coupling reaction of ammoniated PLLA (SPD ammonia) and HA (HATU activation)
  • Example 4 Other steps are the same as Example 4, the only difference is that after adding 50 mg HATU, the reaction is stirred at 55°C for 18 hours.
  • the grafting efficiency of PLLA and HA was calculated to be about 65%. Its median micelle size in aqueous solution is approximately 420 nm.
  • Comparative Example 1 First ammoniate hyaluronic acid and then perform coupling reaction with PLLA-NHS activated ester
  • the water is removed by freeze-drying.
  • the sponge-like product is placed in Soak in 40 ml of methylene chloride for 1 hour, pour off the solvent, repeat the soaking three times, and then vacuum-dry the product to remove the methylene chloride to obtain HA-b-PLLA.
  • the median particle size of the chloroform solution of PLLA-NHS active ester is 250nm, and stir at 35°C. React for 24 hours.

Abstract

本发明公开了一种接枝聚合物材料及其制备方法与应用。本发明通过以内源性多胺为偶联剂，通过纳米分散非均相偶联技术(Nano Disperse Heterogeneous Coupling，NDHC)，在非均相条件下实现左旋聚乳酸(PLLA)与透明质酸(HA)或其盐的偶联，得到HA-b-PLLA接枝共聚物。所制备的HA-b-PLLA接枝共聚物接枝效率高，具有两亲性，可应用于药物载体、组织工程材料以及软组织修复等领域。

Description

一种接枝聚合物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，具体涉及一种接枝聚合物材料及其制备方法与应用。
背景技术
透明质酸(hyaluronic acid，简称HA)或透明质酸钠，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。HA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，可通过两种方式对透明质酸进行改性以达到交联的目的，其一为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。其二为对透明质酸侧链进行疏水化改性，降低透明质酸酶对透明质酸的亲和性，可有效透明质酸酶对透明质酸的接触概率，从而提高透明质酸在体内的停留时间。
目前，透明质酸与可吸收聚酯的偶联复合物也有报道，其中透明质酸与乳酸-乙醇酸共聚物(PLGA)的接枝共聚物报道较多。由于PLGA优良的溶解性能，可在各种极性溶剂中溶解，在PLGA与HA进行反应时，可使用水与极性有机溶剂的混合溶剂进行均相反应，接枝率较高，反应条件容易控制。同时，由于PLGA熔点较低，可在较低温度下进行熔融接枝反应，使PLGA与HA进行偶联。中国专利文献CN107286608A公开了一种通过熔融法将PLGA与HA进行偶联的方法，其中，PLGA与HA之间通过偶联剂胱胺进行偶联。中国专利CN104162169B公开了一种胱胺衍生的PLGA在N,N-二甲基甲酰胺(DMF)或二甲基亚砜(DMSO)与水的混合溶剂中与HA进行偶联。Jung Kyu Park等人(Park J K,Yeom J,Oh E J,et al.Guided bone regeneration by poly(lactic-co-glycolic acid)grafted hyaluronic acid bi-layer films for periodontal barrier applications[J].Acta Biomaterialia,2009,5(9):3394-3403.)公开了以己二酸二酰肼为偶联剂，将HA与PLGA进行偶联的方法，该方法也是通过现用己二酸二酰肼将透明质酸进行氨化，再在DMSO单一溶液中偶联PLGA的羟基琥珀酰亚胺(NHS)活性酯。
另外，透明质酸与聚乳酸(PLA，消旋)的接枝共聚物也有较多报道，由于消旋PLA为无定型聚合物，溶解性较好，可在四氢呋喃、丙酮、DMSO等有机溶剂中溶解，在与HA接枝时亦可采用水与有机溶剂的混合溶剂，HA与PLA发生均相偶联反应，接枝率较高，反应条件也较容易控制。中国专利CN104056275B中公开了通过将HA的端醛基进行氨化，在DMSO与水混合溶剂中，与PLA进行偶联，得到HA-PLA接枝共聚物。Fabio Salvatore Palumbo等人(Palumbo F S,Pitarresi G,Mandracchia D,et al.New graft copolymers of hyaluronic acid and polylactic acid:Synthesis and characterization[J].Carbohydrate Polymers,2006,66(3):379-385.)公开了先通过将HA进行季铵盐化，再通过DMSO与二氯甲烷复合溶液中进行PLA与HA的偶联反应，反应结束后通过阳离子交换树脂去除季铵盐。Giovanna Pitarresi 等人(Pitarresi G,Palumbo F S,Fiorica C,et al.Injectable in situ forming microgels of hyaluronic acid-g-polylactic acid for methylprednisolone release[J].European Polymer Journal,2013,49(3):718-725.)公开了季铵化的HA在DMSO与二氯甲烷混合溶液中与PLA进行偶联反应得到HA-PLA接枝共聚物。
综上可知，目前报道的HA与聚酯的接枝聚合物的制备包括两种方式，一为采用将透明质酸氨化后与聚酯的NHS活化酯进行偶联，二为利用透明质酸的羟基与聚酯的NHS活化酯直接进行偶联。其中，第二种方法由于羟基较低的反应活性，导致反应效率较低。而第一种方法中使用的均为外源性多氨，而外源性多氨均具有一定的生物毒性，因此该方法制备的接枝聚合物存在一定的安全隐患，并且聚酯的NHS活化酯在水中易发生水解，其与只能溶解在水中的高分子量透明质酸(分子量大于100KDa)的偶联反应，偶联反应效率低下。
聚左旋乳酸(PLLA)为左旋乳酸的聚合物，其在体内可降解为左旋乳酸，为人体多糖的代谢产物之一，因此PLLA具有良好的组织相容性，被广泛的应用于骨钉、骨板，以及医疗美容等领域。PLLA由于其优良的结晶性能，导致PLLA的溶解性较差，仅能溶解于卤代有机物(如氯仿、二氯甲烷、六氟异丙醇等)等有限的有机溶剂中。而透明质酸HA或其盐在分子量超过100KDa时，在有机溶剂中的溶解度大幅下降，仅能溶解在水中，而上述可溶解PLLA的卤代有机物均难溶与水，因此，PLLA与透明质酸HA或其盐的偶联反应比较困难，并且接枝通常只能发生在表面，接枝效率低，产物不稳定。
发明内容
为了解决上述问题，并提高接枝效率，本发明提供一种接枝聚合物材料及其制备方法与应用。通过利用纳米分散非均相偶联技术(Nano Disperse Heterogeneous Coupling，NDHC)，将氨化后的聚酯与透明质酸或其盐进行偶联，制备得到接枝效率高的HA-b-PLLA接枝聚合物。
在本发明第一方面，提供一种接枝聚合物，其主要由PLLA与HA或其盐通过氨化试剂偶联得到，所述氨化试剂包括精胺(SM)和/或亚精胺(SPD)。
在本发明的一个实施例中，所述氨化试剂为精胺(SM)。
在本发明另一个实施例中，所述氨化试剂为亚精胺(SPD)。
具体地，PLLA的分子量为3-200KDa(例如3、5、10、20、40、50、60、80、100、120、140、150、160、180、200KD)。
具体地，PLLA的比旋光度范围为-155°至-160°。
具体地，PLLA的一端为羧基封端，另一端可以为氢或烷基(例如甲基)封端。
具体地，HA或其盐的分子量为100-3000KDa(例如100、200、400、500、600、800、900、1000、1500、2000、2500、3000KDa)。
具体地，HA的盐可以为其钠盐、钾盐、钙盐，特别是钠盐，即透明质酸钠。
具体地，该接枝聚合物的分子量为103-5000KDa(例如103、200、400、500、600、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000KDa)。
具体地，该接枝聚合物具有如下结构：
Figure PCTCN2022094065-appb-000001
其中，R为
Figure PCTCN2022094065-appb-000002
Figure PCTCN2022094065-appb-000003
R'为H或C 1-6烷基；
n为250-8500的整数；
m为40-3000的整数。
具体地，该接枝聚合物在水溶液中呈纳米胶束粒子状态，纳米粒子的粒径范围为10-900nm(例如10、50、100、200、300、400、500、600、700、800、900nm)。
在本发明的一些实施例中，R'为H或甲基。
在本发明第二方面，提供一种接枝聚合物的制备方法，其包括如下步骤：
(1)PLLA在活化剂存在的情况下与羟基化合物反应，得到PLLA-活性酯；
(2)使步骤(1)所得PLLA-活性酯与氨化试剂反应，得到如下所示的氨化PLLA；
Figure PCTCN2022094065-appb-000004
(3)使步骤(2)所得的氨化PLLA与HA或其盐反应，得到接枝聚合物，如式Ⅰ所示。
具体地，步骤(1)中所述羟基化合物选自：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。如此制备的PLLA-活性酯即为PLLA-NHS活性酯、PLLA-Sulfo-NHS活性酯、PLLA-HOBt活性酯或PLLA-叔丁醇活性酯中的一种或多种。
以NHS为例，在本发明的一个实施方式中，步骤(1)中所述的PLLA活性酯可为PLLA-NHS活性酯，其结构可以如下所示：
Figure PCTCN2022094065-appb-000005
具体地，R'为H或C 1-6烷基，特别是H或甲基。
具体地，步骤(1)包括：
(1-1)配制PLLA的溶液，将羟基化合物与活化剂加入该溶液中，反应；
任选地，步骤(1)还包括纯化步骤(1-2)：将步骤(1-1)所得反应产物加入沉淀剂中，分离沉淀，去除溶剂。
具体地，步骤(1-1)在惰性气体保护下进行；更具体地，所述惰性气体例如氮气、氦气。
具体地，步骤(1-1)中，投入反应时，PLLA与羟基化合物的摩尔比为1:1-20(例如1:1、1:2、1:3、1:4、1:5、1:6、1:8、1:10、1:15、1:20)，特别是1:1-5，其中PLLA摩尔量由投入的质量与其重均分子量的比值计算获得。
具体地，步骤(1-1)中，羟基化合物与活化剂的摩尔比为1:1-20(例如1:1、1:2、1:3、1:4、1:5、1:6、1:8、1:10、1:15、1:20)，特别是1:1-5。
具体地，步骤(1-1)中需要使用溶剂将PLLA溶解并进行均相反应，所使用的溶剂为卤代有机物，例如二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇等。PLLA在卤代有机物中的浓度为50-200mg/ml(质量体积浓度，例如50、60、80、90、100、110、120、140、160、180、200mg/ml)。
具体地，步骤(1-1)中的反应温度范围为15-70℃(例如15、20、25、30、35、40、45、50、55、60、65、70℃)，特别是30-50℃。
具体地，步骤(1-1)中的反应时间10-24小时(例如10、12、14、16、18、20、22、24小时)。
具体地，步骤(1-1)中所述活化剂选自：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。
具体地，水溶性碳二亚胺可以选自：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。
具体地，三苯基膦与溴化物形成的溴化鏻盐可以选自：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。
具体地，该溴化鏻盐可通过已知方法，由三苯基膦与溴化物在例如二氯甲烷的溶剂中反应获得。
具体地，碳鎓盐可以选自：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)及所述碳鎓盐中的一种或几种的混合物。
具体地，步骤(1-2)中所述沉淀剂为醚，特别是C 2-10的醚，例如乙醚、叔丁基甲基醚、甲乙醚等。
具体地，步骤(1-2)中所述沉淀剂与反应产物的体积比为1-10:1(例如1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1)。
具体地，步骤(1-2)中所述将步骤(1-1)所得反应产物加入沉淀剂中的方式为滴加。
具体地，步骤(1-2)中所述分离沉淀的方式可以采用任意合适的方式，例如抽滤。
在本发明的一些实施例中，步骤(1-2)中所述去除溶剂的方式为将分离的沉淀真空干燥；具体地，干燥温度可以为35-45℃(例如40℃)；具体地，干燥时间可以为6-48小时，真空度范围为-0.06至-0.1MPa(例如-0.08MPa)。
以NHS为例，PLLA与NHS反应得到的PLLA-NHS活性酯的反应式如下所示：
Figure PCTCN2022094065-appb-000006
具体地，R'为H或C 1-6烷基，特别是H或甲基。
在本发明的一个实施方式中，步骤(2)中所述PLLA-活性酯为PLLA-NHS活性酯，其结构可以如下所示，
Figure PCTCN2022094065-appb-000007
具体地，步骤(2)包括：
(2-1)分别配制PLLA-活性酯和氨化试剂的溶液，将PLLA-活性酯溶液加入氨化试剂溶液中，反应；
任选地，步骤(2)还包括纯化步骤：(2-2)将步骤(2-1)所得反应产物加入沉淀剂中，分离沉淀，去除溶剂。
具体地，步骤(2-1)在惰性气体保护下进行；更具体地，所述惰性气体例如氮气、氦气。
具体地，步骤(2-1)中所述PLLA-活性酯溶液的溶剂A为有机溶剂，特别是卤代有机物，例如二氯甲烷、三氯甲烷、二氯乙烷、六氟异丙醇等。
具体地，步骤(2-1)中所述氨化试剂溶液的溶剂B为有机溶剂，特别是卤代有机物，例如二氯甲烷、三氯甲烷、二氯乙烷、六氟异丙醇等。
具体地，步骤(2-1)中所述PLLA-活性酯溶液的质量百分比浓度可以为0.1-50％(例如0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.8％、1％、2％、3％、4％、5％、6％、8％、10％、20％、30％、40％、50％)。
具体地，步骤(2-1)中所述氨化试剂溶液的浓度可以为1-100mg/ml(质量体积浓度，例如1、5、10、15、20、25、30、35、40、45、50、60、70、80、90、100mg/ml)，例如5-50mg/ml。
具体地，步骤(2-1)中所述PLLA-活性酯与氨化试剂(SM或SPD)的质量比为1:0.01-15(例如1:0.01、1:0.02、1:0.04、1:0.05、1:0.06、1:0.075、1:0.08、1:0.1、1:0.2、1:0.4、1:0.5、1:0.6、1:0.8、1:1、、1:2、1:4、1:5、1:6、1:8、1:10、1:12、1:15)，特别是1:0.01-1。
具体地，步骤(2-1)中所述将PLLA-活性酯溶液加入氨化试剂溶液中的方式为滴加。
具体地，步骤(2-1)中所述反应的温度为10-70℃(例如10、20、30、40、50、60、70℃)，特别是40-70℃。
具体地，步骤(2-1)中所述反应的时间为6-48小时(例如6、12、18、24、30、36、42、48小时)，特别是18-30小时。
具体地，步骤(2-2)中所述沉淀剂为醚或醇，特别是C 2-10的醚或C 1-6的醇，例如乙醚、叔丁基甲基醚、甲乙醚，或甲醇、乙醇、异丙醇等。
具体地，步骤(2-2)中所述沉淀剂与反应产物的体积比为1-10:1(例如1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1)。
具体地，步骤(2-2)中所述将步骤(2-1)所得反应产物加入沉淀剂中的方式为滴加。
具体地，步骤(2-2)中所述分离沉淀的方式可以采用任意合适的方式，例如抽滤。
在本发明的一些实施例中，步骤(2-2)中所述去除溶剂的方式为将分离的沉淀真空干燥；具体地，干燥温度可以为35-45℃；具体地，干燥时间可以为6-48小时，真空度范围为-0.06至-0.1MPa。
具体地，步骤(3)包括：
(3-1)分别配制HA或其盐的溶液和步骤(2)所得氨化PLLA的溶液，将氨化PLLA的溶液加入到HA或其盐的溶液中；
(3-2)将步骤(3-1)所得混合物超声处理；
(3-3)向步骤(3-2)所得体系中加入活化剂(以及助剂，如果必要的话)，反应；
任选地，步骤(3)还包括纯化步骤：(3-4)将步骤(3-3)所得反应产物(例如通过透析或超滤)去除小分子反应物，冷冻干燥；
任选地，步骤(3)进一步包括精制步骤：(3-5)将步骤(3-4)所得产物浸入溶剂E中，除去溶剂E(可重复多次)，真空干燥。
具体地，步骤(3-1)中所述HA或其盐的溶液的溶剂C为水，即该HA或其盐的溶液为HA或其盐的水溶液。
具体地，步骤(3-1)中所述HA或其盐的溶液的质量百分比浓度为0.1-50％(例如0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.8％、1％、2％、3％、4％、5％、6％、8％、10％、20％、30％、40％、50％)。
具体地，步骤(3-1)中所述HA或其盐的溶液还包括高分子乳化剂，例如聚乙二醇、聚乙烯醇；更具体地，聚乙二醇分子量可以为1000-50000Da(例如1000、2000、4000、6000、8000、10000、15000、20000、30000、40000、50000Da)，特别是1000-20000Da；更具体地，聚乙烯醇分子量可以为5000-500000Da(例如5000、10000、20000、40000、50000、60000、80000、100000、200000、300000、400000、500000Da)，特别是5000-50000Da，醇解度可以为60-100％(例如78％、88％、98％)，特别是87-89％。
更具体地，高分子乳化剂占HA或其盐溶液总质量的0.1-30％(例如0.1％、0.5％、1％、2％、3％、4％、5％、6％、8％、10％、20％、30％)，特别是0.5-10％。
具体地，步骤(3-1)中所述HA或其盐的溶液的pH为5.0-6.5(例如5.0、5.5、6.0、6.5)，该pH可通过例如盐酸调节得到。
具体地，步骤(3-1)中所述氨化PLLA溶液的溶剂D为有机溶剂，特别是卤代有机物，例如二氯甲烷、三氯甲烷、二氯乙烷、六氟异丙醇等。
具体地，溶剂A、溶剂B和溶剂D可以相同或不同。
具体地，步骤(3-1)中所述氨化PLLA溶液的质量百分比浓度为0.1-50％(例如0.1％、0.2％、0.3％、0.4％、0.5％、0.6％、0.8％、1％、2％、3％、4％、5％、6％、8％、10％、20％、30％、40％、50％)。
具体地，步骤(3-1)中HA或其盐与氨化PLLA的质量比为1:0.01-2(例如1:0.01、1:0.05、1:0.1、1:0.2、1:0.3、1:0.4、1:0.5、1:0.6、1:0.7、1:0.8、1:0.9、1:1、1:1.5、1:2)，特别是1:0.1-1。
具体地，步骤(3-2)中所述超声处理的时间可以为5-60分钟(例如5、10、20、30、40、50、60分钟)。
具体地，步骤(3-2)中所述超声处理的功率可以为1000-5000W(例如1000、2000、3000、4000、5000W)。
具体地，步骤(3-2)中超声处理后，氨化PLLA溶液在透明质酸水溶液中分散成纳米液珠，该纳米液珠的粒径范围为10-900nm(例如10、50、100、150、200、250、300、400、500、600、700、800、900nm)。
具体地，步骤(3-3)中所述活化剂加入量为氨化PLLA质量的0.01-100％(例如0.01％、0.1％、5％、10％、20％、30％、40％、50％、60％、70％、80％、90％、100％)。
具体地，步骤(3-3)中所述活化剂选自：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。
具体地，水溶性碳二亚胺可以选自：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。当使用水溶性碳二亚胺活化剂时，可以同时与助剂联合使用以提高交联反应效率。具体地，所述助剂的加入量可以为碳二亚胺质量的10-80％。具体地，所述助剂选自：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。
在本发明的一个实施方式中，步骤(3-3)为：向步骤(3-2)所得体系中加入活化剂和助剂，反应。
在本发明的一个实施例中，活化剂为EDC，助剂为NHS。
具体地，三苯基膦与溴化物形成的溴化鏻盐可以选自：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；在本发明的一个实施例中，溴化鏻盐为三苯基膦与N-溴代琥珀酰亚胺形成的鏻盐。
具体地，该溴化鏻盐可通过已知方法，由三苯基膦与溴化物在例如二氯甲烷的溶剂中反应获得。
具体地，碳鎓盐可以选自：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)及所述碳鎓盐中的一种或几种的混合物；在本发明的一个实施例中，碳鎓盐为HATU。
具体地，步骤(3-3)中所述反应的温度为10-70℃(例如10、20、30、40、50、60、70℃)，特别是30-40℃。
具体地，步骤(3-3)中所述反应的时间为6-48小时(例如6、12、18、24、30、36、42、48小时)，特别是18-30小时。
具体地，步骤(3-4)中所述去除小分子反应物的方式可以为透析或超滤，透析或超滤的时间可以为6-48小时。
具体地，步骤(3-5)中所述溶剂E为有机溶剂，特别是卤代有机物，例如二氯甲烷、三氯甲烷、二氯乙烷、六氟异丙醇等；在本发明的一个实施例中，溶剂E为二氯甲烷。
在本发明的一个实施方式中，接枝聚合物的制备方法包括如下反应路线：
Figure PCTCN2022094065-appb-000008
在本发明第三方面，提供一种氨化PLLA，其具有如下结构，其可作为制备接枝聚合物的中间体：
Figure PCTCN2022094065-appb-000009
其中，R为
Figure PCTCN2022094065-appb-000010
Figure PCTCN2022094065-appb-000011
R'为H或C 1-6烷基；
m为40-3000的整数。
在本发明的一些实施例中，R'为H或甲基。
具体地，氨化PLLA的分子量为3-200KDa(例如3、5、10、20、40、50、60、80、100、120、140、150、160、180、200KD)。
具体地，PLLA的比旋光度范围为-155°至-160°。
在本发明第四方面，提供第三方面所述的氨化PLLA的制备方法，其包括本发明第二方面中所述的步骤(1)和(2)。
在本发明第五方面，提供一方面所述的接枝聚合物、第二方面所述的方法制备的接枝聚合物在制备药物中的应用。
具体地，该药物包含活性成分和所述接枝聚合物作为药物载体，以实现缓释、控释、靶向给药等目的。
在本发明的一个实施方式中，该药物为抗肿瘤药物，其活性成分可以为，例如，伊沙匹隆、丝裂霉素、普卡霉素、博来霉素、匹蒽醌、氨柔比星、伐柔比星、吡柔比星、米托蒽醌、伊达比星、佐柔比星、阿柔比星、表柔比星、柔红霉素、多柔比星、更生霉素；曲贝替定、卡巴他赛、紫杉醇、多西他赛、地美秋霉素、替尼泊苷、依托泊苷、长春福林、长春氟宁、长春瑞滨、长春地辛、长春新碱、长春碱；氟尿苷、曲氟尿苷、替加氟、氟尿嘧啶、地西他滨、阿扎胞苷、卡培他滨、吉西他滨、卡莫氟、阿糖胞苷、奈拉滨、氯法拉滨、氟达拉滨、克拉屈滨、硫鸟嘌呤、巯基嘌呤、普拉曲沙、培美曲塞、雷替曲塞、甲氨蝶呤；达卡巴嗪、替莫唑胺、哌泊溴曼、米托溴醇、依托糖苷、尿嘧啶芥、雷莫司汀、尼莫司汀、氟替莫司汀、链脲佐菌素、司莫司汀、洛莫司汀、卡莫司汀、卡波醌、三嗪醌、噻替哌、甘露硫丹、曲硫丹、白消安、苯达莫司汀、泼尼莫司汀、曲磷酰胺、异环磷酰胺、甲氯乙胺、美法仑、苯丁酸氮芥、环磷酰胺；顺铂、卡铂、奥沙利铂、沙铂、聚铂等。
在本发明第六方面，提供一种药物组合物，其包含活性成分和一方面所述的接枝聚合物、第二方面所述的方法制备的接枝聚合物作为药物载体。
在本发明第七方面，提供一方面所述的接枝聚合物、第二方面所述的方法制备的接枝聚合物在制备组织工程材料中的应用。
具体地，该组织工程材料可以为骨组织工程材料、软骨组织工程材料、角膜组织工程材料、心血管组织工程材料、肝组织工程材料等。
在本发明第八方面，提供一方面所述的接枝聚合物、第二方面所述的方法制备的接枝聚合物在制备软组织填充剂中的应用。
具体地，该软组织填充剂可用于消除皱纹(如眼周皱纹、额头纹、眉间纹、口周纹、鼻唇沟、泪沟、法令纹、颈纹、手部皱纹、妊娠纹等)、抗衰老、疤痕及创伤修复等。
与现有技术相比，本发明具有以下突出特点：
(1)通过以内源性多胺为偶联剂，内源性多胺作为一种无毒无害，更为安全的氨化试剂，通过此类氨化试剂将PLLA进行氨化，由于氨化后的PLLA不易水解，使其更容易在水环境下与透明质酸或其盐发生偶联反应，能有效促进PLLA与HA或其盐进行偶联；同时，还能发挥亚精胺的各种生物活性，保证所有组分均可降解代谢，且降解产物均为人体内源性物质，从而提高了接枝聚合物在制备药物、组织工程材料或软组织填充剂等领域方面的易用性，适于推广应用。
(2)通过纳米分散非均相偶联技术(NDHC)进行接枝反应，具体采用卤代有机物作为溶剂得到氨化PLLA有机溶液，再结合超声的方式将氨化PLLA的有机溶液在透明质酸或其盐的水溶液中分散成纳米液珠，控制纳米液珠的粒径范围为10-900nm，从而扩大两相接触面；另外，纳米液珠中的有机溶剂通过温度选择，控制其挥发，使暴露的氨化PLLA逐步与HA或其盐进行接枝偶联，形成PLLA-HA纳米颗粒；而现有的接枝方式中，通常是PLLA为固相，透明质酸或其盐的溶液为液相的固液两相接枝法，由于固相颗粒大小不可控，无法进行内部接枝，只能在表面接枝；本发明通过双液相接枝体系可将液珠分散为纳米颗粒，更有利于内部接枝，提高接枝效率。
(3)本发明所制备的HA-b-PLLA接枝共聚物，接枝效率高，并且粒径分布均匀，粒径尺寸为50-900nm，该接枝聚合物稳定性好，具有两亲性，可应用在药物载体、组织工程材料以及软组织修复等领域。
附图说明
图1所示为本发明制备的PLLA-NHS活化酯的 1H NMR图谱。
图2所示为本发明制备的氨化PLLA(SPD氨化)的 1H NMR图谱。
图3所示为本发明制备的HA-b-PLLA的 1H NMR图谱。
图4所示为本发明制备的HA-b-PLLA的FT-IR图谱。
图5所示为本发明中制备的HA-b-PLLA在水溶液中的粒径分布图。
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。
本发明中部分物质与其缩写对应如下：
透明质酸  HA
聚左旋乳酸  PLLA
N-羟基琥珀酰亚胺  NHS
聚左旋乳酸-琥珀酰亚胺酯  PLLA-NHS
透明质酸-聚左旋乳酸接枝聚合物  HA-b-PLLA
精胺  SM
亚精胺  SPD
1-乙基-3-(3-二甲基氨基丙基)碳二亚胺  EDC
O-(7-氮杂苯并三氮唑-1-基)-二(二甲氨基)碳鎓六氟磷酸盐  HATU
4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐  DMTMM
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅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作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。
以下实施例中所用的透明质酸或其盐、聚左旋乳酸、NHS、活化剂等主要原料均为市售产品。
实施例1：PLLA-NHS活性酯的制备
称取15.00g PLLA(重均分子量Mw约为50KDa，PDI：2.12，比旋光度：-157°)在氮气保护下加入到三口瓶中，并加入150ml的三氯甲烷溶液，搅拌30min至完全溶解。加入活化剂EDC 93mg，NHS 69mg，在40℃条件下反应16h。反应结束将反应溶液滴加到400ml的乙醚中，然后抽滤，所得滤饼放入40℃真空干燥箱中，-0.08MPa，24h除去残留溶剂，将所得PLLA-NHS干燥保存，产率约90％。PLLA-NHS的 1H NMR图谱如图1所示。
实施例2：PLLA-NHS活性酯与亚精胺的反应
抽取30ml三氯甲烷加入到氮气保护下的三口瓶中，移液枪吸取0.6g亚精胺加入到烧瓶中搅匀，与此同时称取8.00g PLLA-NHS(实施例1制备)在氮气保护下溶解于50ml三氯甲烷中，然后将该溶液滴加到亚精胺溶液中，60℃反应24h。反应结束将反应溶液滴加到200ml的乙醚中，然后抽滤，所得滤饼放入40℃真空干燥箱中，-0.08MPa，24h除去残留溶剂，将所得氨化PLLA干燥保存，产率约75％。氨化PLLA的 1H NMR图谱如图2所示。亚精胺SPD氨化PLLA形成两种氨化连接方式的氨化PLLA，两者 1H NMR图谱的出峰位置基本一致。
实施例3：PLLA-NHS活性酯与精胺的反应
抽取40ml二氯甲烷加入到氮气保护下的三口瓶中，移液枪吸取1.0g精胺加入到烧瓶中搅匀，与此同时称取5.00g PLLA-NHS(实施例1制备)在氮气保护下溶解于50ml 六氟异丙醇中，然后将该溶液滴加到精胺溶液中，20℃反应45h。反应结束将反应溶液滴加到400ml的乙醇中，然后抽滤，所得滤饼放入40℃真空干燥箱中，-0.08MPa，24h除去残留溶剂，将所得氨化PLLA干燥保存，产率约85％。
实施例4：氨化PLLA(SPD氨化)与HA的偶联反应(HATU活化)
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为1,000KDa)0.95g与PEG10000 0.95g，再加95ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至5.0备用。同时，称取0.50g实施例2中获得的氨化PLLA，溶解于10ml六氟异丙醇中，充分溶解。将事先溶解的氨化PLLA溶液加入该透明质酸钠溶液中，随后3.2kW超声15min，此时氨化PLLA的六氟异丙醇溶液液珠粒径中位数为100nm，加入50mg HATU，35℃搅拌反应24h，所得溶液使用截留分子量为30KDa的透析袋透析24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得产物真空干燥除去二氯甲烷即可得到HA-b-PLLA。
该产物标记为HA-b-PLLA1，HA-b-PLLA1的 1H NMR图谱如图3所示(图2中两种氨化PLLA与HA形成的两种HA-b-PLLA，两者 1H NMR图谱的出峰位置基本一致，所以图3选取其中一种作为示意)。图3中显示，化学位移1.8-2.0ppm附近为透明质酸中乙酰氨基上的甲基峰，化学位移4.9-5.1ppm附近为左旋聚乳酸上的次甲基峰，两峰的存在可以证明PLLA与透明质酸成功接枝，并可根据此两处峰的积分面积计算PLLA与HA的接枝效率，计算得到PLLA与HA的接枝效率为80％左右。其FT-IR图谱如图4所示。从图4可以看出，HA-b-PLLA1的红外光谱图中，存在从属PLLA的1760cm -1处的(C＝O)特征伸缩振动峰与从属透明质酸的1630cm -1处的(C＝O)特征伸缩振动峰，这表明PLLA成功接枝到了HA分子上。该产物为雪花状松散粉末，其在水溶液中的胶束粒径分布如图5所示，通过图5的频度曲线可以得到，HA-b-PLLA1的粒径中位数约为520nm，并且从图中可以看出，频度曲线为尖锐的单峰，进一步表明所制备的接枝聚合物粒径非常均匀。
实施例5：氨化PLLA(SPD氨化)与HA的偶联反应(EDC活化)
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为900KDa)2.0g与PEG200001.0g，再加100ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至5.5备用。同时，称取0.50g实施例2中获得的氨化PLLA，溶解于10ml二氯甲烷中，充分溶解。将事先溶解的氨化PLLA溶液加入该透明质酸钠溶液中，随后2.2kW超声30min，此时氨化PLLA的二氯甲烷溶液液珠粒径中位数为200nm，加入100mg EDC与50mg NHS，35℃搅拌反应24h，所得溶液使用截留分子量为30KDa的中空纤维超滤24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得产物真空干燥除去二氯甲烷即可得到HA-b-PLLA。
该产物标记为HA-b-PLLA2，通过 1H NMR图谱中，对化学位移1.8-2.0ppm附近及4.9-5.1ppm附近的峰进行积分，计算得到HA-b-PLLA2中PLLA与HA的接枝效率为25％左右。HA-b-PLLA2的FT-IR图谱如图4所示，从图4可以看出，HA-b-PLLA2的红外光谱图中，存在从属PLLA的1760cm -1处的(C＝O)特征伸缩振动峰与从属透明质酸的1630cm -1处的(C＝O)特征伸缩振动峰，这表明PLLA成功接枝到了HA分子上。其在水溶液中的胶束粒径中位数约为180nm。
实施例6：氨化PLLA(SM氨化)与HA的偶联反应(鏻盐活化)
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。
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为2,000KDa)3.0g与PEG20002.0g，再加100ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至 6.0备用。同时，称取0.50g实施例3中获得的氨化PLLA，溶解于10ml三氯甲烷中，充分溶解。将事先溶解的氨化PLLA溶液加入该透明质酸钠溶液中，随后2.2kW超声30min，此时氨化PLLA的三氯甲烷溶液液珠粒径中位数为150nm，加入200mg溴化琥珀酰亚胺三苯基鏻盐，35℃搅拌反应24h，所得溶液使用截留分子量为10KDa的中空纤维超滤24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得产物真空干燥除去二氯甲烷即可得到HA-b-PLLA。
该产物标记为HA-b-PLLA3，通过 1H NMR图谱中，对化学位移1.8-2.0ppm附近及4.9-5.1ppm附近的峰进行积分，计算得到HA-b-PLLA3中PLLA与HA的接枝效率为56％左右。HA-b-PLLA3的FT-IR图谱如图4所示。从图4可以看出，HA-b-PLLA3的红外光谱图中，存在从属PLLA的1760cm -1处的(C＝O)特征伸缩振动峰与从属透明质酸的1630cm -1处的(C＝O)特征伸缩振动峰，这表明PLLA成功接枝到了HA分子上。其在水溶液中的胶束粒径中位数约为450nm。
实施例7：氨化PLLA(SM氨化)与HA的偶联反应(DMTMM活化)
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为1,500KDa)2.5g与PVA(重均分子量Mw约为10KDa，醇解度88％)2.5g，再加100ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至6.0备用。同时，称取0.50g实施例3中获得的氨化PLLA，溶解于10ml三氯甲烷中，充分溶解。将事先溶解的氨化PLLA溶液加入该透明质酸钠溶液中，随后1.2kW超声60min，此时氨化PLLA的三氯甲烷溶液液珠粒径中位数为250nm，加入300mg DMTMM，35℃搅拌反应24h，所得溶液使用截留分子量为10KDa的中空纤维超滤24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得产物真空干燥除去二氯甲烷即可得到HA-b-PLLA。
通过 1H NMR图谱中，对化学位移1.8-2.0ppm附近及4.9-5.1ppm附近的峰进行积分，计算得到PLLA与HA的接枝效率为42％左右。其在水溶液中的胶束粒径中位数约为240nm。
实施例8：氨化PLLA(SPD氨化)与HA的偶联反应(HATU活化)
其他步骤均与实施例4相同，唯一区别是加入50mg HATU后，在55℃搅拌反应18h。通过 1H NMR图谱中，对化学位移1.8-2.0ppm附近及4.9-5.1ppm附近的峰进行积分，计算得到PLLA与HA的接枝效率为65％左右。其在水溶液中的胶束粒径中位数约为420nm。
对比例1：先氨化透明质酸再与PLLA-NHS活化酯进行偶联反应
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为900KDa)2.0g，再加100ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至5.5备用。同时，称取0.50g亚精胺并溶解于透明质酸水溶液中，加入1.0g EDC与0.2g NHS，35℃搅拌反应24h。所得溶液使用截留分子量为10KDa的中空纤维超滤24h，冷冻干燥除去水分，获得氨化透明质酸。
将实施例1中获得的PLLA-NHS活性酯，溶解于10ml二氯甲烷中，充分溶解。将氨化透明质酸1.5g与PEG2000 1.0g溶解于100ml水中，搅拌均匀，事先溶解的PLLA-NHS活性酯溶液加入该氨化透明质酸钠溶液中，随后2.2kW超声30min，此时PLLA-NHS活性酯的二氯甲烷溶液液珠粒径中位数为200nm，35℃搅拌反应24h，所得溶液使用截留分子量为10KDa的中空纤维超滤24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得产物真空干燥除去二氯甲烷即可得到HA-b-PLLA。通过 1H NMR图谱中，对化学位移1.8-2.0ppm附近及4.9-5.1ppm附近的峰进行积分，计算得到PLLA与HA的接枝效率仅为0.8％左右。
对比例2：透明质酸与PLLA-NHS活化酯直接进行偶联反应
在250ml烧杯中加入透明质酸钠(重均分子量Mw约为1,500KDa)2.5g与PVA(重均分子量Mw约为10KDa，醇解度88％)2.5g，再加100ml水，搅拌均匀，然后放置1-2h使其完全溶解，用盐酸溶液调节pH至6.0备用。同时，称取0.50g实施例1中获得的PLLA-NHS活性酯，溶解于10ml三氯甲烷中，充分溶解。将事先溶解的PLLA-NHS活性酯溶液加入该透明质酸钠溶液中，随后1.2kW超声60min，此时PLLA-NHS活性酯的三氯甲烷溶液液珠粒径中位数为250nm，35℃搅拌反应24h，所得溶液使用截留分子量为10KDa的中空纤维超滤24h，冷冻干燥除去水分，将得到海绵状产物放入40ml二氯甲烷中浸泡1h，倒去溶剂，重复浸泡三次，然后将所得物质真空干燥除去二氯甲烷即可得到产物。通过 1H NMR图谱，未发现4.9-5.1ppm附近的存在PLLA中的次甲基峰，无法进行积分，证明PLLA未能与HA进行偶联，无法得到HA-b-PLLA的接枝共聚物。
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. 一种接枝聚合物，其主要由PLLA与HA或其盐通过氨化试剂偶联得到，其特征在于，所述氨化试剂包括精胺和/或亚精胺；
    优选地，所述接枝聚合物具有如下结构：
    Figure PCTCN2022094065-appb-100001
    其中，R为
    Figure PCTCN2022094065-appb-100002
    Figure PCTCN2022094065-appb-100003
    R'为H或C 1-6烷基；
    n为250-8500的整数；
    m为40-3000的整数；
    优选地，PLLA的分子量为3-200KDa；
    优选地，HA或其盐的分子量为100-3000KDa。
  2. 一种接枝聚合物的制备方法，其包括如下步骤：
    (1)PLLA在活化剂存在的情况下与羟基化合物反应，得到PLLA-活性酯；
    (2)使步骤(1)所得的PLLA-活性酯与氨化试剂反应，得到如下所示的氨化PLLA，所述氨化试剂包括精胺和/或亚精胺；
    Figure PCTCN2022094065-appb-100004
    其中，R为
    Figure PCTCN2022094065-appb-100005
    Figure PCTCN2022094065-appb-100006
    R'为H或C 1-6烷基；
    m为40-3000的整数；
    (3)使步骤(2)所得的氨化PLLA与透明质酸HA或其盐反应，得到接枝聚合物；
    优选地，步骤(1)中所述PLLA-活性酯为PLLA-NHS活性酯、PLLA-Sulfo-NHS活性酯、PLLA-HOBt活性酯或PLLA-叔丁醇活性酯中的一种或多种。
  3. 如权利要求2所述的方法，其特征在于，步骤(1)包括：
    (1-1)配制PLLA的溶液，将羟基化合物与活化剂加入该溶液中，反应；
    优选地，步骤(1)还包括纯化步骤：(1-2)将步骤(1-1)所得反应产物加入沉淀剂中，分离沉淀，去除溶剂。
  4. 如权利要求3所述的方法，其特征在于，步骤(1-1)在惰性气体保护下进行；
    优选地，步骤(1-1)中所述羟基化合物选自：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；
    优选地，步骤(1-1)中，投入反应时，PLLA与羟基化合物的摩尔比为1:1-20；
    优选地，步骤(1-1)中羟基化合物与活化剂的摩尔比为1:1-20；
    优选地，步骤(1-1)中所述PLLA溶液的溶剂为卤代有机物，优选为二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇；
    优选地，步骤(1-1)中的反应温度范围为15-70℃，反应时间10-24小时。
  5. 如权利要求3所述的方法，其特征在于，优选地，步骤(1-2)中所述沉淀剂为醚，优选为C 2-10的醚，进一步优选为乙醚、叔丁基甲基醚或甲乙醚；
    优选地，步骤(1-2)中所述沉淀剂与反应产物的体积比为1-10:1。
  6. 如权利要求2所述的方法，其特征在于，步骤(2)包括：
    (2-1)分别配制PLLA-活性酯和氨化试剂的溶液，将PLLA-活性酯溶液加入氨化试剂溶液中，反应；
    优选地，纯化步骤：(2-2)将步骤(2-1)所得反应产物加入沉淀剂中，分离沉淀，去除溶剂。
  7. 如权利要求6所述的方法，其特征在于，步骤(2-1)在惰性气体保护下进行；
    优选地，步骤(2-1)中所述PLLA-活性酯溶液的溶剂A为卤代有机物，优选为二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇；
    优选地，步骤(2-1)中所述氨化试剂溶液的溶剂B为卤代有机物，优选为二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇；
    优选地，步骤(2-1)中所述PLLA-活性酯与氨化试剂的质量比为1:0.01-15；
    优选地，步骤(2-1)中所述反应的温度为10-70℃，更优选为40-70℃；所述反应时间为6-48h，更优选为18-30h。
  8. 如权利要求6所述的方法，其特征在于，步骤(2-2)中所述沉淀剂为醚或醇，优选为乙醚、叔丁基甲基醚、甲乙醚、甲醇、乙醇或异丙醇；
    优选地，步骤(2-2)中所述沉淀剂与反应产物的体积比为1-10:1。
  9. 如权利要求2所述的方法，其特征在于，步骤(3)包括：
    (3-1)分别配制HA或其盐的溶液和步骤(2)所得氨化PLLA的溶液，将氨化PLLA的溶液加入到HA或其盐溶液中；
    (3-2)将步骤(3-1)所得混合物超声处理；
    (3-3)向步骤(3-2)所得体系中加入活化剂，反应；
    优选地，纯化步骤：(3-4)将步骤(3-3)所得反应产物去除小分子反应物，冷冻干燥；
    更优选地，进一步精制步骤：(3-5)将步骤(3-4)所得产物浸入溶剂E中，除去溶剂E，真空干燥。
  10. 如权利要求9所述的方法，其特征在于，步骤(3-1)中所述HA或其盐的溶液的溶剂C为水；
    优选地，步骤(3-1)中所述HA或其盐的溶液还包括高分子乳化剂；更优选地，所述高分子乳化剂为聚乙二醇或聚乙烯醇；
    优选地，步骤(3-1)中所述HA或其盐的溶液的pH为5.0-6.5；
    优选地，步骤(3-1)中所述氨化PLLA溶液的溶剂D为卤代有机物，优选为二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇；
    优选地，步骤(3-1)中HA或其盐与氨化PLLA的质量比为1:0.01-2。
  11. 如权利要求9所述的方法，其特征在于，步骤(3-2)中所述超声处理的功率为1000-5000W；和/或，步骤(3-2)中所述超声处理的时间为5-60分钟；
    优选地，步骤(3-3)为：向步骤(3-2)所得体系中加入活化剂和助剂，反应，所述助剂选自：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；
    优选地，步骤(3-3)中所述活化剂加入量为氨化PLLA质量的0.01-100％；
    优选地，步骤(3-3)中所述反应的温度为10-70℃，更优选为30-40℃；所述反应时间为6-48h，优选为18-30h；
    优选地，步骤(3-4)中所述去除小分子反应物的方式为透析或超滤；
    优选地，步骤(3-5)中所述溶剂E为卤代有机物，优选为二氯甲烷、三氯甲烷、二氯乙烷或六氟异丙醇。
  12. 如权利要求3或9所述的方法，其特征在于，所述步骤(1-1)或步骤(3-3)中所述活化剂选自：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；
    优选地，所述活化剂为水溶性碳二亚胺，其选自：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。
  13. 如权利要求12所述的方法，其特征在于，所述活化剂为三苯基膦与溴化物形成的溴化鏻盐，其选自：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；
    优选地，步骤(3-3)中所述活化剂为碳鎓盐，其选自：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)及所述碳鎓盐中的一种或几种的混合物。
  14. 权利要求1所述的接枝聚合物、权利要求2-13任一项所述的方法制备的接枝聚合物在制备药物、组织工程材料或软组织填充剂中的应用。
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WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
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US20170043051A1 (en) * 2015-06-11 2017-02-16 Case Western Reserve University Dry Spray on Hemostatic System
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WO2014064632A1 (en) * 2012-10-24 2014-05-01 Teoxane Dermal injectable sterile composition
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