CN102875818B - Polyamino acid grafted copolymer and preparation method thereof - Google Patents

Abstract

The present invention provides a polyamino acid graft copolymer represented by formula (I), the preparation method of which is: react polyamino acid, hydrophilic compound, and hydrophobic compound under the action of a condensation accelerator to obtain polyamino acid graft copolymer represented by formula (I). The polyamino acid graft copolymer shown; the polyamino acid is polyglutamic acid, polyaspartic acid or polyglutamic acid-polyaspartic acid copolymer; the hydrophilic compound is such as formula (5) ~ Any one of the compounds shown in (8); the hydrophobic compound is a hydroxyl-containing polyester, a C8-C30 fatty alcohol, or a biologically active small biomolecule. In an aqueous medium, the polyamino acid graft copolymer prepared by the invention exists stably, is not easy to deposit, can stably wrap medicines, and is suitable for intravenous delivery of medicines. At the same time, the main chain and side chain of the polyamino acid graft copolymer are natural or synthetic molecules with good biocompatibility, so it has good biocompatibility and degradability.

Description

A kind of polyamino acid graft copolymer and preparation method thereof

technical field

The invention relates to the field of polyamino acid, in particular to a polyamino acid graft copolymer and a preparation method thereof.

Background technique

Polyamino acid macromolecules have good side group modification, biodegradability, biocompatibility and regular secondary structure. Wide application prospects. However, due to the limited functionality of a single polyamino acid, its application is also limited.

It is one of the important ways to improve the properties of polyamino acid polymer materials by introducing the second component into polyamino acid polymer materials to prepare copolymers. By adjusting the type of copolymer monomer, the proportion of each component and the molecular weight of the obtained copolymer to control the performance of the copolymer, and to endow the copolymer with new characteristics for each component material, it can greatly expand the application field of polyamino acid materials .

The Chinese patent document with application number 200880019839.2 discloses a class of conjugates of polyglutamic acid and various drugs. This polymer conjugate can be used for multiple drugs, targeting agents, stabilizers and/or imaging agents deliver.

The Chinese patent literature with the application number 03813182.X and the Chinese patent literature 200680009864.3 respectively disclose polyamino acids modified with α-tocopherol and hydrophobic groups, which can form stable colloidal suspensions and can be used for bioactive Protein binding and controlled release in vivo.

Biomaterials (Vol32, p3862-3874, 2011) reported the grafting of paclitaxel and cyclic arginine-glycine-aspartic acid-D-proline-lysine short peptide (cRGDfK ), and studied its inhibitory effects on tumors at the cell and animal levels.

According to the report of Biomaterials (Vol32, p3435-3446, 2011), positively charged polyamino acid nanocarriers are easy to combine with plasma proteins to cause deposition. A large accumulation of parts. Therefore, the polyamino acid copolymer reported above has a large amount of charges under physiological conditions, and lacks the protection of stabilizers, which is easy to cause deposition under physiological conditions, thus limiting its application in vivo, especially in intravenous drug delivery.

Contents of the invention

The technical problem solved by the present invention is to provide an amino acid graft copolymer. This kind of copolymer system can be used to encapsulate drugs, and it is not easy to deposit in vivo environment, so it has a good application prospect in intravenous drugs.

The present invention provides a polyamino acid graft copolymer as shown in formula (I),

Wherein, L is independently selected from methylene or ethylene;

R ₁ is any one of the groups shown in formula (1) to formula (4);

R ₂ is a hydroxyl-containing polyester group other than one hydroxyl group, a C8~C30 alkyl group, an aryl group, a C8~C30 alkenyl group, a C8~C30 alkyne group, or a C8~C30 group containing at least one heteroatom Alkyl groups or groups other than hydroxyl groups of small biomolecules with biological activity;

R ₃ is methyl, allyl or propargyl;

a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000;

n is the degree of polymerization, 10≤n≤500.

Preferably, the _R2 is a group except one hydroxyl group of polylactic acid, a group except one hydroxyl group of polylactic acid-glycolic acid copolymer, a group except one hydroxyl group of polyε-caprolactone, C10~C20 Alkyl group of C8~C20, aryl group of C10~C20, alkyne group of C10~C20, alkyne group of C10~C20, alkyl group of C1~C20 containing at least one heteroatom, group of paclitaxel except one hydroxyl group or camptothecin Groups other than one hydroxyl group.

Preferably, said R _is oleyl alcohol, α-tocopherol except for hydroxyl group and cholesterol group except for hydroxyl group, docetaxel except for hydroxyl group, 10-hydroxycamptothecin except for hydroxyl group The group except the hydroxyl group of topotecan or the group except the hydroxyl group of irinotecan.

Preferably, the ratio of a to a+b+c is 2-49%.

Preferably, the ratio of c to a+b+c is 2-49%.

The invention provides a kind of preparation method of polyamino acid graft copolymer, comprising:

react polyamino acid, hydrophilic compound and hydrophobic compound under the action of condensation accelerator to obtain polyamino acid graft copolymer as shown in formula (I);

The polyamino acid is polyglutamic acid, polyaspartic acid or polyglutamic acid-polyaspartic acid copolymer;

The hydrophilic compound is any one of the compounds shown in formulas (5) to (8);

The hydrophobic compound is a hydroxyl-containing polyester, a C8-C30 fatty alcohol, or a biologically active small biomolecule;

Wherein, L is independently selected from methylene or ethylene;

R ₁ is a group represented by formula (1) ~ formula (4);

R ₂ is a hydroxyl-containing polyester group other than one hydroxyl group, a C8~C30 alkyl group, an aryl group, a C8~C30 alkenyl group, a C8~C30 alkyne group, or a C8~C30 group containing at least one heteroatom Alkyl groups or groups other than hydroxyl groups of small biomolecules with biological activity;

R ₃ is methyl, allyl or propargyl;

a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000;

n is the degree of polymerization, 10≤n≤500.

Preferably, the condensation accelerator is 4-methylaminopyridine.

Preferably, the hydrophobic compound is polylactic acid, polylactic acid-glycolic acid copolymer, polyε-caprolactone, oleyl alcohol, tocopherol, cholesterol, paclitaxel or camptothecin.

Preferably, the number average molecular weight of the hydrophilic compound is 2000-12000.

Preferably, the reaction time is 40-100 h, and the reaction temperature is 0-25°C.

Compared with the prior art, the present invention has prepared a polyamino acid graft copolymer as shown in formula (I), in formula (I), R ₁ is a hydrophilic side chain, and R ₂ is a hydrophobic side chain. In the aqueous medium, the hydrophilic side chain is on the outside of the polyamino acid graft copolymer, which protects the negatively charged polyamino acid main chain and makes the negatively charged poly amino acid main chain less susceptible to protein adsorption, thereby ensuring the polyamino acid The graft copolymer exists stably and is not easy to deposit; the hydrophobic side chain is located inside the polyamino acid graft copolymer, which ensures that the polyamino acid graft copolymer can self-assemble into smaller particles, making it easy to carry drugs and difficult to deposit, Therefore, the amino acid graft copolymer can stably wrap medicines and is suitable for intravenous delivery of medicines. At the same time, the main chain and side chain of the polyamino acid graft copolymer are natural or synthetic molecules with good biocompatibility, so it has good biocompatibility and degradability.

Description of drawings

Fig. 1 is the proton nuclear magnetic resonance spectrum figure of the poly(L-glutamic acid) homopolymer that embodiment 2 provides;

Fig. 2 is the proton nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer that embodiment 14 provides;

Fig. 3 is the infrared spectrogram of the polyamino acid graft copolymer that embodiment 14 provides;

Fig. 4 is the proton nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer that embodiment 19 provides;

Fig. 5 is the infrared spectrogram of the polyamino acid graft copolymer that embodiment 19 provides;

Fig. 6 is the proton nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer that embodiment 25 provides;

Fig. 7 is the infrared spectrogram of the polyamino acid graft copolymer that embodiment 25 provides;

Fig. 8 is the result figure of the investigation of the toxicity of the polyamino acid graft copolymer prepared in Example 19 and the positive control PEI25K to HeLa cells;

Fig. 9 is the pharmacokinetic curve of the polyamino acid graft copolymer prepared in Example 19 loaded with paclitaxel.

Detailed ways

In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the present invention.

The embodiment of the present invention discloses a polyamino acid graft copolymer represented by formula (I),

Wherein, L is independently selected from methylene or ethylene;

R ₁ is any one of the groups shown in formula (1) to formula (4);

R ₂ is a hydroxyl-containing polyester group other than one hydroxyl group, a C8~C30 alkyl group, an aryl group, a C8~C30 alkenyl group, a C8~C30 alkyne group, or a C8~C30 group containing at least one heteroatom Alkyl groups or biologically active small biomolecules except hydroxyl groups; preferably polylactic acid except one hydroxyl group, polylactic acid-glycolic acid copolymer except one hydroxyl group, polyε-caprolactone Groups other than one hydroxyl group, C10~C20 alkyl groups, C8~C20 aryl groups, C10~C20 alkenyl groups, C10~C20 alkyne groups, C1~C20 alkyl groups containing at least one heteroatom, paclitaxel A group other than a hydroxyl group or a group of camptothecin except a hydroxyl group; more preferably oleyl alcohol, alpha-tocopherol except a hydroxyl group and cholesterol except a hydroxyl group, docetaxel except a hydroxyl group Groups other than 10-hydroxycamptothecin except hydroxyl groups, topotecan groups except hydroxyl groups or irinotecan groups except hydroxyl groups.

R ₃ is methyl, allyl or propargyl;

a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000, a, b and c preferably satisfy the following conditions: 50≤a+b+c≤200 , the ratio of a to a+b+c is 2~49%, the ratio of c to a+b+c is 2~49%, the ratio of b to (a+b+c) is 2~96%; a, b and c more preferably meet the following conditions: the ratio of a to a+b+c is 5~20%, the ratio of c to a+b+c is 5~30%, the ratio of b to (a+b+c) 10~50%;

n is the degree of polymerization, 10≤n≤500, preferably 40≤n≤280.

The present invention also provides a kind of preparation method of polyamino acid graft copolymer, comprising:

react polyamino acid, hydrophilic compound and hydrophobic compound under the action of condensation accelerator to obtain polyamino acid graft copolymer as shown in formula (I);

The polyamino acid is polyglutamic acid, polyaspartic acid or polyglutamic acid-polyaspartic acid copolymer;

The hydrophilic compound is any one of the compounds shown in formulas (5) to (8);

The hydrophobic compound is a hydroxyl-containing polyester, a C8-C30 fatty alcohol, or a biologically active small biomolecule;

Wherein, L is independently selected from methylene or ethylene;

R ₁ is a group represented by formula (1) ~ formula (4);

R ₂ is a hydroxyl-containing polyester group other than one hydroxyl group, a C8~C30 alkyl group, an aryl group, a C8~C30 alkenyl group, a C8~C30 alkyne group, or a C8~C30 group containing at least one heteroatom Alkyl groups or biologically active small biomolecules except hydroxyl groups; preferably polylactic acid except one hydroxyl group, polylactic acid-glycolic acid copolymer except one hydroxyl group, polyε-caprolactone Groups other than one hydroxyl group, C10~C20 alkyl groups, C8~C20 aryl groups, C10~C20 alkenyl groups, C10~C20 alkyne groups, C1~C20 alkyl groups containing at least one heteroatom, paclitaxel A group other than a hydroxyl group or a group of camptothecin except a hydroxyl group; more preferably oleyl alcohol, alpha-tocopherol except a hydroxyl group and cholesterol except a hydroxyl group, docetaxel except a hydroxyl group Groups other than 10-hydroxycamptothecin except hydroxyl groups, topotecan groups except hydroxyl groups or irinotecan groups except hydroxyl groups.

R ₃ is methyl, allyl or propargyl;

a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000, a, b and c preferably satisfy the following conditions: 50≤a+b+c≤200 , the ratio of a to a+b+c is 2~49%, the ratio of c to a+b+c is 2~49%, the ratio of b to (a+b+c) is 2~96%; a, b and c more preferably meet the following conditions: the ratio of a to a+b+c is 5~20%, the ratio of c to a+b+c is 5~30%, the ratio of b to (a+b+c) 10~50%;

n is the degree of polymerization, 10≤n≤500, 40≤n≤280.

In the present invention, polyamino acids, hydrophilic compounds, and hydrophobic compounds are used as raw materials for the reaction. The polyamino acid is polyaspartic acid, polyglutamic acid or polyaspartic acid-polyglutamic acid copolymer. The number average molecular weight of the polyamino acid is preferably 1,000-150,000. The present invention has no special restrictions on the preparation method of the polyamino acid, and it can be prepared in a manner well known to those skilled in the art, such as the following method:

The amino acid-N-internal carboxylic acid anhydride is initiated by n-hexylamine, undergoes a ring-opening polymerization reaction in an organic solvent, and deprotects to obtain a polyamino acid after the reaction. The amino acid-N-internal carboxylic acid anhydride is γ-benzyl-L-glutamic acid ester, γ-benzyl-L-aspartic acid ester or a mixture of both. The reaction time is preferably 60-80 hours, the reaction temperature is preferably 20-30°C, and the organic solvent is preferably N,N-dimethylformamide.

In the present invention, the hydrophilic compound is any one of the compounds shown in formulas (5) to (8). In the present invention, there is no special limitation on the source of the hydrophilic compound, which can be prepared in a manner known to those skilled in the art, or can be purchased from the market. The number average molecular weight of the hydrophilic compound is preferably 2000-12000. In order to improve the performance of the final polyamino acid copolymer, it is preferable to modify the R ₃ group in the compounds represented by formulas (5) to (8), such as modification with galactose or cRGDfk. The present invention has no special limitation on the modification method, and it can be carried out in a manner known to those skilled in the art.

The hydrophobic compound is a hydroxyl-containing polyester, a C8-C30 fatty alcohol, or a biologically active small molecule, preferably polylactic acid, polylactic acid-glycolic acid copolymer, polyε-caprolactone, oleyl alcohol, Tocopherol, cholesterol, paclitaxel or camptothecin, more preferably polyε-caprolactone with a number average molecular weight of 500-5000, α-tocopherol, docetaxel, 10-hydroxycamptothecin, topotecan, i Litecan. The present invention has no special limitation on the source of the hydrophobic compound, which can be purchased from the market.

In the present invention, the polyamino acid graft copolymer shown in formula (I) can be obtained by reacting polyamino acid, hydrophilic compound, and hydrophobic compound under the action of a condensation accelerator. The condensation accelerator is preferably 4-methylaminopyridine. The solvent for the reaction is preferably dicyclohexylcarbodiimide and anhydrous dimethylsulfoxide. The reaction time is preferably 40~100h, and the reaction temperature is preferably 0~25°C. After the reaction is finished, it is preferably filtered, and the filtrate is dialyzed with N,N-dimethylformamide for 20 to 30 hours, dialyzed with water once, and freeze-dried to obtain the polyamino acid graft copolymer shown in formula (I). The polyamino acid graft copolymer exists in a powder state. The reaction can be completed in one step or in two steps. When one step is completed, the polyamino acid, hydrophilic compound, and hydrophobic compound are added to react simultaneously; when it is completed in two steps, the polyamino acid and the hydrophilic compound are first reacted Then react with hydrophobic compounds.

The polyamino acid graft copolymer prepared by the present invention is co-cultured with HeLa cells, and the result shows that the survival rate of the cells is high, which shows that it has good biocompatibility.

The polyamino acid graft copolymer of the present invention is used to load the hydrophobic drug paclitaxel, its surface potential is measured, and the metabolism of the drug loading system in blood is investigated. The results show that it can prolong the metabolism time of paclitaxel in blood.

In order to further understand the present invention, the following examples illustrate the polyamino acid graft copolymer provided by the present invention and its preparation method, and the protection scope of the present invention is not limited by the following examples. In the following examples, the raw materials used are purchased from the market or prepared according to conventional methods, and the yield of each product=actually obtained product quality/theoretical obtained product quality×100%.

Example 1~3

Add 2.63g (0.01mol) of γ-benzyl-L-glutamate-N-internal carboxylic acid anhydride monomer and 30mL of anhydrous N,N-dimethylformamide into the reaction flask respectively, and stir to dissolve. 0.0002 mol, 0.0001 mol and 0.00005 mol of n-hexylamine were added under stirring conditions, and the reaction was continued for 72 hours under stirring at 25°C. After the reaction, the reaction mixture was settled with 300 mL ether, filtered, washed three times with ether, and vacuum-dried at room temperature for 24 hours to obtain intermediate products, namely poly(γ-benzyl-L-glutamate).

The poly(γ-benzyl-L-glutamate) was analyzed by nuclear magnetic resonance with deuterated trifluoroacetic acid as a solvent, and its number-average molecular weight and average degree of polymerization were calculated according to the nuclear magnetic resonance spectrum. For the results, see Table 1.

Table 1 Number average molecular weight, average degree of polymerization and productive rate of the intermediate product provided by the embodiment of the present invention 1～3

Dissolve 1 g of the above-mentioned poly(γ-benzyl-L-glutamate) in 10 mL of dichloroacetic acid, and add 3 mL of hydrogen bromide/glacial acetic acid solution with a mass content of 33% under stirring to obtain a reaction mixture , after stirring the reaction mixture at 25° C. for 1 h, the obtained product was settled with 150 mL of ether, filtered, dialyzed with water once, and freeze-dried to obtain poly(L-glutamic acid) homopolymer.

Using deuterated water as a solvent to carry out nuclear magnetic resonance analysis on the poly(L-glutamic acid) homopolymer respectively, calculate the number average molecular weight and The average degree of polymerization, the results are shown in Table 2.

Table 2 Number average molecular weight, average degree of polymerization and yield of the poly(L-glutamic acid) homopolymers provided by Examples 1 to 3 of the present invention

NMR analysis was carried out on the poly(L-glutamic acid) homopolymers prepared in Examples 1-3 respectively, and the results showed that poly(L-glutamic acid) homopolymers were prepared in Examples 1-3. Figure 1 is the proton nuclear magnetic resonance spectrum of the poly(L-glutamic acid) homopolymer provided in Example 2. In Figure 1, the chemical shift 4.43ppm is the signal peak of the methine group on the main chain, 2.21ppm is the signal peak of the methylene group connected to the carbonyl group on the side group, and 1.91ppm and 1.71ppm are the signal peaks connected to the main chain on the side group The signal peak of the methylene group.

Embodiment 4~6

Add 2.49g (0.01mol) of γ-benzyl-L-aspartic acid ester-N-internal carboxylic acid anhydride monomer and 30mL of anhydrous N,N-dimethylformamide to the three reaction flasks respectively, Stir to dissolve. Under the condition of stirring, 0.0002 mol, 0.0001 mol and 0.00005 mol of n-hexylamine were respectively added to the three reaction flasks, and the reaction was continued for 72 hours under stirring at 25°C. After the reaction, the reaction mixture was settled with 300 mL ether, filtered, washed three times with ether, and dried in vacuum at room temperature for 24 hours to obtain intermediate products, namely poly(γ-benzyl-L-aspartic acid ester).

Dissolve 1 g of the above-mentioned poly(γ-benzyl-L-aspartic acid ester) in 10 mL of dichloroacetic acid, and add 3 mL of hydrogen bromide/glacial acetic acid solution with a mass content of 33% under stirring conditions to obtain a reaction mixture After the reaction mixture was stirred at 25°C for 1 h, the obtained product was settled with 150 mL of ether, filtered, dialyzed with water once, and freeze-dried to obtain poly(L-aspartic acid) homopolymer.

Using deuterated water as a solvent to carry out nuclear magnetic resonance analysis on the poly(L-aspartic acid) homopolymer respectively, and calculate the molecular weight of the poly(L-aspartic acid) homopolymer according to the nuclear magnetic resonance spectrum. Number average molecular weight and average degree of polymerization, the results are shown in Table 3.

Table 3 Number average molecular weight, average degree of polymerization and yield of poly(L-aspartic acid) homopolymers provided by Examples 4 to 6 of the present invention

Embodiment 7~9

Add 0.526g (0.002mol), 1.315g (0.005mol), 2.104g (0.008mol) of γ-benzyl-L-glutamic acid ester-N-internal carboxylic acid anhydride monomer and Add 1.992g (0.008mol), 1.245g (0.005mol), 0.498g (0.002mol) of γ-benzyl-L-aspartic acid ester-N-internal carboxylic acid anhydride monomer and 30mL of anhydrous N,N-Dimethylformamide, stirred to dissolve. Under the condition of stirring, 0.0001mol of n-hexylamine was added respectively, and the reaction was continued for 72h under stirring at 25°C. After the reaction, the reaction mixture was settled with 300 mL of ether, filtered, washed three times with ether, and dried in vacuum at room temperature for 24 hours to obtain an intermediate product, poly(γ-benzyl-L-aspartic acid ester-co- gamma-benzyl-L-aspartate).

Dissolve 1 g of the above-mentioned poly(γ-benzyl-L-aspartate-co-γ-benzyl-L-aspartate) in 10 mL of dichloroacetic acid, and add 3 mL of mass A hydrogen bromide/glacial acetic acid solution with a content of 33% was used to obtain a reaction mixture. After stirring the reaction mixture for 1 h at 25° C., the obtained product was settled with 150 mL of ether, filtered, dialyzed with water once, and freeze-dried. A poly(L-glutamic acid-co-L-aspartic acid) copolymer was obtained.

Using deuterated water as a solvent, the poly(L-glutamic acid-co-L-aspartic acid) copolymer is subjected to nuclear magnetic resonance analysis, and the poly(L-glutamine) is calculated according to the nuclear magnetic resonance spectrum. acid-co-L-aspartic acid) copolymer number average molecular weight and average degree of polymerization, the results are shown in Table 4.

Table 4 The number average molecular weight, average degree of polymerization and yield of poly(L-glutamic acid-co-L-aspartic acid) copolymers provided by Examples 7 to 9 of the present invention

Example 10

The poly(L-glutamic acid) used in Example 10 was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction bottle, then add 0.20g (0.1mmol) polyethylene glycol monomethyl ether, and 0.23g (0.1mmol) poly(L-glutamic acid) For ε-caprolactone, add 82.5 mg (0.4 mmol) of dicyclohexylcarbodiimide (DCC), 2.4 mg (0.02 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 11

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction bottle, then add 0.40g (0.2mmol) polyethylene glycol monomethyl ether, and 0.23g (0.1mmol) poly For ε-caprolactone, add 124 mg (0.6 mmol) of dicyclohexylcarbodiimide (DCC), 3.7 mg (0.03 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 12

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction bottle, then add 0.80g (0.4mmol) polyethylene glycol monomethyl ether, and 0.23g (0.1mmol) poly(L-glutamic acid) For ε-caprolactone, add 206 mg (1.0 mmol) of dicyclohexylcarbodiimide (DCC), 6.1 mg (0.05 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, nuclear magnetic resonance analysis was carried out on the above-mentioned polyamino acid graft copolymers, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 13

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) to each dry reaction bottle, then add 0.20g (0.1mmol) of polyethylene glycol monomethyl ether, and 0.46g (0.2mmol) of poly(L-glutamic acid) For ε-caprolactone, add 124 mg (0.6 mmol) of dicyclohexylcarbodiimide (DCC), 3.7 mg (0.03 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 14

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) to each dry reaction bottle, then add 0.40g (0.2mmol) of polyethylene glycol monomethyl ether, and 0.46g (0.2mmol) of poly For ε-caprolactone, add 165 mg (0.8 mmol) of dicyclohexylcarbodiimide (DCC), 4.9 mg (0.04 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Figure 2 is the H NMR spectrum of the polyamino acid graft copolymer provided in Example 14 of the present invention. In Figure 2, the chemical shift of 3.65ppm is the methylene signal peak on the polyethylene glycol graft chain; The methylene signal peak on the chain; several signal peaks around 2.2ppm are the signal peaks on the poly(L-glutamic acid) main chain (partially shielded by the grafted chain).

Figure 3 is the infrared spectrum of the polyamino acid graft copolymer provided in Example 14 of the present invention. In Figure 3, the peak at wave number 1734 cm ^-1 is the stretching vibration absorption peak of poly(L-glutamic acid) amide carbonyl (ν _C=O ); the peak at wave number 1108 cm ^-1 is polyethylene glycol The absorption peak of stretching vibration of branched ether bonds (δ _CO ); the peak at wavenumber 1697cm ^-1 is the absorption peak of stretching vibration of poly(ε-caprolactone) ester bond carbonyl group (ν _C=O ); wavenumber is 2946cm ^- The peak that appears in ¹ is the stretching vibration absorption peak (ν _CH ) of the alkyl carbon-hydrogen bond.

It can be seen from Figure 2 and Figure 3 that poly(L-glutamic acid) reacted with polyethylene glycol and polyε-caprolactone to form a graft copolymer.

Example 15

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction flask, then add 0.80g (0.4mmol) polyethylene glycol monomethyl ether, and 0.46g (0.2mmol) poly(L-glutamic acid) To ε-caprolactone, add 248 mg (1.2 mmol) of dicyclohexylcarbodiimide (DCC), 7.3 mg (0.06 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 16

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction bottle, then add 0.20g (0.2mmol) polyethylene glycol monomethyl ether, and 0.92g (0.4mmol) poly(L-glutamic acid) To ε-caprolactone, add 248 mg (1.2 mmol) of dicyclohexylcarbodiimide (DCC), 7.3 mg (0.06 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, nuclear magnetic resonance analysis was carried out on the above-mentioned polyamino acid graft copolymers, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 17

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) to each dry reaction bottle, then add 0.40g (0.2mmol) of polyethylene glycol monomethyl ether, and 0.46g (0.2mmol) of poly For ε-caprolactone, add 165 mg (0.8 mmol) of dicyclohexylcarbodiimide (DCC), 4.9 mg (0.04 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Example 18

The poly(L-glutamic acid) used in this example was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da; the polyε-caprolactone used had a number average molecular weight of 2300 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to each dry reaction flask, then add 0.80g (0.4mmol) polyethylene glycol monomethyl ether, and 0.46g (0.2mmol) poly(L-glutamic acid) To ε-caprolactone, add 248 mg (1.2 mmol) of dicyclohexylcarbodiimide (DCC), 7.3 mg (0.06 mmol) of 4-methylaminopyridine (DMAP), and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated trifluoroacetic acid as a solvent, the polyamino acid graft copolymer was subjected to nuclear magnetic resonance analysis, and the results are shown in Table 5. Table 5 shows the number of graft chains, number average molecular weight and yield of the polyamino acid graft copolymers provided in Examples 10-18 of the present invention.

Table 5 The number of graft chains, molecular weight and yield of the polyamino acid graft copolymers provided by Examples 10 to 18 of the present invention

Example 19~21

The poly(L-glutamic acid) used in Examples 19-21 was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to the dry reaction bottle, then add 0.40g (0.2mmol) polyethylene glycol monomethyl ether, and 0.2mmol, 0.4mmol, 0.6mmol α-tocopherol, respectively add 165mg (0.8mmol), 248mg (1.2mmol), 330mg (1.6mmol) of dicyclohexylcarbodiimide (DCC), 4.9mg (0.04mmol), 7.3mg (0.06mmol), 9.8 mg (0.08 mmol) of 4-methylaminopyridine (DMAP), 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was carried out on the above-mentioned polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 6 for the results.

Table 6 The number of grafted chains, molecular weight and yield of the amino acid graft copolymers provided by Examples 19 to 21 of the present invention

Figure 4 is the H NMR spectrum of the polyamino acid graft copolymer provided in Example 19 of the present invention. In Fig. 4, the chemical shift 3.65ppm is the methylene signal peak on the polyethylene glycol graft chain; the signal peak of the chemical shift 0.86ppm is the methyl signal peak on the α-tocopherol graft chain; 2.21ppm, 1.91ppm and 1.71ppm are the signal peaks of the methylene group of the poly(L-glutamic acid) main chain structure.

Figure 5 is the infrared spectrum of the polyamino acid graft copolymer provided in Example 19 of the present invention. In Figure 5, the peak at wave number 1749 cm ^-1 is the stretching vibration absorption peak of poly(L-glutamic acid) amide carbonyl (ν _C=O ); the peak at wave number 1108 cm ^-1 is polyethylene glycol The absorption peak of stretching vibration of branched ether bond (δ _CO ); the peak at wavenumber 1697cm ^-1 is the absorption peak of stretching vibration of ester bond link carbonyl group (ν _C=O ); the peak at wavenumber of 2890cm ^-1 is alkane The stretching vibration absorption peak of the carbon-hydrogen bond (ν _CH ).

It can be seen from Figure 4 and Figure 5 that poly(L-glutamic acid) reacted with polyethylene glycol and α-tocopherol to form a graft copolymer.

Example 22~24

The poly(L-glutamic acid) used in Examples 22-24 was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000 Da.

Add 0.26g (0.02mmol) poly(L-glutamic acid) to the dry reaction bottle, then add 0.40g (0.2mmol) polyethylene glycol monomethyl ether, and 0.2mmol, 0.4mmol, 0.6mmol Cholesterol α-tocopherol, add 165mg (0.8mmol), 248mg (1.2mmol), 330mg (1.6mmol) of dicyclohexylcarbodiimide (DCC), 4.9mg (0.04mmol), 7.3mg (0.06mmol) respectively , 9.8 mg (0.08 mmol) of 4-methylaminopyridine (DMAP), 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was carried out on the above polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 7 for the results.

Table 7 The number of graft chains, molecular weight and yield of amino acid graft copolymers provided by Examples 22 to 24 of the present invention

Example 25~30

The poly(L-glutamic acid) used in Examples 25 to 30 was synthesized in Example 2; the polyethylene glycol used was polyethylene glycol monomethyl ether with a number average molecular weight of 2000Da; the biologically active small molecules used in Examples 25 to 30 were as follows: Paclitaxel, docetaxel, camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) to the dry reaction bottle, then add 0.4g (0.2mmol) of polyethylene glycol monomethyl ether and 0.2mmol of biologically active small molecules, Add 165 mg (0.8 mmol) of dicyclohexylcarbodiimide, 4.9 mg (0.04 mmol) of 4-methylaminopyridine, and 10 mL of anhydrous dimethyl sulfoxide, respectively. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was carried out on the above-mentioned polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 8 for the results.

Table 8 The number of graft chains, molecular weight and yield of amino acid graft copolymers provided by Examples 25 to 30 of the present invention

Figure 6 is the H NMR spectrum of the polyamino acid graft copolymer provided in Example 25 of the present invention. In Fig. 6, the chemical shift 3.65ppm is the methylene signal peak on the polyethylene glycol graft chain; the signal peak between the chemical shift 7-8ppm is the aromatic ring signal peak on the paclitaxel graft chain; 2.21ppm, 1.91ppm and 1.71ppm are the signal peaks of the methylene group of the poly(L-glutamic acid) main chain structure.

Figure 7 is the infrared spectrum of the polyamino acid graft copolymer provided in Example 25 of the present invention. In Figure 7, the peak at wave number 1748 cm ^-1 is the stretching vibration absorption peak of poly(L-glutamic acid) amide carbonyl (ν _C=O ); the peak at wave number 1108 cm ^-1 is polyethylene glycol The absorption peak of the stretching vibration of the branched ether bond (δ _CO ); the peak at the wave number 1697cm ^-1 is the absorption peak of the stretching vibration of the carbonyl bonded by the ester bond (ν _C=O ); the peak at the wave number of 2884cm ^-1 is the alkane The stretching vibration absorption peak of the radical carbon-hydrogen bond (ν _CH ); the peak at wave number 3345cm ^-1 is the stretching vibration absorption peak of the hydroxyl group on paclitaxel (ν _OH ).

It can be seen from Figure 6 and Figure 7 that poly(L-glutamic acid) reacted with polyethylene glycol and paclitaxel to form a graft copolymer.

Example 31

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2 to the dry reaction bottle, and then add 0.40g (0.2mmol) of poly(L-glutamic acid) as shown in formula (I), _R3 is allyl Allyl polyethylene glycol, 0.2 mmol of α-tocopherol, 165 mg (0.8 mmol) of dicyclohexylcarbodiimide, 4.9 mg (0.04 mmol) of 4-methylaminopyridine and 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, and then dialyzed with water once, and freeze-dried to obtain a polyamino acid graft copolymer. Yield 95.2%.

Example 32

Add 0.22g (0.1mmol) of the compound shown in formula (I), in formula (I), _R3 is propargyl, 30mg (0.11mmol) of azidogalactose, 10mL of N,N-di Methylformamide, _N2 sparged for 30 min. Add 14.3 mg (0.1 mmol) of cuprous bromide and 31.2 mg (0.2 mmol) of bipyridine, continue purging for 10 minutes, seal, and react at 40°C for 24 hours. After the reaction was completed, the solid was collected by settling twice with 100 mL of glacial ether, and dried under vacuum at room temperature for 24 hours. It was dialyzed for 48 hours with a dialysis bag with a molecular weight cut-off of 1000 Da, and then freeze-dried to obtain galactose-modified polyethylene glycol. Yield 0.25g, yield 99%.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2 to the dry reaction bottle, then add 0.46g (0.2mmol) of galactose-modified polyethylene glycol, and 0.2mmol of To α-tocopherol, add 165 mg (0.8 mmol) of dicyclohexylcarbodiimide, 4.9 mg (0.04 mmol) of 4-methylaminopyridine, and 10 mL of anhydrous dimethyl sulfoxide, respectively. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a lactose-modified polyamino acid graft copolymer. Yield 93.1%.

Example 33

Using azobisisobutyronitrile (AIBN) as a thermal initiator, add 0.98g (0.4mmol) of a compound represented by formula (I) into an ampoule, where R ₃ in formula (I) is an allyl group, 0.11g ( 1mmol) mercaptopropionic acid, 65.7mg (0.4mmol) azobisisobutyronitrile, add 10mL N,N-dimethylformamide, freeze in liquid nitrogen-vacuumize three times, seal, and react at 80°C for 24 hours. After the reaction, settle twice with 100 mL of glacial ether, collect the solid, and dry it under vacuum at room temperature for 24 hours to obtain mercaptopropionic acid-modified polyethylene glycol. Yield 0.982g, yield 96.2%.

The mercaptopropionic acid-modified polyethylene glycol was N-hydroxysuccinimide (NHS) activated. Add 0.767g (0.3mmol) of the above-mentioned mercaptopropionic acid-modified polyethylene glycol, 82.5mg (0.4mmol) of dicyclohexylcarbodiimide (DCC), 20mL of anhydrous dichloromethane into a dry reaction flask, and stir at room temperature After 15 minutes, 46.0mg (0.4mmol) of N-hydroxysuccinimide was added and reacted at 25°C for 24 hours. After the reaction was completed, the solids were settled twice with 100 mL of ether, and the solids were collected and vacuum-dried at room temperature for 24 hours to obtain N-hydroxysuccinimide-activated polyethylene glycol. Yield 0.759g, yield 95.3%.

cRGDfk modification on NHS-activated PEG. Add 0.24 g (0.1 mmol) of NHS-activated polyethylene glycol, 10 mL of water, and 60.4 mg (0.1 mmol) of cRGDfk into a round bottom flask, and stir at room temperature for 24 h. It was dialyzed with a dialysis bag with a molecular weight cut-off of 1000Da for 48 hours, and then freeze-dried to obtain cRGDfk-modified polyethylene glycol. Yield 0.3g, yield 99%.

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2 to the dry reaction bottle, then add 0.60g (0.2mmol) of cRGDfk modified polyethylene glycol, and 0.2mmol of α -Tocopherol, add 165 mg (0.8 mmol) of dicyclohexylcarbodiimide, 4.9 mg (0.04 mmol) of 4-methylaminopyridine, and 10 mL of anhydrous dimethyl sulfoxide, respectively. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polyamino acid graft copolymer modified by cRGDfk. Yield 92.5%.

Example 34

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2, 45.3mg (0.22mmol) of dicyclohexylcarbodiimide (DCC), and 10mL of dimethylimide to the dry reaction flask sulfone, and after stirring for 15 minutes, 23 mg (0.2 mmol) of N-hydroxysuccinimide (NHS) were added. Stirring was continued for 6 hours, and 0.40 g (0.2 mmol) of aminated polyethylene glycol monomethyl ether represented by formula (II), R ₃ was methyl (the number of aminated polyethylene glycol monomethyl ether) was added The average molecular weight is 2000Da). Seal the reaction for 48 hours. After filtration, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried.

Add the above product and 0.083g (0.2mmol) of α-tocopherol to the dry reaction flask, add 165mg (0.8mmol) of dicyclohexylcarbodiimide (DCC), 4.9mg (0.04mmol) of 4-methylamino Pyridine (DMAP), 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was performed on the above-mentioned polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 9 for the results.

Example 35

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2, 45.3mg (0.22mmol) of dicyclohexylcarbodiimide (DCC), and 10mL of dimethylimide to the dry reaction flask sulfone, and after stirring for 15 minutes, 23 mg (0.2 mmol) of N-hydroxysuccinimide (NHS) were added. Stirring was continued for 6 hours, and 0.40 g (0.2 mmol) of aminated polyethylene glycol monomethyl ether as shown in formula (III), R ₃ was methyl (the number of aminated polyethylene glycol monomethyl ether) was added The average molecular weight is 2000Da). Seal the reaction for 48 hours. After filtration, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried.

Add the above product and 0.083g (0.2mmol) of α-tocopherol to the dry reaction flask, add 165mg (0.8mmol) of dicyclohexylcarbodiimide (DCC), 4.9mg (0.04mmol) of 4-methylamino Pyridine (DMAP), 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was performed on the above-mentioned polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 9 for the results.

Example 36

Add 0.26g (0.02mmol) of poly(L-glutamic acid) prepared in Example 2, 45.3mg (0.22mmol) of dicyclohexylcarbodiimide (DCC), and 10mL of dimethylimide to the dry reaction flask sulfone, and after stirring for 15 minutes, 23 mg (0.2 mmol) of N-hydroxysuccinimide (NHS) were added. Stirring was continued for 6 hours, and 0.40 g (0.2 mmol) of aminated polyethylene glycol monomethyl ether as shown in formula (IV) was added, R ₃ being methyl (the number of aminated polyethylene glycol monomethyl ether The average molecular weight is 2000Da). Seal the reaction for 48 hours. After filtration, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried.

Add the above product and 0.083g (0.2mmol) of α-tocopherol to the dry reaction flask, add 165mg (0.8mmol) of dicyclohexylcarbodiimide (DCC), 4.9mg (0.04mmol) of 4-methylamino Pyridine (DMAP), 10 mL of anhydrous dimethyl sulfoxide. React at 25°C for 48 hours after sealing. After filtering, the filtrate was dialyzed with N,N-dimethylformamide for 24 hours, then dialyzed with water once, and freeze-dried to obtain a polygraft copolymer.

Using deuterated heavy water as a solvent, NMR analysis was performed on the above-mentioned polyamino acid graft copolymers, and the number of grafted chains and number average molecular weight of different chains were calculated. See Table 9 for the results.

Table 9 The number of graft chains, molecular weight and yield of the polyamino acid graft copolymers provided by Examples 34 to 36 of the present invention

Example 37

Collect logarithmic phase HeLa (human cervical cancer) cells, adjust the cell concentration, inoculate into a 96-well plate, and each well contains 100 μL (about 10 ⁴ ) cells;

Cultivate at 37°C, saturated humidity, 5% CO ₂ cell incubator for 24 hours, discard the culture medium;

Dilute the block copolymer prepared in Example 19 with the culture medium into solutions of 7 concentrations: 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL, and 7.8125 μg/mL Sample; polyethylene glycol with a molecular weight of 25000K (PEI25K) was used as a positive control, and polyethylene glycol with a molecular weight of 25000K was diluted to 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 Solution samples with 7 concentrations of μg/mL, 15.625μg/mL, and 7.8125μg/mL;

Add each solution sample into a 96-well plate, add 200 μL to each well, and have 6 replicate wells for each concentration;

Incubate at 37°C, saturated humidity, 5% CO ₂ cell incubator for 24h;

After 24 hours, add 20 μL of 5 mg/mL 3-(4,5-dimethylthiazole-2)-2,5-dimethyltetrazolium bromide salt solution to each well, and continue to incubate for 4 hours;

Terminate the culture, suck off the culture medium in the wells, add 150 μL dimethyl sulfoxide to each well, shake at a low speed for 10 minutes, use a microplate reader to detect the absorption value of each well at 492 nm, and convert the block copolymers and positive controls at various concentrations See Figure 8 for the cell viability of PEI25K. Fig. 8 is the result figure of the investigation of the toxicity of the polyamino acid graft copolymer prepared in Example 19 and the positive control PEI25K to HeLa cells, Toxicity of positive control PEI25K to HeLa cells, For the toxicity of polyamino acid graft copolymers to HeLa cells, the results show that the cell survival rate under the polyamino acid graft copolymers of various concentrations is basically the same, and close to 100%. The substance has good biocompatibility and is basically non-toxic to cells.

Example 38

Dissolve 50 mg of the block copolymer prepared in Example 19 in 5 mL of dimethyl sulfoxide, add 5 mg of paclitaxel, add 5 mL of deionized water dropwise under stirring, continue stirring at room temperature for 12 hours, dialyze with deionized water for 48 hours, and change the water 6 times to remove Dimethyl sulfoxide to obtain paclitaxel nanoparticle micelles; the nanoparticle micelles were quickly frozen under aseptic conditions, and freeze-dried to obtain paclitaxel nanoparticle lyophilized powder with a mass ratio of carrier to paclitaxel of 10:1, which produced The rate is 96.3%.

The concentration of paclitaxel in the obtained paclitaxel nanoparticles was measured by high performance liquid chromatography, and the encapsulation efficiency (DLE) and encapsulation amount (DLC) of paclitaxel in the nanoparticles were calculated by the following formula;

The encapsulation efficiency of the obtained paclitaxel nanoparticles was 97.02%, and the encapsulation amount was 8.8%.

The paclitaxel nanoparticle freeze-dried powder obtained was reconstituted, and the potential test was performed on the formed nanoparticle micelles, and the Zeta potential was -23.9±5.7mV.

Example 39

Six healthy rats were weighed and divided into two groups. According to the weight, 0.5-1.0 mL of paclitaxel and paclitaxel nanoparticles were injected into the tail vein, and 0.5 mL of blood was collected from the orbit at 3 min, 0.5 h, 1 h, 4 h, 10 h, and 24 h. Anticoagulant treatment and centrifugation to obtain plasma.

Take 0.2mL plasma, add 20μL internal standard diphenhydramine, 0.6mL acetonitrile, vortex for 1min, and sonicate for 10min. Then centrifuge at 1200rpm for 10min, take the supernatant, and blow dry with nitrogen. HPLC test, reverse phase C18 column, mobile phase is acetonitrile/water=4:1. First test the standard curve, and then test the samples one by one to obtain the drug concentration of each sample.

Fig. 9 is the pharmacokinetic curve of the polyamino acid graft copolymer prepared in Example 19 loaded with paclitaxel. Curve A is the metabolic kinetic curve of the small molecule drug paclitaxel, and curve B is the pharmacokinetic curve of the polyamino acid graft copolymer prepared in Example 19 loaded with paclitaxel. The results showed that after loading paclitaxel on the polyamino acid graft copolymer, the metabolism time of paclitaxel in plasma could be significantly prolonged. It can be seen that, using the polyamino acid graft copolymer provided by the present invention for drug loading can maintain a long circulation time in the blood stably, and has a better application prospect for intravenous drug delivery.

The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. a polyamino acid graft copolymer as shown in formula (I), Wherein, L is independently selected from methylene or ethylene; R _is any one of the groups shown in formula (1) to formula (4); _R2 is a polylactic acid group except one hydroxyl group, a polylactic acid-glycolic acid copolymer except one hydroxyl group, a polyε-caprolactone group except one hydroxyl group, a C10-C20 alkyl group, a C8 ~C20 aryl group, C10~C20 alkenyl group, C10~C20 alkyne group, C1~C20 alkyl group containing at least one heteroatom, taxol except one hydroxyl group or camptothecin except one hydroxyl group group; R ₃ is methyl, allyl or propargyl; a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000; n is the degree of polymerization, 10≤n≤500. 2. polyamino acid graft copolymer according to claim 1 is characterized in that, described R ₂ is oleyl alcohol group, α-tocopherol removes the group other than hydroxyl group and cholesterol removes the group other than hydroxyl group, Dorsey Paclitaxel except hydroxyl group, 10-hydroxycamptothecin except hydroxyl group, topotecan except hydroxyl group or irinotecan except hydroxyl group. 3. The polyamino acid graft copolymer according to claim 1, characterized in that the ratio of a to a+b+c is 2-49%. 4. The polyamino acid graft copolymer according to claim 1, characterized in that the ratio of c to a+b+c is 2-49%. 5. A preparation method of polyamino acid graft copolymer, comprising: Polyamino acid, hydrophilic compound, hydrophobic compound are reacted under the effect of condensation accelerator, obtain polyamino acid graft copolymer as shown in formula (I); The polyamino acid is polyglutamic acid, polyaspartic acid or polyglutamic acid-polyaspartic acid copolymer; The hydrophilic compound is any one of the compounds shown in formulas (5) to (8); The hydrophobic compound is a hydroxyl-containing polyester, a C8-C30 fatty alcohol, or a biologically active small biomolecule; Wherein, L is independently selected from methylene or ethylene; R ₁ is a group shown in formula (1) to formula (4); _R2 is a polylactic acid group except one hydroxyl group, a polylactic acid-glycolic acid copolymer except one hydroxyl group, a polyε-caprolactone group except one hydroxyl group, a C10-C20 alkyl group, a C8 ~C20 aryl group, C10~C20 alkenyl group, C10~C20 alkyne group, C1~C20 alkyl group containing at least one heteroatom, taxol except one hydroxyl group or camptothecin except one hydroxyl group group; R ₃ is methyl, allyl or propargyl; a, b and c are degrees of polymerization, a>0, b≥0, c>0, 10≤a+b+c≤1000; n is the degree of polymerization, 10≤n≤500. 6. The preparation method according to claim 5, characterized in that, the condensation accelerator is 4-methylaminopyridine. 7. The preparation method according to claim 5, wherein the hydrophobic compound is polylactic acid, polylactic acid-glycolic acid copolymer, polyε-caprolactone, oleyl alcohol, tocopherol, cholesterol, paclitaxel or camptothecin. 8. The preparation method according to claim 5, characterized in that the number average molecular weight of the hydrophilic compound is 2000-12000. 9. The preparation method according to any one of claims 5-8, characterized in that the reaction time is 40-100 h, and the reaction temperature is 0-25°C.