Graft polymer material and preparation method and application thereof

Abstract

The application discloses a grafted polymer material and a preparation method and application thereof. The application realizes coupling of poly-L-lactic acid (PLLA) and hyaluronic acid (HA) or a salt thereof under heterogeneous conditions by taking endogenous polyamine as a coupling agent and through nano-disperse heterogeneous coupling technology (NDHC) to obtain a HA-b-PLLA graft copolymer. The prepared HA-b-PLLA graft copolymer has high grafting efficiency and has amphiphilicity, and can be applied to fields of drug carriers, tissue engineering materials and soft tissue repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a grafted polymer material, its preparation method, and its application. Background Technology

[0002] Hyaluronic acid (HA), also known as sodium hyaluronate, is a glycosaminoglycan composed of D-glucuronic acid linked by a β-1,4 glycosidic bond and N-acetylglucosamine linked by a β-1,3 glycosidic bond. HA is widely used in cosmetics and ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects. Hyaluronic acid is a naturally occurring substance in the body, possessing good biocompatibility and certain biological activity. However, exogenous hyaluronic acid is degraded in vivo by hyaluronidase, resulting in a shorter residence time in the body and thus a shorter treatment effect, requiring multiple injections to achieve the desired therapeutic effect. To prevent hyaluronic acid from being degraded by hyaluronidase, it can be modified in two ways to achieve cross-linking. The first method involves using chemical cross-linking agents to cross-link hyaluronic acid molecules, forming a spatial network structure. This dense, rigid network structure inhibits the degradation of hyaluronidase and other enzymes, prolonging the residence time of exogenous hyaluronic acid in the body and ensuring both biocompatibility and therapeutic efficacy. The second method involves hydrophobic modification of the hyaluronic acid side chains, reducing the affinity of hyaluronidase for hyaluronic acid. This effectively increases the probability of hyaluronidase contact with hyaluronic acid, thereby increasing its residence time in the body.

[0003] Currently, coupling composites of hyaluronic acid and absorbable polyester have also been reported, with graft copolymers of hyaluronic acid and lactic acid-glycolic acid (PLGA) being the most frequently reported. Due to PLGA's excellent solubility in various polar solvents, a homogeneous reaction can be carried out using a mixture of water and polar organic solvents when reacting PLGA with HA, resulting in a high grafting rate and easily controllable reaction conditions. Furthermore, because PLGA has a low melting point, a melt grafting reaction can be performed at a lower temperature, enabling coupling between PLGA and HA. Chinese patent document CN107286608A discloses a method for coupling PLGA and HA via a melt method, where PLGA and HA are coupled using the coupling agent cystamine. Chinese patent CN104162169B discloses the coupling of cystamine-derived PLGA with HA in a mixed solvent of N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and water. Jung Kyu Park et al. (Park JK, Yeom J, Oh EJ, 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.) disclosed a method for coupling HA and PLGA using adipic acid dihydrazide as a coupling agent. This method also involves amination of hyaluronic acid with adipic acid dihydrazide and then coupling the active hydroxysuccinimide (NHS) ester of PLGA in a single DMSO solution.

[0004] In addition, there are many reports on graft copolymers 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. When grafting with HA, a mixed solvent of water and organic solvents can also be used. HA and PLA undergo a homogeneous coupling reaction, resulting in a high grafting rate, and the reaction conditions are relatively easy to control. Chinese patent CN104056275B discloses a method of obtaining an HA-PLA graft copolymer by amination of the terminal aldehyde groups of HA and coupling it with PLA in a mixed solvent of DMSO and water. 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 a method that first quaternizes HA, then carries out the coupling reaction of PLA and HA in a composite solution of DMSO and dichloromethane, and removes the quaternary ammonium salt by cation exchange resin after the reaction. Giovanna Pitarresi et al. (Pitarresi G, Palumbo FS, Fiorica C, et al. Injectable in situforming 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 dichloromethane to obtain HA-PLA graft copolymer.

[0005] In summary, the reported preparation methods for HA-polyester graft polymers currently include two approaches: one involves coupling hyaluronic acid (HA) with the NHS-activated ester of the polyester after amination, and the other involves directly coupling the hydroxyl groups of hyaluronic acid with the NHS-activated ester of the polyester. The second method suffers from low reaction efficiency due to the low reactivity of the hydroxyl groups. The first method uses exogenous polyamines, which are inherently biotoxic, posing safety risks to the graft polymers prepared by this method. Furthermore, the NHS-activated ester of the polyester is prone to hydrolysis in water, resulting in low coupling efficiency when coupled with high molecular weight hyaluronic acid (molecular weight greater than 100 kDa), which is only soluble in water.

[0006] Poly-L-lactic acid (PLLA) is a polymer of L-lactic acid, which can be degraded into L-lactic acid in vivo. As a metabolite of human polysaccharides, PLLA exhibits good tissue compatibility and is widely used in bone screws, bone plates, and medical aesthetics. Due to its excellent crystallinity, PLLA has poor solubility, dissolving only in a limited number of organic solvents, such as halogenated organic compounds (e.g., chloroform, dichloromethane, hexafluoroisopropanol). Hyaluronic acid (HA) or its salts exhibit significantly reduced solubility in organic solvents when their molecular weight exceeds 100 kDa, dissolving only in water. Since the aforementioned halogenated organic compounds that can dissolve PLLA are all poorly soluble in water, the coupling reaction between PLLA and hyaluronic acid (HA) or its salts is difficult, and grafting typically occurs only on the surface, resulting in low grafting efficiency and unstable products. Summary of the Invention

[0007] To address the aforementioned problems and improve grafting efficiency, this invention provides a grafted polymer material, its preparation method, and its applications. By utilizing nano-disperse heterogeneous coupling (NDHC) technology, an amination-treated polyester is coupled with hyaluronic acid or its salt to prepare a HA-b-PLLA grafted polymer with high grafting efficiency.

[0008] In a first aspect of the invention, a graft polymer is provided, which is mainly obtained by coupling PLLA with HA or its salt via an amination agent, said amination agent including spermine (SM) and / or spermidine (SPD).

[0009] In one embodiment of the present invention, the amination reagent is spermine (SM).

[0010] In another embodiment of the present invention, the amination reagent is spermidine (SPD).

[0011] Specifically, the molecular weight of PLLA is 3-200 kDa (e.g., 3, 5, 10, 20, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200 kDa).

[0012] Specifically, the specific rotation of PLLA ranges from -155° to -160°.

[0013] Specifically, one end of PLLA is capped with a carboxyl group, and the other end can be capped with hydrogen or an alkyl group (e.g., methyl).

[0014] Specifically, the molecular weight of HA or its salts is 100-3000 kDa (e.g., 100, 200, 400, 500, 600, 800, 900, 1000, 1500, 2000, 2500, 3000 kDa).

[0015] Specifically, HA can be a sodium salt, potassium salt, or calcium salt, especially a sodium salt, namely sodium hyaluronate.

[0016] Specifically, the molecular weight of the graft polymer is 103-5000 kDa (e.g., 103, 200, 400, 500, 600, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 kDa).

[0017] Specifically, the grafted polymer has the following structure:

[0018]

[0019] Where R is

[0020] R' is H or C 1-6 alkyl;

[0021] n is an integer between 250 and 8500;

[0022] m is an integer between 40 and 3000.

[0023] Specifically, the grafted polymer exists in aqueous solution as nanomicelle particles with a particle size range of 10-900 nm (e.g., 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 nm).

[0024] In some embodiments of the present invention, R' is H or methyl.

[0025] In a second aspect of the present invention, a method for preparing a grafted polymer is provided, comprising the following steps:

[0026] (1) PLLA reacts with hydroxyl compounds in the presence of an activator to obtain PLLA-active ester;

[0027] (2) React the PLLA-active ester obtained in step (1) with an amination reagent to obtain the amination PLLA shown below;

[0028]

[0029] (3) React the aminated PLLA obtained in step (2) with HA or its salt to obtain a grafted polymer, as shown in Formula I.

[0030] Specifically, the hydroxyl compound in step (1) is selected from one or more of the following: N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt). The PLLA-active ester thus prepared is one or more of the following: PLLA-NHS active ester, PLLA-Sulfo-NHS active ester, PLLA-HOBt active ester, or PLLA-tert-butanol active ester.

[0031] Taking NHS as an example, in one embodiment of the present invention, the PLLA active ester mentioned in step (1) can be a PLLA-NHS active ester, the structure of which can be as follows:

[0032]

[0033] Specifically, R' is H or C. 1-6 Alkyl groups, especially H or methyl groups.

[0034] Specifically, step (1) includes:

[0035] (1-1) Prepare a solution of PLLA by adding the hydroxyl compound and activator to the solution and reacting.

[0036] Optionally, step (1) further includes a purification step (1-2): the reaction product obtained in step (1-1) is added to a precipitant, the precipitate is separated, and the solvent is removed.

[0037] Specifically, step (1-1) is performed under the protection of an inert gas; more specifically, the inert gas is, for example, nitrogen or helium.

[0038] Specifically, in step (1-1), when the reaction is initiated, the molar ratio of PLLA to the hydroxyl compound is 1:1-20 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:15, 1:20), especially 1:1-5, wherein the molar amount of PLLA is calculated from the ratio of the mass of PLLA initiated to its weight-average molecular weight.

[0039] Specifically, in step (1-1), the molar ratio of the hydroxyl compound to the activator is 1:1-20 (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:8, 1:10, 1:15, 1:20), especially 1:1-5.

[0040] Specifically, in step (1-1), PLLA needs to be dissolved in a solvent and subjected to a homogeneous reaction. The solvent used is a halogenated organic compound, such as dichloromethane, trichloromethane, dichloroethane, or hexafluoroisopropanol. The concentration of PLLA in the halogenated organic compound is 50-200 mg / ml (mass-volume concentration, for example, 50, 60, 80, 90, 100, 110, 120, 140, 160, 180, 200 mg / ml).

[0041] Specifically, the reaction temperature range in step (1-1) is 15-70°C (e.g., 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70°C), especially 30-50°C.

[0042] Specifically, the reaction time in step (1-1) is 10-24 hours (e.g., 10, 12, 14, 16, 18, 20, 22, 24 hours).

[0043] Specifically, the activator in step (1-1) is selected from one or more of the following: water-soluble carbodiimide, phosphonium bromide formed by triphenylphosphine and bromide, carbomonium salt, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0044] Specifically, the water-soluble carbodiimide may 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[bis(methoxymethyl)methyl]carbodiimide, salts thereof, and mixtures thereof.

[0045] Specifically, 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, etc.

[0046] Specifically, the phosphonium bromide salt can be obtained by reacting triphenylphosphine with a bromide in a solvent such as dichloromethane using known methods.

[0047] Specifically, the caronium salt may be selected from: O-(7-azabenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HCTU), O-(benzotriazol-1-yl)-di(dimethylamino)caronium tetrafluoroborate (TBTU), O-(N-succinimide)-di(dimethylamino)caronium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroborate (TNTU), and one or a mixture of several of the caronium salts selected from these.

[0048] Specifically, the precipitant mentioned in steps (1-2) is an ether, especially C. 2-10 Ethers, such as diethyl ether, tert-butyl methyl ether, methyl ethyl ether, etc.

[0049] Specifically, the volume ratio of the precipitant to the reaction product in step (1-2) is 1-10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1).

[0050] Specifically, the method of adding the reaction product obtained in step (1-1) to the precipitant as described in step (1-2) is dropwise addition.

[0051] Specifically, the method of separating the precipitate described in steps (1-2) can be any suitable method, such as vacuum filtration.

[0052] In some embodiments of the present invention, the solvent removal method in step (1-2) is to vacuum dry the separated precipitate; specifically, the drying temperature can be 35-45°C (e.g., 40°C); specifically, the drying time can be 6-48 hours, and the vacuum degree range is -0.06 to -0.1 MPa (e.g., -0.08 MPa).

[0053] Taking NHS as an example, the reaction formula for the PLLA-NHS active ester obtained by reacting PLLA with NHS is shown below:

[0054]

[0055] Specifically, R' is H or C. 1-6 Alkyl groups, especially H or methyl groups.

[0056] In one embodiment of the present invention, the PLLA-active ester in step (2) is a PLLA-NHS active ester, the structure of which may be as follows.

[0057]

[0058] Specifically, step (2) includes:

[0059] (2-1) Prepare solutions of PLLA-active ester and amination reagent respectively, add the PLLA-active ester solution to the amination reagent solution, and react;

[0060] Optionally, step (2) further includes a purification step: (2-2) adding the reaction product obtained in step (2-1) to a precipitant, separating the precipitate, and removing the solvent.

[0061] Specifically, step (2-1) is carried out under the protection of an inert gas; more specifically, the inert gas is, for example, nitrogen or helium.

[0062] Specifically, 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, trichloromethane, dichloroethane, hexafluoroisopropanol, etc.

[0063] Specifically, the solvent B of the amination reagent solution in step (2-1) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, trichloromethane, dichloroethane, hexafluoroisopropanol, etc.

[0064] Specifically, the mass percentage concentration of the PLLA-active ester solution in step (2-1) can be 0.1-50% (e.g., 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%).

[0065] Specifically, the concentration of the amination 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, 100 mg / ml), for example 5-50 mg / ml.

[0066] Specifically, the mass ratio of PLLA-active ester to amination reagent (SM or SPD) in step (2-1) is 1:0.01-15 (e.g., 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), particularly 1:0.01-1.

[0067] Specifically, the method of adding the PLLA-active ester solution to the amination reagent solution in step (2-1) is dropwise addition.

[0068] Specifically, the temperature of the reaction described in step (2-1) is 10-70°C (e.g., 10, 20, 30, 40, 50, 60, 70°C), especially 40-70°C.

[0069] Specifically, the reaction time described in step (2-1) is 6-48 hours (e.g., 6, 12, 18, 24, 30, 36, 42, 48 hours), especially 18-30 hours.

[0070] Specifically, the precipitant mentioned in step (2-2) is an ether or an alcohol, especially C. 2-10 ether or C 1-6 Alcohols, such as diethyl ether, tert-butyl methyl ether, methyl ethyl ether, or methanol, ethanol, isopropanol, etc.

[0071] Specifically, the volume ratio of the precipitant to the reaction product in step (2-2) is 1-10:1 (e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1).

[0072] Specifically, the method of adding the reaction product obtained in step (2-1) to the precipitant as described in step (2-2) is dropwise addition.

[0073] Specifically, the method of separating the precipitate described in step (2-2) can be any suitable method, such as vacuum filtration.

[0074] In some embodiments of the present invention, the solvent removal method 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, and the vacuum degree range is -0.06 to -0.1 MPa.

[0075] Specifically, step (3) includes:

[0076] (3-1) Prepare solutions of HA or its salt and the aminated PLLA obtained in step (2) respectively, and add the aminated PLLA solution to the solution of HA or its salt.

[0077] (3-2) The mixture obtained in step (3-1) is ultrasonically treated;

[0078] (3-3) Add an activator (and auxiliaries, if necessary) to the system obtained in step (3-2) and react;

[0079] Optionally, step (3) further includes a purification step: (3-4) removing small molecule reactants from the reaction product obtained in step (3-3) (e.g., by dialysis or ultrafiltration) and freeze-drying;

[0080] Optionally, step (3) further includes a purification step: (3-5) immersing the product obtained in step (3-4) in solvent E, removing solvent E (which can be repeated multiple times), and vacuum drying.

[0081] Specifically, 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.

[0082] Specifically, the mass percentage concentration of the solution of HA or its salt in step (3-1) is 0.1-50% (e.g., 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%).

[0083] Specifically, the solution of HA or its salt described in step (3-1) further includes a polymeric emulsifier, such as polyethylene glycol or polyvinyl alcohol; more specifically, the molecular weight of polyethylene glycol can be 1000-50000 Da (e.g., 1000, 2000, 4000, 6000, 8000, 10000, 15000, 20000, 30000, 40000, 50000 Da), particularly 1000-20000 Da; more specifically, Polyvinyl alcohol can have a molecular weight of 5,000-500,000 Da (e.g., 5,000, 10,000, 20,000, 40,000, 50,000, 60,000, 80,000, 100,000, 200,000, 300,000, 400,000, 500,000 Da), especially 5,000-50,000 Da, and a degree of alcoholysis of 60-100% (e.g., 78%, 88%, 98%), especially 87-89%.

[0084] More specifically, the polymeric emulsifier comprises 0.1-30% (e.g., 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 20%, 30%), particularly 0.5-10%, of the total mass of HA or its salt solution.

[0085] Specifically, the pH of the solution of HA or its salt in step (3-1) is 5.0-6.5 (e.g., 5.0, 5.5, 6.0, 6.5), which can be obtained by adjusting, for example, hydrochloric acid.

[0086] Specifically, the solvent D of the ammoniated PLLA solution in step (3-1) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, trichloromethane, dichloroethane, hexafluoroisopropanol, etc.

[0087] Specifically, solvent A, solvent B, and solvent D can be the same or different.

[0088] Specifically, the mass percentage concentration of the ammoniated PLLA solution in step (3-1) is 0.1-50% (e.g., 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%).

[0089] Specifically, in step (3-1), the mass ratio of HA or its salt to aminated PLLA is 1:0.01-2 (e.g., 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), particularly 1:0.1-1.

[0090] Specifically, the duration of the ultrasonic treatment in step (3-2) can be 5-60 minutes (e.g., 5, 10, 20, 30, 40, 50, 60 minutes).

[0091] Specifically, the power of the ultrasonic treatment in step (3-2) can be 1000-5000W (e.g., 1000, 2000, 3000, 4000, 5000W).

[0092] Specifically, after ultrasonic treatment in step (3-2), the ammoniated PLLA solution is dispersed into nanoparticles in an aqueous hyaluronic acid solution, and the particle size range of the nanoparticles is 10-900 nm (e.g., 10, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900 nm).

[0093] Specifically, the amount of activator added in step (3-3) is 0.01-100% of the mass of ammoniated PLLA (e.g., 0.01%, 0.1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%).

[0094] Specifically, the activator in step (3-3) is selected from one or more of the following: water-soluble carbodiimide, phosphonium bromide formed by triphenylphosphine and bromide, carbomonium salt, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM).

[0095] Specifically, 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, or salts thereof, and mixtures thereof. When using a water-soluble carbodiimide activator, it can be used in conjunction with an auxiliary agent to improve the efficiency of the crosslinking reaction. Specifically, the amount of the auxiliary agent added can be 10-80% of the mass of the carbodiimide. Specifically, the auxiliary agent is selected from any one or more of: N-hydroxysuccinimide (NHS), sulfonated N-hydroxysuccinimide (Sulfo-NHS), tert-butanol, and 1-hydroxybenzotriazole (HOBt).

[0096] In one embodiment of the present invention, step (3-3) is: adding an activator and an auxiliary agent to the system obtained in step (3-2) and reacting.

[0097] In one embodiment of the present invention, the activator is EDC and the auxiliary agent is NHS.

[0098] Specifically, 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, etc.; in one embodiment of the present invention, the phosphonium bromide salt is the phosphonium salt formed by triphenylphosphine and N-bromosuccinimide.

[0099] Specifically, the phosphonium bromide salt can be obtained by reacting triphenylphosphine with a bromide in a solvent such as dichloromethane using known methods.

[0100] Specifically, the caronium salt may be selected from: O-(7-azabenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HBTU), O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate (HCTU), O-(benzotriazol-1-yl)-di(dimethylamino)caronium tetrafluoroborate (TBTU), O-(N-succinimide)-di(dimethylamino)caronium tetrafluoroborate (TSTU), 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroborate (TNTU), and one or a mixture of several of the caronium salts; in one embodiment of the present invention, the caronium salt is HATU.

[0101] Specifically, the reaction temperature described in step (3-3) is 10-70°C (e.g., 10, 20, 30, 40, 50, 60, 70°C), especially 30-40°C.

[0102] Specifically, the reaction time described in step (3-3) is 6-48 hours (e.g., 6, 12, 18, 24, 30, 36, 42, 48 hours), especially 18-30 hours.

[0103] Specifically, the method for removing small molecule reactants in step (3-4) can be dialysis or ultrafiltration, and the dialysis or ultrafiltration time can be 6-48 hours.

[0104] Specifically, the solvent E mentioned in steps (3-5) is an organic solvent, especially a halogenated organic compound, such as dichloromethane, trichloromethane, dichloroethane, hexafluoroisopropanol, etc.; in one embodiment of the present invention, solvent E is dichloromethane.

[0105] In one embodiment of the present invention, the method for preparing the grafted polymer includes the following reaction route:

[0106]

[0107] In a third aspect of the invention, an amination-modified PLLA is provided, having the following structure, which can be used as an intermediate in the preparation of grafted polymers:

[0108]

[0109] Where R is

[0110] R' is H or C 1-6 alkyl;

[0111] m is an integer between 40 and 3000.

[0112] In some embodiments of the present invention, R' is H or methyl.

[0113] Specifically, the molecular weight of aminated PLLA is 3-200 kDa (e.g., 3, 5, 10, 20, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200 kDa).

[0114] Specifically, the specific rotation of PLLA ranges from -155° to -160°.

[0115] In a fourth aspect of the present invention, a method for preparing amination PLLA as described in the third aspect is provided, comprising steps (1) and (2) as described in the second aspect of the present invention.

[0116] In a fifth aspect of the invention, the use of the grafted polymer described in one aspect and the grafted polymer prepared by the method described in the second aspect in the preparation of a pharmaceutical product is provided.

[0117] Specifically, the drug contains an active ingredient and the grafted polymer as a drug carrier to achieve purposes such as sustained release, controlled release, and targeted drug delivery.

[0118] In one embodiment of the present invention, the drug is an antitumor drug, and its active ingredient may be, for example, ixaspirin, mitomycin, procainamide, bleomycin, pinantrone, amararubicin, vararubicin, pirarubicin, mitoxantrone, idarubicin, zorubicin, ararubicin, epirubicin, daunorubicin, doxorubicin, daunorubicin; trabectedin, carbamate, paclitaxel, docetaxel, demetocin, teniposide, etoposide, vinblastine, vinorelbine, vinorelbine, vindesine, vincristine, vinblastine; fluorouracil, trifluridine, tegafur, fluorouracil, decitabine, azacitidine, capecitabine, gemcitabine. Tadalafil, carmoflurane, cytarabine, nerabine, clofarabine, fludarabine, cladribine, thioguanine, mercaptopurine, pralatrexate, pemetrexed, raltitrexed, methotrexate; dacarbazine, temozolomide, piperobroman, mitoxobromol, etoricoside, uracil mustard, ramustine, nimustine, flutimustine, streptozotocin, semustine, lomustine, carmustine, carboquinone, triazinon, thiotepa, mannitol, trothion, busulfan, bendamustine, prednimustine, trophosphatamide, ifosfamide, methyl chloroethylamine, melphalan, chlorambucil, cyclophosphamide; cisplatin, carboplatin, oxaliplatin, saxaplatin, polyplatin, etc.

[0119] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising an active ingredient and the grafted polymer described in one aspect, or the grafted polymer prepared by the method described in the second aspect, as a drug carrier.

[0120] In a seventh aspect of the invention, the application of the grafted polymer described in one aspect and the grafted polymer prepared by the method described in the second aspect in the preparation of tissue engineering materials is provided.

[0121] Specifically, the tissue engineering material can be bone tissue engineering material, cartilage tissue engineering material, corneal tissue engineering material, cardiovascular tissue engineering material, liver tissue engineering material, etc.

[0122] In an eighth aspect of the invention, the use of the grafted polymer described in one aspect and the grafted polymer prepared by the method described in the second aspect in the preparation of soft tissue fillers is provided.

[0123] Specifically, this soft tissue filler can be used to eliminate wrinkles (such as wrinkles around the eyes, forehead wrinkles, frown lines, perioral wrinkles, nasolabial folds, tear troughs, nasolabial folds, neck wrinkles, hand wrinkles, stretch marks, etc.), for anti-aging, scar and wound repair, etc.

[0124] Compared with the prior art, the present invention has the following outstanding features:

[0125] (1) By using endogenous polyamines as coupling agents, endogenous polyamines are a non-toxic, harmless, and safer ammoniation reagent. PLLA is ammonified by such ammoniation reagents. Since ammonified PLLA is not easily hydrolyzed, it is easier for it to undergo coupling reaction with hyaluronic acid or its salt in an aqueous environment, which can effectively promote the coupling of PLLA with HA or its salt. At the same time, it can also exert various biological activities of spermidine, ensuring that all components can be degraded and metabolized, and the degradation products are all endogenous substances in the human body. This improves the usability of grafted polymers in the fields of drug preparation, tissue engineering materials or soft tissue fillers, and is suitable for promotion and application.

[0126] (2) Grafting reaction is carried out through nanodispersion heterogeneous coupling technology (NDHC). Specifically, a halogenated organic compound is used as a solvent to obtain an aminated PLLA organic solution. Then, the aminated PLLA organic solution is dispersed into nanoparticles in an aqueous solution of hyaluronic acid or its salt by ultrasound. The particle size of the nanoparticles is controlled within the range of 10-900 nm, thereby expanding the contact surface between the two phases. In addition, the organic solvent in the nanoparticles is controlled to volatilize by temperature selection, so that the exposed aminated PLLA gradually grafts and couples with HA or its salt to form PLLA-HA nanoparticles. In the existing grafting methods, PLLA is usually the solid phase and the solution of hyaluronic acid or its salt is the liquid phase, which is a solid-liquid two-phase grafting method. Since the size of the solid phase particles is uncontrollable, internal grafting is not possible, and only surface grafting is possible. This invention can disperse the liquid droplets into nanoparticles through a two-liquid-phase grafting system, which is more conducive to internal grafting and improves grafting efficiency.

[0127] (3) 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 50-900nm. The graft polymer has good stability and is amphiphilic, and can be applied in the fields of drug carrier, tissue engineering material and soft tissue repair. Attached Figure Description

[0128] Figure 1 The image shows the PLLA-NHS activated ester prepared according to the present invention. 1 H NMR spectrum.

[0129] Figure 2 The image shows the amination of PLLA (SPD amination) prepared according to the present invention. 1 H NMR spectrum.

[0130] Figure 3 The image shows the HA-b-PLLA prepared according to this invention. 1 H NMR spectrum.

[0131] Figure 4 The image shows the FT-IR spectrum of the HA-b-PLLA prepared in this invention.

[0132] Figure 5 The figure shows the particle size distribution of HA-b-PLLA prepared in this invention in aqueous solution. Detailed Implementation

[0133] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0134] The following are the abbreviations for some of the substances in this invention:

[0135] Hyaluronic acid (HA)

[0136] Poly-L-lactic acid (PLLA)

[0137] N-hydroxysuccinimide NHS

[0138] Poly(L-lactic acid-succinimide) PLLA-NHS

[0139] Hyaluronic acid-poly-L-lactic acid grafted polymer HA-b-PLLA

[0140] Spermine SM

[0141] Spermine SPD

[0142] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC

[0143] O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbomony hexafluorophosphate HATU

[0144] 4-(4,6-Dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride DMTMM

[0145] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0146] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0147] The main raw materials used in the following examples, such as hyaluronic acid or its salts, poly-L-lactic acid, NHS, and activators, are all commercially available products.

[0148] Example 1: Preparation of PLLA-NHS active ester

[0149] 15.00 g of PLLA (weight-average molecular weight Mw approximately 50 kDa, PDI: 2.12, specific rotation: -157°) was weighed and added to a three-necked flask under nitrogen protection. 150 mL of chloroform solution was added, and the mixture was stirred for 30 min until completely dissolved. 93 mg of activator EDC and 69 mg of NHS were added, and the reaction was carried out at 40 °C for 16 h. After the reaction was complete, the reaction solution was added dropwise to 400 mL of diethyl ether, then filtered. The resulting filter cake was placed in a vacuum drying oven at -0.08 MPa for 24 h at 40 °C to remove residual solvent. The obtained PLLA-NHS was dried and stored, with a yield of approximately 90%. 1 H NMR spectrum as follows Figure 1 As shown.

[0150] Example 2: Reaction of PLLA-NHS active ester with spermidine

[0151] 30 ml of chloroform was added to a three-necked flask under nitrogen protection. 0.6 g of spermidine was pipetted into the flask and stirred. Simultaneously, 8.00 g of PLLA-NHS (prepared in Example 1) was weighed and dissolved in 50 ml of chloroform under nitrogen protection. This solution was then added dropwise to the spermidine solution, and the reaction was carried out at 60°C for 24 h. After the reaction was complete, the reaction solution was added dropwise to 200 ml of diethyl ether, and then filtered. The resulting filter cake was placed in a vacuum drying oven at 40°C and -0.08 MPa for 24 h to remove residual solvent. The resulting ammoniated PLLA was dried and stored, with a yield of approximately 75%. The ammoniated PLLA... 1 H NMR spectrum as follows Figure 2 As shown. Spermine SPD amination of PLLA forms a PLLA with two amination linkage modes, both of which... 1 The peak positions of the H NMR spectra are basically the same.

[0152] Example 3: Reaction of PLLA-NHS active ester with spermine

[0153] 40 ml of dichloromethane was added to a three-necked flask under nitrogen protection. 1.0 g of spermine was pipetted into the flask and stirred until homogeneous. Simultaneously, 5.00 g of PLLA-NHS (prepared in Example 1) was weighed and dissolved in 50 ml of hexafluoroisopropanol under nitrogen protection. This solution was then added dropwise to the spermine solution, and the reaction was carried out at 20°C for 45 h. After the reaction was complete, the reaction solution was added dropwise to 400 ml of ethanol, and then filtered. The resulting filter cake was placed in a vacuum drying oven at 40°C and -0.08 MPa for 24 h to remove residual solvent. The resulting ammoniated PLLA was dried and stored, with a yield of approximately 85%.

[0154] Example 4: Coupling reaction of amination PLLA (SPD amination) with HA (HATU activation)

[0155] Add 0.95g of sodium hyaluronate (weight-average molecular weight Mw approximately 1,000 kDa) and 0.95g of PEG10000 to a 250ml beaker, then add 95ml of water, stir well, and let stand for 1-2 hours to allow complete dissolution. Adjust the pH to 5.0 with hydrochloric acid solution for later use. Simultaneously, weigh 0.50g of the amination-modified PLLA obtained in Example 2 and dissolve it in 10ml of hexafluoroisopropanol until fully dissolved. The pre-dissolved aminated PLLA solution was added to the sodium hyaluronate solution, followed by sonication at 3.2 kW for 15 min. At this point, the median droplet size of the aminated PLLA hexafluoroisopropanol solution was 100 nm. 50 mg HATU was added, and the mixture was stirred at 35 °C for 24 h. The resulting solution was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 30 kDa. The solution was then freeze-dried to remove moisture. The resulting spongy product was soaked in 40 ml of dichloromethane for 1 h. The solvent was discarded, and the soaking was repeated three times. Finally, the product was vacuum-dried to remove the dichloromethane, yielding HA-b-PLLA.

[0156] The product is labeled HA-b-PLLA1. 1 H NMR spectrum as follows Figure 3 As shown ( Figure 2 Two types of aminated PLLA react with HA to form two types of HA-b-PLLA. 1 The peak positions of the H NMR spectra are basically the same, so Figure 3 (One of them is selected as an illustration). Figure 3 The data shows that the methyl peak on the acetylamino group of hyaluronic acid is located near the chemical shift of 1.8-2.0 ppm, and the methylene peak on polylactic acid (PLLA) is located near the chemical shift of 4.9-5.1 ppm. The presence of these two peaks proves that PLLA and hyaluronic acid were successfully grafted. The grafting efficiency of PLLA and HA can be calculated based on the integrated area of ​​these two peaks, and the calculated grafting efficiency is approximately 80%. The FT-IR spectrum is shown below. Figure 4 As shown. From Figure 4 It can be seen that in the infrared spectrum of HA-b-PLLA1, there is a 1760 cm⁻¹ region belonging to PLLA. -1 The characteristic stretching vibration peak at (C=O) and the peak at 1630 cm⁻¹ belonging to hyaluronic acid are similar. -1 The characteristic stretching vibration peak at (C=O) indicates that PLLA was successfully grafted onto the HA molecule. The product is a snowflake-like loose powder, and its micelle size distribution in aqueous solution is as follows: Figure 5 As shown, through Figure 5 The frequency curves show that the median particle size of HA-b-PLLA1 is approximately 520 nm. Furthermore, the frequency curves exhibit a sharp single peak, indicating that the prepared grafted polymer has a very uniform particle size.

[0157] Example 5: Coupling reaction of amination PLLA (SPD amination) with HA (EDC activation)

[0158] Add 2.0 g of sodium hyaluronate (weight-average molecular weight Mw approximately 900 kDa) and 1.0 g of PEG20000 to a 250 ml beaker, then add 100 ml of water, stir well, and let stand for 1-2 hours to allow it to completely dissolve. Adjust the pH to 5.5 with hydrochloric acid solution for later use. Meanwhile, weigh 0.50 g of the amination PLLA obtained in Example 2, dissolve it in 10 ml of dichloromethane, and allow it to dissolve completely. The pre-dissolved aminated PLLA solution was added to the sodium hyaluronate solution, followed by sonication at 2.2 kW for 30 min. At this point, the median droplet size of the aminated PLLA solution in dichloromethane was 200 nm. 100 mg EDC and 50 mg NHS were added, and the mixture was stirred at 35 °C for 24 h. The resulting solution was ultrafiltered using a hollow fiber with a molecular weight cutoff of 30 kDa for 24 h, and then freeze-dried to remove moisture. The resulting spongy product was then soaked in 40 ml of dichloromethane for 1 h, the solvent was discarded, and the soaking was repeated three times. Finally, the product was vacuum-dried to remove the dichloromethane, yielding HA-b-PLLA.

[0159] The product was labeled HA-b-PLLA2, and was obtained through... 1 In the 1H NMR spectrum, integrating the peaks near chemical shifts of 1.8–2.0 ppm and 4.9–5.1 ppm, the grafting efficiency of PLLA to HA in HA-b-PLLA2 was calculated to be approximately 25%. The FT-IR spectrum of HA-b-PLLA2 is shown below. Figure 4 As shown, from Figure 4 It can be seen that in the infrared spectrum of HA-b-PLLA2, there is a 1760 cm⁻¹ region belonging to PLLA. -1 The characteristic stretching vibration peak at (C=O) and the peak at 1630 cm⁻¹ belonging to hyaluronic acid are similar. -1 The characteristic stretching vibration peak at (C=O) indicates that PLLA was successfully grafted onto the HA molecule. Its median micelle size in aqueous solution is approximately 180 nm.

[0160] Example 6: Coupling reaction of aminated PLLA (SM aminated) with HA (phosphonium salt activation)

[0161] 0.2 mol of triphenylphosphine and 0.2 mol of N-bromosuccinimide were dissolved in 1000 mL of dichloromethane and stirred at 20-25 °C for 24 h. After the reaction was completed, the dichloromethane was removed using a rotary evaporator to obtain triphenylphosphine bromide succinimide salt, which was then sealed and stored for later use.

[0162] Add 3.0 g of sodium hyaluronate (weight-average molecular weight Mw approximately 2,000 kDa) and 2.0 g of PEG2000 to a 250 ml beaker, then add 100 ml of water, stir well, and let stand for 1-2 hours to allow it to completely dissolve. Adjust the pH to 6.0 with hydrochloric acid solution for later use. Meanwhile, weigh 0.50 g of the amination PLLA obtained in Example 3, dissolve it in 10 ml of chloroform, and allow it to dissolve completely. The pre-dissolved aminated PLLA solution was added to the sodium hyaluronate solution, followed by sonication at 2.2 kW for 30 min. At this point, the median droplet size of the aminated PLLA solution in chloroform was 150 nm. 200 mg of succinimide triphenylphosphonium bromide was added, and the mixture was stirred at 35 °C for 24 h. The resulting solution was ultrafiltered using a hollow fiber with a molecular weight cutoff of 10 kDa for 24 h, and then freeze-dried to remove moisture. The resulting sponge-like product was then soaked in 40 ml of dichloromethane for 1 h, the solvent was discarded, and the soaking was repeated three times. Finally, the product was vacuum-dried to remove the dichloromethane, yielding HA-b-PLLA.

[0163] The product was labeled HA-b-PLLA3, and was obtained through... 1 In the 1H NMR spectrum, integrating the peaks near chemical shifts of 1.8–2.0 ppm and 4.9–5.1 ppm, the grafting efficiency of PLLA to HA in HA-b-PLLA3 was calculated to be approximately 56%. The FT-IR spectrum of HA-b-PLLA3 is shown below. Figure 4 As shown. From Figure 4 It can be seen that in the infrared spectrum of HA-b-PLLA3, there is a 1760 cm⁻¹ region belonging to PLLA. -1 The characteristic stretching vibration peak at (C=O) and the peak at 1630 cm⁻¹ belonging to hyaluronic acid are similar. -1 The characteristic stretching vibration peak at (C=O) indicates that PLLA was successfully grafted onto the HA molecule. Its median micelle size in aqueous solution is approximately 450 nm.

[0164] Example 7: Coupling reaction of amination PLLA (SM amination) with HA (DMTMM activation)

[0165] Add 2.5g of sodium hyaluronate (weight-average molecular weight Mw approximately 1,500 kDa) and 2.5g of PVA (weight-average molecular weight Mw approximately 10 kDa, degree of alcoholysis 88%) to a 250ml beaker, then add 100ml of water, stir well, and let stand for 1-2 hours to allow it to completely dissolve. Adjust the pH to 6.0 with hydrochloric acid solution for later use. Simultaneously, weigh 0.50g of the amination-modified PLLA obtained in Example 3, dissolve it in 10ml of chloroform, and allow it to dissolve completely. The pre-dissolved aminated PLLA solution was added to the sodium hyaluronate solution, followed by sonication at 1.2 kW for 60 min. At this point, the median droplet size of the aminated PLLA solution in chloroform was 250 nm. 300 mg of DMTMM was added, and the mixture was stirred at 35 °C for 24 h. The resulting solution was ultrafiltered for 24 h using hollow fiber with a molecular weight cutoff of 10 kDa. The solution was then freeze-dried to remove moisture. The resulting spongy product was soaked in 40 ml of dichloromethane for 1 h. The solvent was then discarded, and the soaking process was repeated three times. Finally, the product was vacuum-dried to remove the dichloromethane, yielding HA-b-PLLA.

[0166] pass 1 In the 1H NMR spectrum, integrating the peaks near chemical shifts of 1.8–2.0 ppm and 4.9–5.1 ppm, the grafting efficiency of PLLA to HA was calculated to be approximately 42%. The median micelle size in aqueous solution is approximately 240 nm.

[0167] Example 8: Coupling reaction of amination PLLA (SPD amination) with HA (HATU activation)

[0168] All other steps were the same as in Example 4, the only difference being that after adding 50 mg of HATU, the reaction was stirred at 55°C for 18 hours. 1 In the 1H NMR spectrum, integrating the peaks near chemical shifts of 1.8–2.0 ppm and 4.9–5.1 ppm, the grafting efficiency of PLLA to HA was calculated to be approximately 65%. The median micelle size in aqueous solution is approximately 420 nm.

[0169] Comparative Example 1: Hyaluronic acid was first amination-treated and then coupled with PLLA-NHS activated ester.

[0170] Add 2.0 g of sodium hyaluronate (weight-average molecular weight Mw approximately 900 kDa) to a 250 ml beaker, then add 100 ml of water, stir well, and let stand for 1-2 hours to allow it to completely dissolve. Adjust the pH to 5.5 with hydrochloric acid solution. Simultaneously, weigh 0.50 g of spermidine and dissolve it in the hyaluronic acid aqueous solution, add 1.0 g of EDC and 0.2 g of NHS, and stir at 35°C for 24 hours. Ultrafilter the resulting solution using a hollow fiber with a molecular weight cutoff of 10 kDa for 24 hours, then freeze-dry to remove moisture to obtain ammoniated hyaluronic acid.

[0171] The PLLA-NHS active ester obtained in Example 1 was dissolved in 10 ml of dichloromethane until fully dissolved. 1.5 g of ammoniated hyaluronic acid and 1.0 g of PEG2000 were dissolved in 100 ml of water and stirred until homogeneous. The pre-dissolved PLLA-NHS active ester solution was added to this ammoniated hyaluronic acid solution, followed by sonication at 2.2 kW for 30 min. At this point, the median droplet size of the PLLA-NHS active ester solution in dichloromethane was 200 nm. The reaction was stirred at 35°C for 24 h. The resulting solution was ultrafiltered using a hollow fiber with a molecular weight cutoff of 10 kDa for 24 h, and then freeze-dried to remove moisture. The resulting sponge-like product was soaked in 40 ml of dichloromethane for 1 h, the solvent was discarded, and the soaking was repeated three times. Finally, the product was vacuum-dried to remove the dichloromethane, yielding HA-b-PLLA. 1 By integrating the peaks near chemical shifts of 1.8-2.0 ppm and 4.9-5.1 ppm in the 1H NMR spectrum, the grafting efficiency of PLLA to HA was calculated to be only about 0.8%.

[0172] Comparative Example 2: Hyaluronic acid and PLLA-NHS activated ester undergo direct coupling reaction.

[0173] Add 2.5g of sodium hyaluronate (weight-average molecular weight Mw approximately 1,500 kDa) and 2.5g of PVA (weight-average molecular weight Mw approximately 10 kDa, degree of alcoholysis 88%) to a 250ml beaker, then add 100ml of water, stir well, and let stand for 1-2 hours to allow complete dissolution. Adjust the pH to 6.0 with hydrochloric acid solution for later use. Simultaneously, weigh 0.50g of the PLLA-NHS active ester obtained in Example 1 and dissolve it in 10ml of chloroform until fully dissolved. The pre-dissolved PLLA-NHS active ester solution was added to the sodium hyaluronate solution, followed by sonication at 1.2 kW for 60 min. At this point, the median droplet size of the PLLA-NHS active ester solution in chloroform was 250 nm. The reaction was stirred at 35 °C for 24 h. The resulting solution was ultrafiltered using a hollow fiber with a molecular weight cutoff of 10 kDa for 24 h, and then freeze-dried to remove moisture. The resulting sponge-like product was then soaked in 40 ml of dichloromethane for 1 h, the solvent was discarded, and the soaking was repeated three times. Finally, the obtained substance was vacuum-dried to remove the dichloromethane, yielding the final product. 1 The 1H NMR spectrum did not show a methylene peak in the 4.9-5.1 ppm range, indicating that PLLA could not be integrated, thus proving that PLLA failed to couple with HA and that a HA-b-PLLA graft copolymer could not be obtained.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A graft polymer, mainly obtained by coupling PLLA with HA or its salt via an ammonifying agent, characterized in that, The amination reagent includes spermine and / or spermidine; The grafted polymer is prepared by a method comprising the following steps: (1) PLLA reacts with hydroxyl compounds in the presence of an activator to obtain PLLA-active esters; (2) React the PLLA-active ester obtained in step (1) with an amination reagent to obtain the amination PLLA shown below, wherein the amination reagent includes spermine and / or spermidine; （Ⅱ） Where R is , ; R' is H or C 1-6 alkyl; m is an integer between 40 and 3000; (3) React the ammoniated PLLA obtained in step (2) with hyaluronic acid HA or its salt to obtain the grafted polymer; Step (3) includes: (3-1) Prepare solutions of HA or its salt and the aminated PLLA solution obtained in step (2) respectively, and add the aminated PLLA solution to the HA or its salt solution; (3-2) The mixture obtained in step (3-1) is ultrasonically treated; (3-3) Add an activator to the system obtained in step (3-2) and react; The solvent C of the solution of HA or its salt in step (3-1) is water; The solution of HA or its salt in step (3-1) further includes a polymeric emulsifier; the polymeric emulsifier is polyethylene glycol or polyvinyl alcohol; The solvent D of the ammoniated PLLA solution in step (3-1) is dichloromethane, trichloromethane, dichloroethane or hexafluoroisopropanol.

2. The grafted polymer as claimed in claim 1, characterized in that, The molecular weight of PLLA is 3-200 kDa.

3. The grafted polymer as described in claim 1, characterized in that, HA or its salts have a molecular weight of 100-3000 kDa.

4. A method for preparing a grafted polymer, comprising the following steps: (1) PLLA reacts with hydroxyl compounds in the presence of an activator to obtain PLLA-active esters; (2) React the PLLA-active ester obtained in step (1) with an amination reagent to obtain the amination PLLA shown below, wherein the amination reagent includes spermine and / or spermidine; （Ⅱ） in, R is , ; R' is H or C 1-6 alkyl; m is an integer between 40 and 3000; (3) React the ammoniated PLLA obtained in step (2) with hyaluronic acid HA or its salt to obtain the grafted polymer; Step (3) includes: (3-1) Prepare solutions of HA or its salt and the aminated PLLA solution obtained in step (2) respectively, and add the aminated PLLA solution to the HA or its salt solution; (3-2) The mixture obtained in step (3-1) is ultrasonically treated; (3-3) Add an activator to the system obtained in step (3-2) and react; The solvent C of the solution of HA or its salt in step (3-1) is water; The solution of HA or its salt in step (3-1) further includes a polymeric emulsifier; the polymeric emulsifier is polyethylene glycol or polyvinyl alcohol; The solvent D of the ammoniated PLLA solution in step (3-1) is dichloromethane, trichloromethane, dichloroethane or hexafluoroisopropanol.

5. The method as described in claim 4, characterized in that, The PLLA-active ester mentioned in step (1) is one or more of PLLA-NHS active ester, PLLA-Sulfo-NHS active ester, and PLLA-HOBt active ester.

6. The method as described in claim 4, characterized in that, Step (1) includes: (1-1) Prepare a solution of PLLA by adding the hydroxyl compound and activator to the solution and reacting. Purification step (1-2): Add the reaction product obtained in step (1-1) to the precipitant, separate the precipitate, and remove the solvent.

7. The method as described in claim 6, characterized in that, Step (1-1) is carried out under inert gas protection.

8. The method as described in claim 6, characterized in that, The hydroxy compound mentioned in step (1-1) is selected from any one or more of N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, and 1-hydroxybenzotriazole.

9. The method as described in claim 6, characterized in that, In step (1-1), the molar ratio of PLLA to the hydroxyl compound during the reaction is 1:1-20.

10. The method as described in claim 6, characterized in that, In step (1-1), the molar ratio of the hydroxyl compound to the activator is 1:1-20.

11. The method as described in claim 6, characterized in that, The solvent for the PLLA solution in step (1-1) is a halogenated organic compound.

12. The method as described in claim 11, characterized in that, The halogenated organic compound is dichloromethane, trichloromethane, dichloroethane, or hexafluoroisopropanol.

13. The method as described in claim 6, characterized in that, The reaction temperature range in step (1-1) is 15-70℃, and the reaction time is 10-24 hours.

14. The method as described in claim 6, characterized in that, The precipitant mentioned in steps (1-2) is ether.

15. The method as described in claim 14, characterized in that, The ether is C 2-10 ether.

16. The method as described in claim 15, characterized in that, The C 2-10 The ether is diethyl ether, tert-butyl methyl ether, or methyl ethyl ether.

17. The method as described in claim 6, characterized in that, The volume ratio of the precipitant to the reaction product in step (1-2) is 1-10:

1.

18. The method as described in claim 4, characterized in that, Step (2) includes: (2-1) Prepare solutions of PLLA-active ester and amination reagent respectively, add the PLLA-active ester solution to the amination reagent solution, and react; Purification step (2-2): Add the reaction product obtained in step (2-1) to the precipitant, separate the precipitate, and remove the solvent.

19. The method as described in claim 18, characterized in that, Step (2-1) is carried out under inert gas protection.

20. The method as described in claim 18, characterized in that, The solvent A of the PLLA-active ester solution in step (2-1) is a halogenated organic compound.

21. The method as described in claim 20, characterized in that, The halogenated organic compound is dichloromethane, trichloromethane, dichloroethane, or hexafluoroisopropanol.

22. The method as described in claim 18, characterized in that, The solvent B in the amination reagent solution described in step (2-1) is a halogenated organic compound.

23. The method as described in claim 22, characterized in that, The halogenated organic compound is dichloromethane, trichloromethane, dichloroethane, or hexafluoroisopropanol.

24. The method as described in claim 18, characterized in that, The mass ratio of PLLA-active ester to amination reagent in step (2-1) is 1:0.01-15.

25. The method as described in claim 18, characterized in that, The reaction temperature in step (2-1) is 10-70℃; the reaction time is 6-48h.

26. The method as described in claim 25, characterized in that, The reaction temperature in step (2-1) is 40-70℃; the reaction time is 18-30h.

27. The method as described in claim 18, characterized in that, The precipitant mentioned in step (2-2) is an ether or an alcohol.

28. The method as described in claim 27, characterized in that, The ether or alcohol is diethyl ether, tert-butyl methyl ether, methyl ethyl ether, methanol, ethanol or isopropanol.

29. The method as described in claim 18, characterized in that, The volume ratio of the precipitant to the reaction product in step (2-2) is 1-10:

1.

30. The method as described in claim 4, characterized in that, Step (3) also includes: Purification step (3-4): Remove small molecule reactants from the reaction product obtained in step (3-3) and freeze-dry.

31. The method as described in claim 30, characterized in that, Step (3) also includes: Further refining step (3-5): Immerse the product obtained in step (3-4) in solvent E, remove solvent E, and vacuum dry.

32. The method as described in claim 4, characterized in that, The polymeric emulsifier accounts for 0.1-30% of the total mass of HA or its salt solution.

33. The method as described in claim 4, characterized in that, The pH of the solution of HA or its salt mentioned in step (3-1) is 5.0-6.

5.

34. The method as described in claim 4, characterized in that, In step (3-1), the mass ratio of HA or its salt to aminated PLLA is 1:0.01-2.

35. The method as described in claim 4, characterized in that, The power of the ultrasonic treatment in step (3-2) is 1000-5000W; and / or the time of the ultrasonic treatment in step (3-2) is 5-60 minutes.

36. The method as described in claim 4, characterized in that, Step (3-3) is as follows: add an activator and an auxiliary agent to the system obtained in step (3-2) and react. The auxiliary agent is selected from any one or more of N-hydroxysuccinimide, sulfonated N-hydroxysuccinimide, and 1-hydroxybenzotriazole.

37. The method as described in claim 4, characterized in that, The amount of activator added in step (3-3) is 0.01-100% of the mass of ammoniated PLLA.

38. The method as described in claim 4, characterized in that, The reaction temperature in step (3-3) is 10-70℃; the reaction time is 6-48h.

39. The method as described in claim 38, characterized in that, The reaction temperature in step (3-3) is 30-40℃; the reaction time is 18-30h.

40. The method as described in claim 30, characterized in that, The method for removing small molecule reactants described in steps (3-4) is dialysis or ultrafiltration.

41. The method as described in claim 31, characterized in that, The solvent E mentioned in steps (3-5) is a halogenated organic compound.

42. The method as described in claim 41, characterized in that, The halogenated organic compound is dichloromethane, trichloromethane, dichloroethane, or hexafluoroisopropanol.

43. The method as described in claim 4 or 6, characterized in that, The activator in step (1-1) or step (3-3) is selected from one or more of the following: water-soluble carbodiimide, phosphonium bromide formed by triphenylphosphine and bromide, carbomonium salt, and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride.

44. The method as described in claim 43, characterized in that, The activator is a water-soluble carbodiimide selected from: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide or its salts, and a mixture of one or more of them.

45. The method as described in claim 43, characterized in that, The activator is a phosphonium bromide salt formed by triphenylphosphine and bromide, selected from: phosphonium salt formed by triphenylphosphine and carbon tetrabromide, and phosphonium salt formed by triphenylphosphine and N-bromosuccinimide.

46. ​​The method as described in claim 43, characterized in that, The activator mentioned in step (3-3) is a caronium salt, which is selected from: O-(7-azabenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate, O-(benzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate, O-(5-chlorobenzotriazol-1-yl)-di(dimethylamino)caronium hexafluorophosphate, O-(benzotriazol-1-yl)-di(dimethylamino)caronium tetrafluoroborate, O-(N-succinimide)-di(dimethylamino)caronium tetrafluoroborate, 2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethylurea tetrafluoroborate, and one or a mixture of several of the caronium salts.

47. The use of the grafted polymer according to any one of claims 1-3, or the grafted polymer prepared by the method according to any one of claims 4-46, in the preparation of pharmaceuticals, tissue engineering materials, or soft tissue fillers.

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