CN104045837B - A kind of three-arm star-shaped hydrophilic copolymers and synthetic method thereof and application - Google Patents

Abstract

A three-armed star-shaped hydrophilic copolymer and its synthesis method and application, using the inner core of tertiary primary amine to initiate the ring-opening polymerization reaction of γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride in the ring, using carbon two The imine method bonds the small molecule RAFT polymeric chain transfer agent to the three terminal amino groups of the star polymer to obtain the macromolecular RAFT chain transfer agent; Amide and N-methylolacrylamide are used as monomers, and RAFT polymerization is carried out. Finally, the hydroxyl group of the N-methylolacrylamide component in the copolymer is used to react with the isocyanated methylated polyethylene glycol to achieve a disulfide bond. The desired three-arm star-shaped hydrophilic copolymer is obtained after linking polyethylene glycol and hydrazinolysis treatment. This method can flexibly control the molecular weight and chain length of each polymer component, the reaction conditions are mild, and the raw materials are easy to obtain. The synthesized polymer can improve drug efficacy and reduce toxic side effects. At the same time, doxorubicin and gene drugs are loaded to achieve synergistic anti-tumor treatment effect.

Description

A kind of three-arm star-shaped hydrophilic copolymer and its synthesis method and application

technical field

The invention belongs to the field of biomedical materials, and in particular relates to a three-arm star-shaped hydrophilic copolymer and its synthesis method and application.

Background technique

Chemotherapy based on cytotoxic drugs is one of the main strategies for the treatment of malignant tumors. However, cytotoxic drugs usually have high cytotoxicity, poor water solubility and no bioselectivity, and can kill tumor cells and normal cells at the same time, with serious side effects. Since the curative effect and side effects of chemotherapy are strongly positively correlated with the drug dose, it is difficult to improve the therapeutic effect by increasing the drug concentration. What's more serious is that almost all tumor cells will develop multidrug resistance during chemotherapy with cytotoxic drugs. These have become the main obstacles restricting the success of chemotherapy. Many studies in recent years have shown that the use of biological materials such as biocompatible and biodegradable polymer materials to load chemotherapy drugs can effectively increase the local concentration of drugs in tumor tissues, improve the bioavailability of drugs, achieve enhanced therapeutic effects, and reduce toxicity to normal cells. purpose of side effects. At present, the most researched drug carrier materials are biocompatible and biodegradable polyester materials, such as polylactide, polyglycolide, polycaprolactone, etc. These hydrophobic polyester materials can effectively load hydrophobic cytotoxic drugs, but they mainly release drugs through hydrolytic degradation, which is slow and affects drug efficacy. It can be seen that they are not ideal drug carrier materials. Therefore, the synthesis of new drug carrier materials and the development of new drug release mechanisms have always been research hotspots. In recent years, polyamino acids such as polyglutamic acid and polylysine have attracted interest in the field of drug delivery due to their good biocompatibility and biodegradability. Polyamino acids are a class of homopolymeric or copolymeric polymer materials composed of amino acids and their derivatives linked by peptide bonds. Different amino acids exhibit different side groups, charging properties, and hydrophilic and hydrophobic properties, which can flexibly adjust the physical and chemical properties of polyamino acids, such as degradation performance, biocompatibility, and self-assembly behavior. In addition, the degradation products of polyamino acids are near-neutral amino acids, which can avoid side effects such as inflammatory reactions caused by acidic degradation products of traditional polyester materials. It can be seen that polyamino acids and their derivatives have broad development prospects in drug delivery. For example, the Chinese patent application number 201110132965.2 discloses a method for preparing an amphiphilic block copolymer by using amino-terminated polyethylene glycol to initiate L-tryptophan polymerization. load. Another example is that the Chinese patent application number 201110200449.9 discloses a reduction-sensitive purine-polyaspartimide prodrug, which contains a disulfide bond that can be degraded under the reduction of glutathione in tumor cells, releasing The medicine has the functions of reducing toxic and side effects and improving curative effect.

Whether polyester or polyamino acid is used as a chemotherapy drug carrier, it must be combined with hydrophilic polyethylene glycol to form an amphiphilic block copolymer, so that it can self-assemble in water to have a hydrophobic core and a hydrophilic shell of nanomicelles. Among them, the hydrophobic inner core is used to wrap hydrophobic chemotherapy drugs, and the hydrophilic outer shell can stabilize the nanocarriers, prevent micelles from aggregating, avoid the conditioning effect of the immune system, and realize long-term circulation in the body. Compared with traditional small-molecule liposome micelles, amphiphilic block copolymers have lower critical micelle concentration, better stability in blood, and special 'enhanced penetration' through tumor tissue and retention effect' to achieve passive targeting, effectively enrich the drug inside the tumor tissue, and promote the uptake of the carrier-drug by tumor cells. However, nanomicelles formed solely by the self-assembly of amphiphilic block copolymers still have the risk of micelles dissociation and early drug release due to the dilution effect after injection into the body.

In recent years, gene therapy has been regarded as a promising new strategy for the treatment of malignant tumors. It introduces exogenous therapeutic genes into tumor cells to correct defective genes or inhibit/promote the expression of certain genes/proteins, thereby achieving purpose of treating disease. At the same time, gene therapy is also an effective way to overcome the multidrug resistance of tumor cells in chemotherapy. In order to overcome the safety, limited loading capacity, potential immune response and carcinogenesis of viral vectors in gene therapy, research on non-viral vectors has become a new focus. Non-viral vectors have the advantages of high safety, low immunogenicity, large carrying capacity, and easy mass production, and mainly include cationic liposomes and cationic polymers. Among them, the cationic polymer is the subject of research, which can form a stable complex with the genetic material through electrostatic interaction, effectively compress the genetic material, and assist the gene to pass through the cell membrane. Polyethyleneimine is the most classic cationic polymer carrier, which has the advantages of easy availability of raw materials, high compression ratio, strong buffer capacity and high transfection efficiency, and the research is the most concentrated. However, polyethyleneimine is non-degradable and highly cytotoxic, which restricts its application range.

At present, it has become a development trend to use different mechanisms of action and different types of drugs to treat malignant tumors in combination, especially the combination therapy based on nanocarriers has high hopes. However, the biggest bottleneck of nanocarrier-based combination therapy is the synthesis of ideal carrier materials that can simultaneously load different drugs. The Chinese patent application number 201210114241.X discloses a triblock copolymer composed of polyethylene glycol, polylactic acid and polyarginine, in which the polyester segment and the polyarginine segment are used to load hydrophobic drugs and Genetic material with good biosafety and drug delivery capabilities. However, the segments of the block copolymer are connected by ester bonds and urethane bonds, there is no stimuli-responsive dissociation mechanism, and the drug release rate is slow, making it difficult to give full play to the curative effect of the drug.

Contents of the invention

The purpose of the present invention is to provide a three-arm star-shaped water-soluble copolymer and its synthesis method and application. The method can flexibly control the molecular weight and chain length of each polymer component, the reaction conditions are mild, and the raw materials are easy to obtain. Drugs can improve drug efficacy, reduce toxic and side effects, and simultaneously load doxorubicin and gene drugs to achieve synergistic anti-tumor therapeutic effects.

In order to achieve the above object, the synthetic method of three-arm star-shaped hydrophilic copolymer of the present invention is characterized in that, comprises the steps:

1) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

Dissolve the three-armed poly(L-glutamic acid-γ-benzyl ester) after water removal in an anhydrous organic solvent, and then add a RAFT chain transfer agent with a carboxyl group at 0°C to 4°C under a nitrogen atmosphere, 4 -Dimethylaminopyridine and dicyclohexylcarbodiimide, react at room temperature for 24 hours to 72 hours, then precipitate with cold ether, collect the precipitate and dry it in vacuum to obtain three-arm poly(L-glutamic acid-γ-benzyl ester ) Macromolecular RAFT chain transfer agent; wherein, poly(L-glutamic acid-γ-benzyl ester) after water removal, RAFT chain transfer agent with a carboxyl group, 4-dimethylaminopyridine and dicyclohexylcarbodiethylene The molar ratio of amine is 1:(6~20):3:(6~60);

2) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide):

Dissolve three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent, N-(3-dimethylaminopropyl)methacrylamide, N-methylolacrylamide in anhydrous In dioxane, then add the initiator in an oxygen-free environment, react at 60 ° C ~ 65 ° C for 36 hours ~ 72 hours, after the reaction is completed, concentrate the obtained reaction system, then dissolve it in dichloromethane and precipitate it in cold ether , the precipitate was collected and dried in vacuo to obtain three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide); among them, the three-armed poly The molar ratio of (L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent, N-(3-dimethylaminopropyl)methacrylamide and N-methylolacrylamide is 1:(72 ~290): (3.6~14.4);

3) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Dissolve the three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) after water removal in anhydrous chloroform, and then add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol, react at 60°C to 65°C for 36 hours to 72 hours, concentrate the obtained reaction system after the reaction, and precipitate with ether, then Dialyzed in distilled water, and finally freeze-dried to obtain three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene bismuth Alcohol copolymer; wherein, isocyanated end-methoxy polyethylene glycol and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl base) methacrylamide) molar ratio is (9 ~ 36): 1;

4) Synthesis of three-armed star-shaped hydrophilic copolymer: the three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide )-Polyethylene glycol copolymer hydrazinolysis removes the benzyl group to obtain a three-armed star-shaped hydrophilic copolymer.

The poly(L-glutamic acid-γ-benzyl ester) in the described step 1) is prepared by the following method: anhydrous tris(2-aminoethyl)amine is dissolved in anhydrous organic solvent, Add γ-benzyl ester-L-glutamic acid-N-carboxyl internal anhydride under nitrogen atmosphere, stir to form a homogeneous solution, and then react at 25°C to 40°C for 48 hours to 72 hours. After the reaction, the obtained reaction solution Precipitate with cold ether and wash with ethanol, and obtain poly(L-glutamic acid-γ-benzyl ester) after vacuum drying; wherein, anhydrous tris(2-aminoethyl)amine and γ-benzyl ester-L-glutamine The molar ratio of the acid anhydride in the acid-N-carboxyl ring is (1:30)～(1:120), and the anhydrous organic solvent in step 1) and step 4) is anhydrous dimethyl formamide, anhydrous chloroform or anhydrous tetrahydrofuran.

The anhydrous tris(2-aminoethyl)amine is obtained by refluxing calcium hydride and distilling under reduced pressure under a nitrogen atmosphere.

The γ-benzyl ester-L-glutamic acid-N-carboxyl ring anhydride is synthesized by the following steps: L-glutamic acid-γ-benzyl ester is added in anhydrous tetrahydrofuran, and then triphosgene is added under a nitrogen atmosphere , followed by reacting at 50°C until a clear solution was formed and concentrated, and finally adding anhydrous n-hexane to the reaction system obtained after concentration to precipitate, and recrystallizing the obtained precipitate with anhydrous n-hexane to obtain γ-benzyl ester-L- Glutamic acid-N-carboxyl ring anhydride; wherein, the molar ratio of L-glutamic acid-γ-benzyl ester to triphosgene is 1:(0.35～0.4).

The method of step 4) hydrazinolysis to remove benzyl group is: three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl) methacrylic acid Amide)-polyethylene glycol copolymer is dissolved in anhydrous organic solvent, after adding anhydrous hydrazine dropwise, react at 30 ℃ ~ 40 ℃ for 18 hours ~ 48 hours, after the reaction is completed, the reaction system obtained is 0.25% in mass concentration Dialyzed in aqueous ammonia, and finally freeze-dried to obtain a three-armed star-shaped hydrophilic copolymer; wherein, anhydrous hydrazine and three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3 - dimethylaminopropyl) methacrylamide) - the molar ratio of the benzyl protecting group in the polyethylene glycol copolymer is (3-20):1.

The RAFT chain transfer agent with a carboxyl group in the step 1) is S-1-dodecyl-S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate, 4 -Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-cyano-4-(thiobenzoyl)pentanoic acid, or 4-cyanopentanoic acid dithio Benzoic acid.

The initiator in the step 2) is azobisisobutyronitrile or 4,4'-azobis(cyanovaleric acid), and the three-arm poly(L-glutamic acid-γ-benzyl ester) is large The molar ratio of molecular RAFT chain transfer agent to initiator is 1: (0.36-1.44).

The methyl-terminated polyethylene glycol modified with 2,2'-dithiodiethylisocyanate in the step 3) is synthesized by the following method: the methoxy-terminated polyethylene glycol with a molecular weight of 800-4000Da Alcohol, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate were dissolved in anhydrous toluene, and reacted for 48 hours at 85°C under a nitrogen atmosphere, and anhydrous n-hexane was added to the obtained reaction system to precipitate , collect the precipitate and dry it in vacuum to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, dibutyltin dilaurate and 2, The molar ratio of 2'-dithiodiethylisocyanate is 1:(0.02～0.08):(3～8).

A three-arm star-shaped hydrophilic copolymer that adopts the synthesis of the method, and its chemical name is three-arm star-shaped poly(L-glutamic acid-γ-hydrazide)-poly(N-(3-dimethyl Aminopropyl) methacrylamide)-polyethylene glycol copolymer, the chemical structure is:

The application of the three-arm star-shaped hydrophilic copolymer as a carrier loaded with doxorubicin and gene medicine.

Dissolve 1g to 6g of poly(L-glutamic acid-γ-benzyl ester) in every 100 milliliters of anhydrous organic solvent;

In step 2), 2 g to 10 g of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent is added to every 100 milliliters of anhydrous dioxane;

Dissolve 2g to 5g of tPBLG-b-PDMAPMA after dehydration in every 100ml of anhydrous chloroform in step 3);

Dissolve 5g-15g of L-glutamic acid-γ-benzyl ester per 100ml of anhydrous tetrahydrofuran.

Dissolve 5g to 15g of gamma-benzyl ester-L-glutamic acid-N-carboxyl internal anhydride in every 100 milliliters of anhydrous organic solvent;

Dissolve 5g-30g of methyl-terminated polyethylene glycol per 100ml of anhydrous toluene.

The molar ratio of the three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent to the initiator is 1: (0.36-1.44);

The gene drug is Pgp siRNA, bcl-2 siRNA or EGF siRNA, and the loading method is to mix the gene drug and the three-arm star-shaped water-soluble copolymer loaded with doxorubicin, and form a complex by mixing with a vortex shaker, Realize the loading of gene medicine.

Dissolve 5g～30g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl) per 100 ml of anhydrous organic solvent in the step 5) methacrylamide)-polyethylene glycol copolymer.

Compared with prior art, the beneficial effect of the present invention is:

(1) The three-arm star-shaped water-soluble copolymer synthesized by the present invention is composed of poly(L-glutamic acid-γ-hydrazide) of poly(L-glutamic acid-γ-hydrazide) which realizes the stealth effect in vivo, and is bonded with the chemotherapeutic drug doxorubicin by a hydrazone bond. Segment and artificially synthesized cationic poly(N-(3-dimethylaminopropyl)methacrylamide) composed of electrostatically charged gene substances, therefore, the three-arm star-shaped water-soluble copolymer synthesized by the present invention can simultaneously load chemotherapy Drug doxorubicin and genetic material to achieve synergistic anti-tumor therapeutic effect.

Among them, the functional bridge connecting polyethylene glycol contains a disulfide bond, so that when one end of the carrier loaded with doxorubicin and gene drug enters the tumor cell, it can be broken in response to the reducing environment in the tumor cell, and the polyethylene glycol can be removed. The cationic layer of the gene-loaded drug is exposed after alcohol, and the exposed cationic layer can promote the escape of the drug-carrier from the endosome to the cytoplasm, release the loaded drug, and avoid the enzymatic hydrolysis of the lysosome, thereby improving drug efficacy and reducing side effects . The poly(L-glutamic acid-γ-hydrazide) segment of the present invention is prepared by a ring-opening polymerization method, and its final degradation product is glutamic acid, which can be absorbed and utilized by the human body without causing disadvantages of acidic degradation products of polyester materials. influences. The non-degradable cationic poly(N-(3-dimethylaminopropyl)methacrylamide) segment is synthesized by RAFT polymerization method, the molecular weight distribution is narrow and the molecular weight is controllable, by effectively controlling its molecular weight, it is possible to ensure The loading effect of genetic material can also ensure that it is excreted through the excretory system to avoid its accumulation in the body.

(2) The synthesis method of the present invention can flexibly control the molecular weight and chain length of each polymer component, the reaction conditions are mild, and the raw materials are easy to get. The three-arm star-shaped water-soluble copolymer synthesized has water solubility and is loaded with the chemotherapeutic drug doxorubicin Afterwards, it can self-assemble in aqueous solution to form single-molecule nanomicelles, which are passively targeted by tumor tissue through the "enhanced penetration and retention effect" and enriched in tumor tissues, promoting the uptake of carrier-drugs by tumor cells, improving Drug bioavailability, thereby improving efficacy and reducing side effects.

At the same time, self-assembled unimolecular nanomicelles have a star-shaped structure, and the formed nanomicelles will have higher stability, lower critical micelle concentration, and longer circulation time in vivo than linear amphiphilic copolymers. Therefore, the three-armed star-shaped water-soluble copolymer has higher stability in vivo after drug loading, has passive targeting effect, can improve drug efficacy and reduce toxic and side effects.

(3) The number of polyethylene glycol segments on the three-arm star-shaped water-soluble copolymer can be flexibly adjusted by the number of N-methylolacrylamide in the cationic segment polymer, and a polyethylene glycol segment can be connected to a single arm. It presents a tri-block copolymer molecular structure, and can also link several polyethylene glycol segments to present a molecular structure combining a block and a comb structure, which makes the adjustment of the physical and chemical properties of the hydrophilic shell of the nano-micelle more flexible.

Description of drawings

Fig. 1 is the synthetic route synoptic diagram of three-arm star-shaped hydrophilic copolymer of the present invention;

Fig. 2 is the three-arm star poly(L-glutamic acid-γ-hydrazide)-poly(N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol water-soluble compound synthesized in Example 1. The ¹ H-NMR spectrum of the copolymer.

Fig. 3 is a particle size distribution diagram of self-assembled nanoparticles loaded with doxorubicin on the carrier synthesized in Example 1.

Fig. 4 is the laser confocal fluorescence photo of the three-arm star-shaped hydrophilic copolymer synthesized in Example 1 co-loading doxorubicin and FAM-labeled siRNA nanoparticles into the breast cancer cell MCF-7; wherein, a is DAPI staining The fluorescent photo of the cell nucleus, b is the fluorescent photo of doxorubicin at the nucleus, c is the fluorescent photo of FAM-labeled siRNA after entering the cell, and d is the combination of a-c.

detailed description

Referring to Fig. 1, the present invention uses three (2-aminoethyl) amines as the inner core to initiate the ring-opening polymerization of γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride to form three-armed poly(L-glutamic acid -γ-benzyl ester) molecule; then, use the carbodiimide method to connect a RAFT chain transfer agent with a carboxyl group on its terminal amino group, and implement RAFT polymerization to synthesize a cationic polymer segment; finally, bond the isocyanated terminal methyl group Polyethylene glycol, and after hydrazinolysis treatment to obtain the desired three-armed star-shaped hydrophilic copolymer, can be used for the simultaneous loading and delivery of the chemotherapeutic drug doxorubicin and genetic material.

The present invention will be further described below by way of examples, but the present invention is not limited thereto.

Example 1:

1) Synthesis of γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA):

Add L-glutamic acid-γ-benzyl ester into anhydrous tetrahydrofuran, then add triphosgene under nitrogen atmosphere, then react at 50°C until a clear solution is formed, then concentrate under reduced pressure to remove most of the solvent to concentrate the solvent, and finally to Add anhydrous n-hexane to the reaction system obtained after concentration to precipitate, and recrystallize the obtained precipitate with anhydrous n-hexane to obtain γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA) ; Wherein, the molar ratio of L-glutamic acid-γ-benzyl ester to triphosgene is 1:0.35, and 10g of L-glutamic acid-γ-benzyl ester is dissolved in every 100 milliliters of anhydrous tetrahydrofuran;

2) Synthesis of three-arm poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous dimethylformamide, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and then react at 25°C for 72 hours; after the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and dried in vacuo After that, white powdery three-arm poly(L-glutamic acid-γ-benzyl ester) (tPBLG) was obtained; wherein, the molar ratio of anhydrous tris(2-aminoethyl)amine and BLG-NCA was 1:30 , dissolve 15g of BLG-NCA per 100ml of anhydrous dimethylformamide;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

tPBLG was dewatered by azeotropic toluene under nitrogen to obtain tPBLG after dehydration, and the three-armed poly(L-glutamic acid-γ-benzyl ester) after dehydration was dissolved in anhydrous chloroform, and then in a nitrogen atmosphere Add S-1-dodecyl-S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT) and 4-dimethylaminopyridine (DMAP) at 0°C and dicyclohexylcarbodiimide (DCC), then precipitated with cold ether after reacting at room temperature for 48 hours, collected the precipitate and vacuum-dried to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) Molecular RAFT chain transfer agent, that is, tPBLG-CTA; wherein, the molar ratio of tPBLG, DDACT, DMAP and DCC after water removal is 1:6:3:12, and 3g of tPBLG after water removal is dissolved in 100 ml of anhydrous chloroform , the reflux temperature during azeotropic water removal is 120°C, and the water removal time is 2 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add azo Diisobutyronitrile was reacted at 60°C for 48 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and dried in vacuo to obtain an arm star polymer (L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide), that is, tPBLG-b-PDMAPMA; among them, tPBLG-CTA, DMAPMA, N-hydroxy The molar ratio of methacrylamide and azobisisobutyronitrile is 1:72:3.6:0.36, and 20g of tPBLG-CTA is dissolved in every 100ml of anhydrous dioxane;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 800Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.03:5, and 5g of methoxy-terminated polyethylene glycol is dissolved in 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, and then Add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 60°C for 36 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water for 48 Hours, three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization; Among them, 2,2'-dithiodiethylisocyanate-modified end-methyl polyethylene glycol and three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N- The molar ratio of (3-dimethylaminopropyl) methacrylamide) is 9: 1, and the tPBLG-b-PDMAPMA after dissolving 2g dehydration in every 100 milliliters of anhydrous chloroforms, the reflux temperature when azeotropic dehydration is 110℃, water removal time is 6 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In dimethylformamide, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star Hydrophilic copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-poly The molar ratio of benzyl protecting group in the ethylene glycol copolymer is 5:1; Dissolve 10g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

The chemical name of the three-arm star-shaped hydrophilic copolymer synthesized in this example is three-arm star poly(L-glutamic acid-γ-hydrazide)-poly(N-(3-dimethylaminopropyl) methyl Acrylamide)-polyethylene glycol hydrophilic copolymer, its structural formula is

See Figure 2 for the ¹ H-NMR spectrum. It can be seen from Figure 2 that the chemical shifts of the protons of each copolymer component were well identified, indicating that the synthesis of the copolymer was successful.

The three-armed star-shaped hydrophilic copolymer carrier synthesized in this example exhibits amphiphilic properties after being loaded with doxorubicin, and can self-assemble into nanoparticles in water. The particle size distribution diagram measured by dynamic light scattering method is shown in Figure 3 . It can be seen from Figure 3 that the particle size distribution range is 50 nm to 110 nm, and the particle size distribution range is narrow, which is ideal.

The three-armed star-shaped hydrophilic copolymer synthesized in this example is co-loaded with doxorubicin and green fluorescent agent FAM-labeled siRNA to form nanoparticles that can effectively enter the cell through endocytosis, and its distribution in the cell is shown in the figure 4. B in Figure 4a is the fluorescence emitted by DAPI-stained nuclei, R in Figure 4b is the fluorescence emitted by doxorubicin at the nucleus, and G in Figure 4c is after FAM-labeled bcl-2siRNA enters breast cancer cell MCF-7 Figure 4d was obtained after combining Figures 4a-4c. It can be seen from Figure 4d that both doxorubicin and bcl-2 siRNA can effectively enter the breast cancer cell MCF-7 and distribute around the nucleus. It can be seen that the synthesis The vector can be used for co-loading and delivery of doxorubicin and bcl-2 siRNA.

Example 2:

1) Same as step 1) in Example 1.

2) Synthesis of three-arm poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous chloroform, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and react at 40°C for 60 hours; after the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and vacuum-dried to obtain a white powder tPBLG; wherein, the molar ratio of anhydrous tris(2-aminoethyl)amine and BLG-NCA is 1:60, and 5g of BLG-NCA is dissolved in every 100 milliliters of anhydrous chloroform;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was azeotropically dewatered with toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous dimethylformamide, and then S-1-deca was added at 0°C under nitrogen atmosphere. Dialkyl-S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC ), then precipitated with cold ether after reacting at room temperature for 48 hours, collected the precipitate and vacuum-dried to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, i.e. tPBLG-CTA Among them, the molar ratio of tPBLG, DDACT, DMAP and DCC after water removal is 1:12:3:30, and 6g of tPBLG after water removal is dissolved in every 100 ml of anhydrous dimethylformamide; The reflux temperature is 120°C, and the water removal time is 2 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add 4, 4'-Azobis(cyanovaleric acid) was reacted at 60°C for 48 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and Dry in vacuo to obtain three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide), i.e. tPBLG-b-PDMAPMA; wherein, The molar ratio of tPBLG-CTA, DMAPMA, N-methylolacrylamide and 4,4'-azobis(cyanovaleric acid) is 1:290:14.4:0.36, per 100 ml of anhydrous dioxane Dissolve 10g of tPBLG-CTA;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol: dry methoxy-terminated polyethylene glycol with a molecular weight of 4000Da, dibutyltin dilaurate and 2 , 2'-dithiodiethylisocyanate was dissolved in anhydrous toluene, and reacted for 48 hours at 85°C under a nitrogen atmosphere, and anhydrous n-hexane was added to the obtained reaction system to precipitate to obtain 2,2'-disulfide Methyl-terminated polyethylene glycol modified with substituted diethylisocyanate; wherein, the molar ratio of methyl-terminated polyethylene glycol, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate is 1: 0.02:6, dissolve 10g of methoxy-terminated polyethylene glycol in every 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, and then Add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 60°C for 72 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water for 48 Hours, three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization; Among them, 2,2'-dithiodiethylisocyanate-modified end-methyl polyethylene glycol and three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N- The molar ratio of (3-dimethylaminopropyl) methacrylamide) is 12:1; Dissolve 2g of tPBLG-b-PDMAPMA after removing water in every 100 milliliters of anhydrous chloroform; The reflux temperature during azeotropic water removal is 115℃, water removal time is 4 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In dimethylformamide, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star Hydrophilic copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-poly The molar ratio of the benzyl protecting group in the ethylene glycol copolymer is 8:1, dissolve 30g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

Example 3:

1) Same as step 1) in Example 1.

2) Synthesis of three-arm poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous tetrahydrofuran, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and react at 40°C for 48 hours; after the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and vacuum-dried to obtain a white powder tPBLG; wherein, the molar ratio of anhydrous tris(2-aminoethyl)amine and BLG-NCA is 1:45, and 5g of BLG-NCA is dissolved in every 100 milliliters of anhydrous tetrahydrofuran;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was azeotropically dewatered with toluene under nitrogen to obtain tPBLG after dehydration, and the tPBLG after dehydration was dissolved in anhydrous chloroform; then S-1-dodecyl- S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), followed by After reacting at room temperature for 48 hours, precipitate with cold ether, collect the precipitate and dry it in vacuo to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent; wherein, tPBLG after removing water The molar ratio of DDACT, DMAP and DCC is 1:20:3:60, and 3g of tPBLG after water removal is dissolved in 100 ml of anhydrous chloroform; the reflux temperature is 110°C during azeotropic water removal, and the water removal time is 6 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and add azobis Isobutyronitrile, reacted at 60°C for 48 hours, removed the solvent under reduced pressure after the reaction, dissolved and concentrated the reaction system obtained in dichloromethane to remove the solvent, dissolved in dichloromethane and precipitated in cold ether, collected the precipitate and vacuumed Dry to obtain three-arm star poly(L-glutamate-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl) methacrylamide), namely tPBLG-b-PDMAPMA; wherein, tPBLG - The molar ratio of CTA, DMAPMA, N-methylolacrylamide and 4,4'-azobis(cyanovaleric acid) is 1:144:7.2:0.64, dissolved in 100 ml of anhydrous dioxane 4g of tPBLG-CTA;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol: dry methoxy-terminated polyethylene glycol with a molecular weight of 2000Da, dibutyltin dilaurate and 2 , 2'-dithiodiethylisocyanate was dissolved in anhydrous toluene, and reacted for 48 hours at 85°C under a nitrogen atmosphere, and anhydrous n-hexane was added to the obtained reaction system to precipitate to obtain 2,2'-disulfide Methyl-terminated polyethylene glycol modified with substituted diethylisocyanate; wherein, the molar ratio of methoxy-terminated polyethylene glycol, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate is 1 : 0.04:6, dissolve 5g of methoxy-terminated polyethylene glycol in every 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, Then add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 60°C for 48 hours. After the reaction is completed, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water After 48 hours, three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization ; Among them, 2,2'-dithiodiethylisocyanate-modified terminal methyl polyethylene glycol and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N -(3-Dimethylaminopropyl) methacrylamide) in a molar ratio of 24:1; 5g of tPBLG-b-PDMAPMA dissolved in 100 ml of anhydrous chloroform; reflux temperature during azeotropic water removal The temperature is 110°C, and the water removal time is 6 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In dimethylformamide, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star Hydrophilic copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-poly The molar ratio of benzyl protecting group in the ethylene glycol copolymer is 10:1, dissolves 25g three-arm star poly(L-glutamate-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

Example 4:

1) Same as step 1) in Example 1.

2) Synthesis of three-arm star poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous dimethylformamide, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and react at 30°C for 60 hours; after the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and vacuum-dried Obtain white powdery tPBLG; wherein, the molar ratio of tris(2-aminoethyl)amine and BLG-NCA is 1:120, dissolve 5g of anhydrous tris(2-aminoethyl) in every 100 milliliters of anhydrous dimethylformamide base) amine;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

tPBLG is dehydrated by azeotropic toluene under nitrogen to obtain tPBLG after dehydration, and the tPBLG after dehydration is dissolved in anhydrous dimethylformamide, followed by S-1-Dodeca at 0°C under nitrogen atmosphere. Alkyl-S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC) , then reacted at room temperature for 48 hours and then precipitated with cold ether, collected the precipitate and dried in vacuo to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, i.e. tPBLG-CTA; Among them, the molar ratio of tPBLG, DDACT, DMAP and DCC after water removal is 1:20:3:60, and 5.5g of tPBLG after water removal is dissolved in every 100 ml of anhydrous dimethylformamide; The reflux temperature is 110°C, and the water removal time is 6 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add azo Diisobutyronitrile was reacted at 60°C for 48 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and dried in vacuo to obtain a three-armed star Poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide), namely tPBLG-b-PDMAPMA; among them, tPBLG-CTA, DMAPMA, N- The molar ratio of methylolacrylamide and azobisisobutyronitrile is 1:216:10.8:1.44, and 7.5g of tPBLG-CTA is dissolved in every 100ml of anhydrous dioxane;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 2000Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.02:8, and 5g of methoxy-terminated polyethylene glycol is dissolved in 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, and then Add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol, and react at 60°C for 60 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water for 48 Hours, three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization; Among them, 2,2'-dithiodiethylisocyanate-modified end-methyl polyethylene glycol and three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N- The molar ratio of (3-dimethylaminopropyl) methacrylamide) is 12:1; Dissolve 5g tPBLG-b-PDMAPMA after removing water in every 100 milliliters of anhydrous chloroforms; The reflux temperature during azeotropic water removal is 120℃, water removal time is 3 hours;

7) Synthesis of three-arm star-shaped three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous Add anhydrous hydrazine dropwise to tetrahydrofuran and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star-shaped hydrophilic copolymer Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymerization The molar ratio of benzyl protecting group in the product is 5:1; Dissolve 15g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethyl aminopropyl)methacrylamide)-polyethylene glycol copolymer.

Example 5:

1) Same as step 1) in Example 1.

2) Synthesis of three-arm poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous tetrahydrofuran, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and react at 40°C for 60 hours; after the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and dried in vacuum to obtain a white powder tPBLG; wherein, the molar ratio of anhydrous tris(2-aminoethyl)amine and BLG-NCA is 1:80, and 8g of BLG-NCA is dissolved in every 100 milliliters of anhydrous tetrahydrofuran;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was dewatered by azeotropic toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous chloroform, and then S-1-dodecyl- S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), followed by After reacting at room temperature for 48 hours, it was precipitated with cold ether, and the precipitate was collected and dried in vacuo to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, i.e. tPBLG-CTA; wherein, The molar ratio of tPBLG, DDACT, DMAP and DCC after water removal is 1:6:3:8, and 5g of tPBLG after water removal is dissolved in every 100 ml of anhydrous chloroform; Water time 4 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add azo Diisobutyronitrile was reacted at 60°C for 48 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and vacuum-dried to obtain a three-armed star Poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide), that is, to obtain tPBLG-b-PDMAPMA; wherein, tPBLG-CTA, DMAPMA, N - The molar ratio of methylolacrylamide and azobisisobutyronitrile is 1:72:7.2:0.36, and 2g of tPBLG-CTA is dissolved in every 100 ml of anhydrous dioxane;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 800Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.05:4; dissolve 12g of methoxy-terminated polyethylene glycol per 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA is removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal is dissolved in anhydrous chloroform, Then add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 60°C for 52 hours. After the reaction is completed, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water After 48 hours, three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization ; Among them, 2,2'-dithiodiethylisocyanate-modified terminal methyl polyethylene glycol and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N -(3-dimethylaminopropyl) methacrylamide) in a molar ratio of 25:1; 2 g of tPBLG-b-PDMAPMA dissolved in 100 ml of anhydrous chloroform; reflux temperature during azeotropic water removal The temperature is 115°C, and the water removal time is 5 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In chloroform, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the completion of the reaction, dialyze the obtained reaction system in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dry to obtain a three-armed star-shaped hydrophilic copolymer Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymerization The molar ratio of benzyl protecting group in the compound is 20:1; Dissolve 8g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethyl aminopropyl)methacrylamide)-polyethylene glycol copolymer.

Embodiment 6:

1) Same as step 1) in Example 1.

2) Synthesis of three-arm star poly(L-glutamate-γ-benzyl ester) (tPBLG):

Reflux tris(2-aminoethyl)amine through calcium hydride for 12 hours, and distill under reduced pressure under nitrogen atmosphere to obtain anhydrous tris(2-aminoethyl)amine, then dissolve anhydrous tris(2-aminoethyl)amine In anhydrous chloroform, add BLG-NCA under a nitrogen atmosphere, stir to form a uniform solution, and then react at 25 ° C for 70 hours; the obtained reaction solution is precipitated with cold ether and washed with ethanol, and vacuum-dried to obtain a white powder. arm poly(L-glutamic acid-γ-benzyl ester) (tPBLG); wherein, the molar ratio of anhydrous tris(2-aminoethyl)amine and BLG-NCA is 1:90, and every 100 ml of anhydrous chloroform Dissolve 10g of anhydrous tris(2-aminoethyl)amine in

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was azeotropically dewatered with toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous dimethylformamide, and then S-1-deca was added at 0°C under nitrogen atmosphere. Dialkyl-S'-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDACT), 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC ), then precipitated with cold ether after reacting at room temperature for 48 hours, collected the precipitate and vacuum-dried to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, i.e. tPBLG-CTA Wherein, the molar ratio of tPBLG, DDACT, DMAP and DCC after water removal is 1:18:3:40, and 6g of tPBLG after water removal is dissolved in every 100 milliliters of anhydrous dimethylformamide; The reflux temperature is 110°C, and the water removal time is 6 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add 4, 4'-Azobis(cyanovaleric acid) was reacted at 60°C for 48 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and Vacuum drying to obtain three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide), namely to obtain tPBLG-b-PDMAPMA; wherein , the molar ratio of tPBLG-CTA, DMAPMA, N-methylolacrylamide, and 4,4'-azobis(cyanovaleric acid) was 1:290:14.4:1.08 per 100 mL of anhydrous dioxane Dissolve 8.5 g of tPBLG-CTA in the medium.

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 800Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.07:8, and 16g of methoxy-terminated polyethylene glycol is dissolved in 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, and then Add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 60°C for 45 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water for 48 Hours, three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization; Among them, 2,2'-dithiodiethylisocyanate-modified end-methyl polyethylene glycol and three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N- (3-Dimethylaminopropyl) methacrylamide) at a molar ratio of 30:1, dissolve 3.5g of tPBLG-b-PDMAPMA after dehydration in every 100 milliliters of anhydrous chloroform; reflux temperature during azeotropic dehydration The temperature is 120°C, and the water removal time is 2 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In dimethylformamide, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star Hydrophilic copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-poly The molar ratio of benzyl protecting group in the ethylene glycol copolymer is 3:1; Dissolve 12g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

Embodiment 7:

1) Synthesis of γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA):

Add L-glutamic acid-γ-benzyl ester into anhydrous tetrahydrofuran, then add triphosgene under nitrogen atmosphere, then react at 50°C until a clear solution is formed, then concentrate under reduced pressure to remove most of the solvent, and finally concentrate to obtain Anhydrous n-hexane is added in the reaction system to precipitate, and the precipitate obtained is recrystallized with anhydrous n-hexane to obtain γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA); wherein, The molar ratio of L-glutamic acid-γ-benzyl ester to triphosgene is 1:0.38, and 15g of L-glutamic acid-γ-benzyl ester is dissolved in every 100 ml of anhydrous tetrahydrofuran;

2) Same as embodiment 6 step 2);

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was azeotropically dewatered with toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous dimethylformamide, and then 4-cyano- 4-[(dodecylsulfanylthiocarbonyl)sulfanyl]valeric acid, 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), and then reacted at room temperature for 24 hours with Precipitate with cold ether, collect the precipitate and vacuum-dry to obtain a yellow solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, i.e. tPBLG-CTA; wherein, tPBLG after water removal, 4 -Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, DMAP and DCC in a molar ratio of 1:6:3:6 per 100 ml of anhydrous dimethylformamide Dissolve 4g of tPBLG after dehydration in the medium; the reflux temperature during azeotropic dehydration is 110°C, and the dehydration time is 6 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add 4, 4'-Azobis(cyanovaleric acid) was reacted at 65°C for 36 hours. After the reaction was completed, the reaction system was concentrated under reduced pressure to remove the solvent, and then dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and Vacuum drying to obtain three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide), namely to obtain tPBLG-b-PDMAPMA; wherein , the molar ratio of tPBLG-CTA, DMAPMA, N-methylolacrylamide, and 4,4'-azobis(cyanovaleric acid) was 1:290:14.4:1.08 per 100 mL of anhydrous dioxane Dissolve 7 g of tPBLG-CTA in the medium.

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 3000Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.08:3, and 24g of methoxy-terminated polyethylene glycol is dissolved in 100ml of anhydrous toluene;

6) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA was removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal was dissolved in anhydrous chloroform, and then Add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 64°C for 45 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water for 57 Hours, three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization; Among them, 2,2'-dithiodiethylisocyanate-modified end-methyl polyethylene glycol and three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N- The mol ratio of (3-dimethylaminopropyl) methacrylamide) is 36:1, dissolves 4g tPBLG-b-PDMAPMA after removing water in every 100 milliliters of anhydrous chloroforms; The reflux temperature during azeotropic water removal is 120℃, water removal time is 2 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In dimethylformamide, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the reaction, the obtained reaction system is dialyzed in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dried to obtain a three-armed star Hydrophilic copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-poly The molar ratio of benzyl protecting group in the ethylene glycol copolymer is 3:1; Dissolve 16g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

Embodiment 8:

1) Synthesis of γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA):

Add L-glutamic acid-γ-benzyl ester into anhydrous tetrahydrofuran, then add triphosgene under nitrogen atmosphere, then react at 50°C until a clear solution is formed, then concentrate under reduced pressure to remove most of the solvent, and finally concentrate to obtain Anhydrous n-hexane is added in the reaction system to precipitate, and the obtained precipitate is recrystallized with anhydrous n-hexane to obtain γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride (BLG-NCA); wherein, The molar ratio of L-glutamic acid-γ-benzyl ester to triphosgene is 1:0.4, and 5g of L-glutamic acid-γ-benzyl ester is dissolved in every 100 ml of anhydrous tetrahydrofuran;

2) same as step 2) of embodiment 6;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

The tPBLG was azeotropically dewatered by toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous chloroform, and then 4-cyanopentanoic acid dithiocarbamate was added at 0°C under nitrogen atmosphere. Benzoic acid, 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), followed by precipitation with cold ether after reacting at room temperature for 72 hours, collected the precipitate and dried in vacuo to obtain a yellow solid product three-arm poly (L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, namely tPBLG-CTA; wherein, the molar ratio of tPBLG, 4-cyanovaleric acid dithiobenzoic acid, DMAP and DCC after water removal 1:6:3:8, dissolve 5g of tPBLG after dehydration in every 100ml of anhydrous chloroform; the reflux temperature during azeotropic dehydration is 115℃, and the dehydration time is 4 hours;

4) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) (tPBLG-b-PDMAPMA):

Dissolve tPBLG-CTA, N-(3-dimethylaminopropyl)methacrylamide (DMAPMA), and N-methylolacrylamide in anhydrous dioxane, and then add azo Diisobutyronitrile was reacted at 61°C for 72 hours. After the reaction, the resulting reaction system was concentrated under reduced pressure to remove the solvent, dissolved in dichloromethane and precipitated in cold ether. The precipitate was collected and vacuum-dried to obtain a three-armed star Poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide), that is, to obtain tPBLG-b-PDMAPMA; wherein, tPBLG-CTA, DMAPMA, N - The molar ratio of methylolacrylamide and azobisisobutyronitrile is 1:72:7.2:0.36, and 2g of tPBLG-CTA is dissolved in every 100 ml of anhydrous dioxane;

5) Synthesis of 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol:

Dissolve dry methoxy-terminated polyethylene glycol with a molecular weight of 800Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate in anhydrous toluene, and react at 85°C under a nitrogen atmosphere for 48 hours , adding anhydrous n-hexane to the obtained reaction system for precipitation to obtain 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol; wherein, methyl-terminated polyethylene glycol, di The molar ratio of dibutyltin laurate to 2,2'-dithiodiethylisocyanate is 1:0.05:4; dissolve 30g of methoxy-terminated polyethylene glycol per 100ml of anhydrous toluene;

6) three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer synthesis:

Under a nitrogen atmosphere, the trace water contained in tPBLG-b-PDMAPMA is removed by toluene azeotropic method to obtain tPBLG-b-PDMAPMA after water removal, and the tPBLG-b-PDMAPMA after water removal is dissolved in anhydrous chloroform, Then add 2,2'-dithiodiethylisocyanate-modified methyl-terminated polyethylene glycol and react at 65°C for 52 hours. After the reaction, the resulting reaction system is concentrated and precipitated with ether, followed by dialysis in distilled water After 48 hours, three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer was obtained after lyophilization ; Among them, 2,2'-dithiodiethylisocyanate-modified terminal methyl polyethylene glycol and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N -(3-dimethylaminopropyl) methacrylamide) in a molar ratio of 25:1; 2 g of tPBLG-b-PDMAPMA dissolved in 100 ml of anhydrous chloroform; reflux temperature during azeotropic water removal The temperature is 115°C, and the water removal time is 5 hours;

7) Synthesis of three-arm star-shaped hydrophilic copolymer:

Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in anhydrous In chloroform, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the completion of the reaction, dialyze the obtained reaction system in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dry to obtain a three-armed star-shaped hydrophilic copolymer Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymerization The molar ratio of benzyl protecting group in the compound is 20:1; Dissolve 22g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethyl aminopropyl)methacrylamide)-polyethylene glycol copolymer.

Embodiment 9:

1) same as step 1) of embodiment 6;

2) same as step 2) of embodiment 6;

3) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent:

tPBLG was dewatered by azeotropic toluene under nitrogen to obtain tPBLG after dehydration. The tPBLG after dehydration was dissolved in anhydrous chloroform, and then 4-cyano-4-(sulfur Benzoyl) valeric acid, 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC), followed by precipitation with cold ether after 72 hours at room temperature, the precipitate was collected and dried in vacuo to give a yellow The solid product three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent, namely tPBLG-CTA; wherein, tPBLG after water removal, 4-cyano-4-(thiobenzoyl ) The molar ratio of valeric acid, DMAP and DCC is 1:6:3:8, and 2.5g of tPBLG after water removal is dissolved in every 100 ml of anhydrous chloroform; the reflux temperature is 115°C during azeotropic water removal, and the water removal time is 4 Hour;

4) same as step 4) of embodiment 6;

5) same as step 5) of embodiment 6;

6) same as step 6) of embodiment 6;

7) Dissolve the dry three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer in In anhydrous chloroform, add anhydrous hydrazine dropwise and react at 40°C for 24 hours. After the completion of the reaction, dialyze the obtained reaction system in ammonia water with a mass concentration of 0.25% for 2 days, and finally freeze-dry to obtain a three-armed star-shaped hydrophilic Sexual copolymer; Among them, anhydrous hydrazine and three-arm star poly (L-glutamic acid-γ-benzyl ester)-poly (N-(3-dimethylaminopropyl) methacrylamide)-polyethylene di The molar ratio of benzyl protecting group in the alcohol copolymer is 20:1; Dissolve 5g three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3- Dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer.

The cold ether temperature in the above-mentioned examples 1-9 is -20°C below zero, the vacuum drying temperature is 30°C, and the freeze-drying temperature is -40°C. When the reaction system is concentrated, it is concentrated to one-third to one-half of the original volume one.

The methoxy-terminated polyethylene glycol modified by 2,2'-dithiodiethylisocyanate in the present invention is the product 2,2'-dithio Diethyl isocyanate modified polyethylene glycol.

Claims (10)

1. a synthetic method of three-arm star-shaped hydrophilic copolymer, is characterized in that, comprises the steps: 1) Synthesis of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecular RAFT chain transfer agent: Dissolve the three-armed poly(L-glutamic acid-γ-benzyl ester) after water removal in an anhydrous organic solvent, and then add a RAFT chain transfer agent with a carboxyl group at 0°C to 4°C under a nitrogen atmosphere, 4 -Dimethylaminopyridine and dicyclohexylcarbodiimide, react at room temperature for 24 hours to 72 hours, then precipitate with cold ether, collect the precipitate and dry it in vacuum to obtain three-arm poly(L-glutamic acid-γ-benzyl ester ) Macromolecular RAFT chain transfer agent; wherein, the three-arm poly(L-glutamic acid-γ-benzyl ester) after water removal, the RAFT chain transfer agent with a carboxyl group, 4-dimethylaminopyridine and dicyclohexyl carbon The molar ratio of diimine is 1:(6~20):3:(6~60); 2) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide): Dissolve three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent, N-(3-dimethylaminopropyl)methacrylamide, N-methylolacrylamide in anhydrous In dioxane, then add the initiator in an oxygen-free environment, react at 60 ° C ~ 65 ° C for 36 hours ~ 72 hours, after the reaction is completed, concentrate the obtained reaction system, then dissolve it in dichloromethane and precipitate it in cold ether , the precipitate was collected and dried in vacuo to obtain three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide); among them, the three-armed poly The molar ratio of (L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent, N-(3-dimethylaminopropyl)methacrylamide and N-methylolacrylamide is 1:(72 ~290): (3.6~14.4); 3) Synthesis of three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer: Dissolve the three-arm star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide) after water removal in anhydrous chloroform, and then add 2,2'-Dithiodiethylisocyanate-modified methoxy-terminated polyethylene glycol, reacted at 60°C-65°C for 36 hours-72 hours, concentrated the reaction system obtained after the reaction, and precipitated with ether, Then dialyzed in distilled water, and finally freeze-dried to obtain three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene Diol copolymer; wherein, isocyanated methoxy-terminated polyethylene glycol and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylamine) after water removal Propyl) methacrylamide) molar ratio is (9 ~ 36): 1; 4) Synthesis of three-armed star-shaped hydrophilic copolymer: the three-armed star-shaped poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide )-polyethylene glycol copolymer is subjected to hydrazinolysis to remove the benzyl group to obtain a three-armed star-shaped hydrophilic copolymer. 2. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 1 is characterized in that, the poly(L-glutamic acid-γ-benzyl ester) in described step 1) adopts following Prepared by the following method: dissolve anhydrous tris(2-aminoethyl)amine in anhydrous organic solvent, add γ-benzyl ester-L-glutamic acid-N-carboxyl ring acid anhydride under nitrogen atmosphere, stir Form a homogeneous solution, and then react at 25°C to 40°C for 48 hours to 72 hours. After the reaction, the obtained reaction solution is precipitated with cold ether and washed with ethanol, and dried in vacuum to obtain poly(L-glutamic acid-γ-benzyl ester ); wherein, the molar ratio of anhydrous tris (2-aminoethyl) amine and gamma-benzyl ester-L-glutamic acid-N-carboxyl ring anhydride is (1:30)～(1:120), And the anhydrous organic solvent in step 1) is anhydrous dimethylformamide, anhydrous chloroform or anhydrous tetrahydrofuran. 3. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 2, is characterized in that: described anhydrous three (2-aminoethyl) amine is to reflux through calcium hydride, under nitrogen atmosphere Distilled under reduced pressure. 4. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 2 is characterized in that, described gamma-benzyl ester-L-glutamic acid-N-carboxyl ring internal acid anhydride is synthesized by the following steps : Add L-glutamic acid-γ-benzyl ester into anhydrous tetrahydrofuran, then add triphosgene under a nitrogen atmosphere, then react at 50°C until a clear solution is formed and concentrate, and finally add no Water n-hexane is used for precipitation, and the precipitate obtained is recrystallized with anhydrous n-hexane to obtain γ-benzyl ester-L-glutamic acid-N-carboxyl anhydride; wherein, L-glutamic acid-γ-benzyl ester and The molar ratio of triphosgene is 1:(0.35～0.4). 5. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 1 is characterized in that, described step 4) the method for removing benzyl by hydrazinolysis is: three-arm star-shaped poly(L- Glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl) methacrylamide)-polyethylene glycol copolymer is dissolved in anhydrous organic solvent, after adding anhydrous hydrazine dropwise React at 30°C to 40°C for 18 hours to 48 hours. After the reaction, dialyze the obtained reaction system in ammonia water with a mass concentration of 0.25%, and finally freeze-dry to obtain a three-armed star-shaped hydrophilic copolymer; wherein, anhydrous Hydrazine and three-armed star poly(L-glutamic acid-γ-benzyl ester)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer with benzyl protecting group The molar ratio is (3-20):1. 6. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 1 is characterized in that: the RAFT chain transfer agent with a carboxyl in the described step 1) is S-1-dodecyl -S'-(α,α'-dimethyl-α"-acetic acid) trithiocarbonate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid , 4-cyano-4-(thiobenzoyl)valeric acid or 4-cyanovaleric acid dithiobenzoic acid. 7. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 1 is characterized in that: the initiator in described step 2) is azobisisobutyronitrile or 4,4'-isobutyronitrile Nitrobis(cyanovaleric acid), and the molar ratio of three-arm poly(L-glutamic acid-γ-benzyl ester) macromolecule RAFT chain transfer agent to initiator is 1:(0.36-1.44). 8. the synthetic method of three-arm star-shaped hydrophilic copolymer according to claim 1, is characterized in that, in described step 3), the terminal armor modified by 2,2'-dithiodiethylisocyanate Oxy-polyethylene glycol is synthesized by the following method: methoxy-polyethylene glycol with a molecular weight of 800-4000Da, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate are dissolved in In water toluene, react under nitrogen atmosphere at 85°C for 48 hours, add anhydrous n-hexane to the obtained reaction system for precipitation, collect the precipitate and dry it in vacuum to obtain 2,2'-dithiodiethylisocyanate modified methoxy-terminated polyethylene glycol; wherein, the molar ratio of methoxy-terminated polyethylene glycol, dibutyltin dilaurate and 2,2'-dithiodiethylisocyanate is 1:(0.02～0.08) : (3～8). 9. A three-arm star-shaped hydrophilic copolymer that is synthesized by the method described in any one of claims 1 to 8, characterized in that its chemical name is three-arm star-shaped poly(L-glutamine Acid-γ-hydrazide)-poly(N-(3-dimethylaminopropyl)methacrylamide)-polyethylene glycol copolymer, the chemical structure is: 10. The application of the three-arm star-shaped hydrophilic copolymer as claimed in claim 9 as a carrier for loading doxorubicin and gene medicine.