CN103524748A - Polyamino acid graft copolymer, preparation method thereof and injectable hydrogel - Google Patents

Abstract

The present invention provides a kind of polyaminoacid graft copolymer and preparation method thereof, syringeability hydrogel, the polyaminoacid graft copolymer is as shown in the formula (I). Compared with existing natural polymer,The present invention using the poly(L-glutamic acid) with formula (V) structure as main chain,It can simulate native protein and polypeptide to a certain extent,With good biocompatibility and degradability; The graft polymers has the branch of formula (II) structure,It contains phenol structure and adjustable different grafting rate,It can be crosslinked under the action of horseradish peroxidase and hydrogen peroxide,Formation condition is mild,Reaction rate is easy to control,Therefore the higher syringeability hydrogel of mechanical strength can be prepared; And the graft copolymer also includes the branch of formula (III) structure,It can also adjust different grafting rates,To make polyaminoacid graft copolymer shown in formula (I) that there is good water solubility.

Description

Polyamino acid graft copolymer and its preparation method, injectable hydrogel

technical field

The invention belongs to the technical fields of biotechnology, biomedical materials and tissue engineering, and in particular relates to a polyamino acid graft copolymer, a preparation method thereof, and an injectable hydrogel.

Background technique

Hydrogel is a kind of polymer with a cross-linked network structure that can quickly absorb and retain a large amount of water. The polymer network structure contains hydrophilic groups or hydrophilic segments that can combine with water in an aqueous environment. , this hydrogel structure allows hydrophilic small molecules to diffuse in it.

Injectable hydrogel is a new type of hydrogel system that has emerged in recent years. It has unique solution-gel transition characteristics. It is a low-viscosity aqueous solution before gelation, which is convenient for injection. , capable of rapid formation of hydrogels in situ.

Injectable hydrogels are convenient for loading drugs, cells and bioactive molecules, and are suitable for repairing wounds with complex shapes and achieving minimally invasive treatment. Therefore, they have a wide range of applications in local drug release and in situ tissue defect repair. Application prospect.

The prior art discloses a variety of injectable hydrogels, such as block copolymer hydrogels of polyethylene glycol and poly(L-lactic acid), which can achieve reversible changes in sol and gel when the temperature changes; The block copolymer formed by ethylene-polyoxypropylene polymer and polyalanine can also form injectable hydrogel at a certain concentration. However, the poor stability and low mechanical strength of the injectable hydrogel prepared by the prior art limit its application in the biomedical field.

Injectable hydrogels based on enzyme-catalyzed crosslinking have attracted increasing attention due to their mild formation conditions, easy control of reaction rate, relatively high mechanical strength, and good biocompatibility. Generally, under the catalysis of horseradish peroxidase (HRP) and hydrogen peroxide (H ₂ O ₂ ), polymers containing phenol or aniline derivatives can form CC and CO bond crosslinks, and then in situ A hydrogel is formed.

At present, hydrogels based on enzyme-catalyzed crosslinking mainly focus on natural polysaccharides and proteins, such as glucose, hyaluronic acid, chitosan, alginate, cellulose, and gelatin. However, natural polymers have a single component and their properties are difficult to control.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a polyamino acid graft copolymer, a preparation method thereof, and an injectable hydrogel, and the graft ratio of the polyamino acid graft copolymer is controllable.

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

The -R ₁ has a structure of formula (II):

The -R ₂ has the structure of formula (III):

Wherein, x, y, z and m are degree of polymerization, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.

Preferably, the range of m is 10≤m≤300.

The present invention also provides a kind of preparation method of polyamino acid graft copolymer, comprises the following steps:

Mix tyramine, polyethylene glycol monomethyl ether with the structure of formula (IV), poly(L-glutamic acid) with the structure of formula (V) and organic solvent, and under the action of coupling agent, condensation reaction occurs , to obtain the polyamino acid graft copolymer shown in formula (I); the -R ₁ has the structure of formula (II); the -R ₂ has the structure of formula (III);

Among them, x, y, z, m and n are the degree of polymerization, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000; n=x+y+z .

Preferably, the molar ratio of the tyramine to the carboxyl group of the poly(L-glutamic acid) having the structure of formula (V) is (0.1-0.4):1.

Preferably, the molar ratio of the polyethylene glycol monomethyl ether having the structure of formula (IV) to the carboxyl group of poly(L-glutamic acid) having the structure of formula (V) is (0.1-0.4):1.

Preferably, the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinate One or more of imides.

The present invention also provides an injectable hydrogel, comprising the polyamino acid graft copolymer represented by formula (I), an aqueous solvent, horseradish peroxidase and hydrogen peroxide;

The -R ₁ has a structure of formula (II):

The -R ₂ has the structure of formula (III):

Wherein, x, y, z and m are degree of polymerization, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.

Preferably, the polyamino acid graft copolymer represented by the formula (I) is mixed with an aqueous solvent to form a first mixed solution containing 2-30 wt% of the polyamino acid graft copolymer.

Preferably, the horseradish peroxidase is mixed with an aqueous solvent to form a second mixed solution of 0.01 mg/mL-2.0 mg/mL.

Preferably, the hydrogen peroxide is mixed with an aqueous solvent to form a third mixed solution of 0.1 mmol/L-200 mmol/L. The invention provides a polyamino acid graft copolymer and its preparation method, and injectable hydrogel, which comprises tyramide, polyethylene glycol monomethyl ether with the structure of formula (IV), poly (L-glutamic acid) is mixed with an organic solvent, and under the action of a coupling agent, a condensation reaction occurs to obtain a polyamino acid graft copolymer represented by formula (I). Compared with existing natural polymers, the present invention uses poly(L-glutamic acid) with the structure of formula (V) as the main chain, which can simulate natural proteins and polypeptides to a certain extent, and has good biocompatibility and degradability; the grafted polymer has a branched chain of formula (II), which contains a phenol structure and can adjust different grafting ratios, and can be cross-linked under the action of horseradish peroxidase and hydrogen peroxide, The formation conditions are mild and the reaction rate is easy to control, so injectable hydrogels with high mechanical strength can be prepared; and the graft copolymer also contains branched chains of the formula (III), and different grafting ratios can also be adjusted , so that the polyamino acid graft copolymer represented by formula (I) has good water solubility.

Description of drawings

Fig. 1 is the H NMR spectrum of the polyamino acid graft copolymer shown in formula (I) prepared in Example 5 of the present invention;

Fig. 2 is the H NMR spectrum of the polyamino acid graft copolymer shown in formula (I) prepared in Example 6 of the present invention;

Fig. 3 is the H NMR spectrum of the polyamino acid graft copolymer shown in formula (I) prepared in Example 7 of the present invention;

Fig. 4 is the H NMR spectrum of the polyamino acid graft copolymer shown in formula (I) prepared in Example 8 of the present invention;

Fig. 5 is a graph showing the gel time of the injectable hydrogel prepared in Example 7 of the present invention as a function of the concentration of horseradish peroxidase;

Figure 6 is a graph showing the gel time of the injectable hydrogel prepared in Example 7 of the present invention as a function of the concentration of horseradish peroxidase;

Figure 7 is a graph showing the gel time of the injectable hydrogel prepared in Example 7 of the present invention as a function of the concentration of horseradish peroxidase;

Figure 8 is a graph showing the gel time of the injectable hydrogel prepared in Example 8 of the present invention as a function of the concentration of horseradish peroxidase;

Fig. 9 is the gel time curve of the injectable hydrogel prepared in Example 8 of the present invention as a function of the concentration of hydrogen peroxide;

Fig. 10 is a dynamic mechanical test diagram of the injectable hydrogel prepared in Example 8 of the present invention.

Detailed ways

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

The -R ₁ has a structure of formula (II):

The -R ₂ has the structure of formula (III):

Wherein, x, y, z and m are degree of polymerization; 3≤x≤400, preferably 6≤x≤300, more preferably 15≤x≤210; 3≤y≤400, preferably 6≤y≤300, More preferably 15≤y≤210; 0≤z≤900, preferably 5≤z≤788, more preferably 10≤z≤570; 30≤x+y+z≤1000, preferably 50≤x+y+ z≤800, more preferably 100≤x+y+z≤600; the range of m is preferably 10≤m≤300, more preferably 10≤m≤227.

According to the present invention, the weight of _R1 is preferably 2% to 15% of the weight of the polyamino acid graft copolymer, more preferably 5% to 15%; the weight of _R2 is preferably the weight of the polyamino acid graft copolymer 50% to 80%, more preferably 60% to 70%.

The polyamino acid graft polymer of the present invention has a branched chain of formula (II), which contains a phenol structure and can adjust different grafting ratios, and can be cross-linked under the action of horseradish peroxidase and hydrogen peroxide to form The conditions are mild and the reaction rate is easy to control, so injectable hydrogels with high mechanical strength can be prepared; and the graft copolymer also contains branched chains of the formula (III), and different grafting ratios can also be adjusted, Therefore, the polyamino acid graft copolymer represented by formula (I) has good water solubility.

The present invention also provides a preparation method of the polyamino acid graft copolymer represented by the above formula (I), comprising the following steps:

Mix tyramine, polyethylene glycol monomethyl ether with the structure of formula (IV), poly(L-glutamic acid) with the structure of formula (V) and organic solvent, and under the action of coupling agent, condensation reaction occurs , to obtain the polyamino acid graft copolymer represented by formula (I).

Wherein, m and n are polymers, and the m is the same as above, and will not be repeated here; the n=x+y+z.

The present invention has no special limitation on the sources of all raw materials, which can be commercially available.

The polyamino acid graft copolymer of the present invention has poly(L-glutamic acid) with the structure of formula (V) as the main chain, which can simulate natural proteins and polypeptides to a certain extent, and has good biocompatibility and degradability . Poly(L-glutamic acid) having structure (V) in the present invention is preferably prepared according to the following method: with n-hexylamine and/or triethylamine as initiator, γ-benzyl-L-glutamic acid ester- The ring-opening polymerization reaction of N-carboxylic acid internal acid anhydride in the first organic solvent, followed by deprotection of benzyl group, obtains poly(L-glutamic acid) having the structure of formula (V).

The γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride is preferably prepared according to the following method: γ-benzyl-L-glutamate is cyclized with bis(trichloromethyl)carbonate reaction to obtain γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride. The cyclization reaction is preferably carried out in a second organic solvent under anhydrous conditions, and the second organic solvent is soluble γ-benzyl-L-glutamate and bis(trichloro The organic solvent of methyl)carbonate gets final product, without special limitation, preferably tetrahydrofuran among the present invention; Described γ-benzyl-L-glutamate, bis(trichloromethyl)carbonate and the second The organic solvent is preferably mixed according to the ratio of 1g: (0.6-0.8) g: (10-15) ml; the temperature of the cyclization reaction is preferably 30°C-70°C, more preferably 40°C-60°C; the ring The time of the cyclization reaction is preferably 0.5～3h, more preferably 1～3h; the cyclization reaction is preferably carried out under the protection of an inert gas; the inert gas can be an inert gas well known to those skilled in the art, and there is no special Limitation, preferably nitrogen in the present invention. In the present invention, the sources of γ-benzyl-L-glutamate, bis(trichloromethyl)carbonate and the second organic solvent are not particularly limited, as long as they are commercially available.

After the cyclization reaction, a purification treatment is preferably performed. The method for the purification is a method well known to those skilled in the art. The present invention preferably uses cold petroleum ether or n-hexane to settle, filters and drains, dissolves the solid with ethyl acetate, and washes it with cold water for many times. Dry magnesium sulfate over night, filter and recrystallize with ethyl acetate and n-hexane, and dry the solid in vacuum for 12-48 hours to obtain γ-benzyl-L-glutamic acid ester-N-carboxylic acid internal anhydride.

In the invention, n-hexylamine and/or triethylamine are used as initiators to undergo ring-opening polymerization reaction of gamma-benzyl-L-glutamic acid ester-N-carboxylic acid internal acid anhydride in a first organic solvent. Wherein, the molar ratio of the initiator to γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride is preferably 1: (50-800), more preferably 1: (100-600); The first organic solvent is an organic solvent known to those skilled in the art that can dissolve n-hexylamine, triethylamine and γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride, and there is no special limitation , preferably one or more of N,N-dimethylformamide, chloroform and dioxane in the present invention; the temperature of the ring-opening polymerization reaction is preferably 0°C to 40°C, more preferably 10°C ℃～30℃; the time of the ring-opening polymerization reaction is preferably 24-72h, more preferably 48-72h; the ring-opening polymerization reaction in the present invention is preferably carried out under the protection of an inert gas, and the inert gas is preferably nitrogen.

After the ring-opening polymerization reaction, a purification treatment is preferably performed. The purification treatment is preferably carried out with diethyl ether for sedimentation and suction filtration to obtain poly(γ-benzyl-L-glutamic acid ester).

After obtaining poly(γ-benzyl-L-glutamate), benzyl deprotection was performed. The method for benzyl deprotection in the present invention is not particularly limited, it can be the method for removing benzyl or benzyloxycarbonyl well known to those skilled in the art, and the present invention preferably adopts the following method: the obtained poly(γ -benzyl-L-glutamic acid ester) is dissolved in dichloroacetic acid, then a mixed solution of hydrobromic acid and acetic acid is added, and reacted at room temperature to obtain poly(L-glutamic acid) having a structure of formula (V). Wherein, the ratio of the mixed solution of poly(γ-benzyl-L-glutamate), dichloroacetic acid, hydrobromic acid and acetic acid is preferably 1 g: (5-15) ml: (1-5) ml; The volume fraction of hydrobromic acid in the mixed solution of hydrobromic acid and acetic acid is preferably 20% to 40%; the time for the reaction at room temperature is preferably 1 to 2h; Settled in the medium, filtered, dissolved the solid in N,N-dimethylsulfoxide, dialyzed in water for three days with a dialysis bag of corresponding size and molecular weight, and obtained poly(L-glucose) with the structure of formula (V) after freeze-drying acid).

Mix poly(L-glutamic acid) with the structure of formula (V), tyramine, polyethylene glycol monomethyl ether with the structure of formula (IV) and organic solvent, and under the action of coupling agent, condensation reaction occurs . The molar ratio of the poly(ethylene glycol monomethyl ether) having the structure of formula (IV) to the carboxyl group of poly(L-glutamic acid) having the structure of formula (V) is preferably (0.1-0.4): 1, more preferably (0.12～0.38): 1, more preferably (0.15～0.35): 1; the molar ratio of the tyramine to the carboxyl group of the poly(L-glutamic acid) having the structure of formula (V) is preferably (0.1～ 0.4): 1, more preferably (0.12-0.38): 1, more preferably (0.15-0.35): 1; the coupling agent and the carboxyl group of poly(L-glutamic acid) having the structure of formula (V) The molar ratio is preferably (0.25-0.8): 1, more preferably (0.3-0.7): 1; in the present invention, it is preferred to use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt One or more of acid salt, N, N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide are coupling agents; the organic solvent is well known to those skilled in the art and can dissolve (V) poly(L-glutamic acid), tyramine, polyethylene glycol monomethyl ether having the structure of formula (IV) and the organic solvent of the coupling agent are sufficient, and there is no special limitation. In the present invention Preferably it is one or more of N,N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone.

In the present invention, the above-mentioned raw materials are preferably carried out in the following order of addition: dissolve poly(L-glutamic acid) having a structure of formula (V) and a coupling agent in an organic solvent, react at room temperature for 4-12 hours, and then add tyramine and Polyethylene glycol monomethyl ether having the structure of formula (IV) undergoes condensation reaction.

The temperature of the condensation reaction in the present invention is preferably 20°C-60°C, more preferably 30°C-50°C; the time of the condensation reaction is preferably 24-72h, more preferably 36-56h.

After the condensation reaction, it is preferred to carry out purification treatment, and the reaction solution is dialyzed in water with a dialysis bag of corresponding size and molecular weight, preferably for 2 to 4 days, and freeze-dried to obtain the polyamino acid graft copolymer represented by formula (I).

In the present invention, tyramine with different feed ratios, polyethylene glycol monomethyl ether with the structure of formula (IV) and poly(L-glutamic acid) with the structure of formula (V) are condensed to obtain the The polyamino acid graft polymer represented by formula (I) can be used as a polymer gel material with enzyme-catalyzed cross-linking function, and has good water solubility, adjustable gelation rate and cross-linking strength. The medical field has broad application prospects.

The present invention also provides an injectable hydrogel, comprising the polyamino acid graft copolymer represented by formula (I), an aqueous solvent, horseradish peroxidase and hydrogen peroxide; wherein, the formula (I ) The polyamino acid graft copolymer shown in ) is the same as described above, and will not be repeated here.

In the injectable hydrogel, the polyamino acid graft copolymer represented by the formula (I) is mixed with an aqueous solvent to form a mixed solution, preferably mixed with an aqueous solvent to form a first mixed solution of 2 to 30 wt%, more preferably 5% to 28%, more preferably 8% to 25%.

In the injectable hydrogel of the present invention, both the horseradish peroxidase and hydrogen peroxide exist in the form of a mixture of aqueous solvents. Wherein, the horseradish peroxidase is preferably mixed with an aqueous solvent to form a second mixed solution of 0.01 mg/mL-2.0 mg/mL, more preferably 0.02 mg/ml-1.5 mg/ml, and more preferably 0.05 mg /ml～1mg/ml; The hydrogen peroxide is preferably mixed with an aqueous solvent to be the third mixed solution of 0.1mmol/L～200mmol/L, more preferably 0.2mmol/L～150mmol/L, more preferably 0.5mmol/L L～100mmol/L.

The aqueous solvent can be an aqueous solvent well-known to those skilled in the art, and there is no special limitation. In the present invention, it is preferably water, physiological saline, buffer solution, tissue culture fluid or body fluid.

In the present invention, the polyamino acid graft copolymer represented by formula (I), horseradish peroxidase and hydrogen peroxide are respectively dissolved in an aqueous solvent to obtain a corresponding aqueous solution, and then the aqueous solutions are mixed to form C-C and The cross-linking point of the C-O bond can quickly form a hydrogel, and can be adjusted by adjusting the grafting rate of tyramine, the concentration of polyamino acid graft copolymer shown in formula (I), the concentration of horseradish peroxidase and the The concentration of hydrogen oxide regulates the gel formation time and the mechanical strength of the gel; at the same time, since the polyamic acid graft copolymer shown in formula (I) contains polyethylene glycol monomethyl ether branched chain, it has good water soluble. In summary, the hydrogel provided by the present invention has the advantages of mild formation conditions, easy control of reaction rate, and easy regulation of mechanical strength, and has biocompatibility and degradability, and can be used in the field of biomedical materials, especially in the field of pharmaceuticals. It has broad application prospects in controlled release and tissue engineering.

In order to further illustrate the present invention, the polyamino acid graft copolymer provided by the present invention, its preparation method, and injectable hydrogel are described in detail below in conjunction with examples.

The reagents used in the following examples are all commercially available.

Example 1

1.1 10g of polyethylene glycol monomethyl ether with a number average molecular weight of 1000 and 100ml of toluene were azeotroped to remove water at 140°C, then the toluene was removed under reduced pressure, and 50ml of dichloromethane was added to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 8ml triethylamine and 16ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

1.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 1.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

1.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

1.4 Dissolve 2 g of γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 1.3 in 20 ml of N,N-dimethylformamide from which water has been removed, and add 25 μL of n-hexylamine as an initiator Under the condition of nitrogen protection, the reaction was stirred at room temperature for 72 hours. After the reaction, it was settled with ether, and the solid was dried in vacuum for 24 hours to obtain poly(γ-benzyl-L-glutamate).

1.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 1.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Calculated by NMR, its molecular weight is 4600.

1.6 Dissolve 1g of the poly(L-glutamic acid) obtained in 1.5 in 50ml of dimethyl sulfoxide, then add 0.35g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl (the point is in the middle, the same below)) and 0.22g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 1g of polyethylene glycol with the structure of formula (IV) obtained in 1.2 Monomethyl ether and 0.16 g of tyramine were reacted at room temperature for 24 hours, and the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

1.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 1.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well to obtain an injectable hydrogel. Use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 1.6 is analyzed by nuclear magnetic resonance, and the result is: the grafting rate of polyethylene glycol monomethyl ether is 12%, and the grafting rate of tyramine is 10%, the yield is 87%.

Example 2

2.1 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 1000 and 100 ml of toluene were removed by azeotropy at 140° C., then the toluene was removed under reduced pressure, and 50 ml of dichloromethane was added to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 8ml triethylamine and 16ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

2.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 2.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

2.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

2.4 Dissolve 2 g of γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 2.3 in 20 ml of N,N-dimethylformamide that has been dehydrated, and add 25 μL of n-hexylamine as an initiator Under the condition of nitrogen protection, the reaction was stirred at room temperature for 72 hours. After the reaction, it was settled with ether, and the solid was dried in vacuum for 24 hours to obtain poly(γ-benzyl-L-glutamate).

2.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 2.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Calculated by NMR, its molecular weight is 4600.

2.6 Dissolve 1 g of the poly(L-glutamic acid) obtained in 2.5 in 50 ml of dimethyl sulfoxide, then add 0.48 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.29g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 1.24g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 2.2 and 0.22g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

2.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 2.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation to obtain an injectable gel. When the small tube is inverted, gelation does not occur within 30s.

The polyamino acid graft copolymer shown in the formula (I) obtained in 2.6 is analyzed by nuclear magnetic resonance, and the results obtained are: the grafting rate of polyethylene glycol monomethyl ether is 15%, and the grafting rate of tyramine is 16%, and the yield was 84%.

Example 3

3.1 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene were removed by azeotropy at 140° C., then the toluene was removed under reduced pressure, and 50 ml of dichloromethane was added to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

3.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 3.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

3.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

3.4 Dissolve 2 g of γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 3.3 in 20 ml of N,N-dimethylformamide from which water has been removed, and add 6 μL of n-hexylamine as an initiator Under the condition of nitrogen protection, the reaction was stirred at room temperature for 72 hours. After the reaction, it was settled with ether, and the solid was dried in vacuum for 24 hours to obtain poly(γ-benzyl-L-glutamate).

3.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 3.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Calculated by NMR, its molecular weight is 21000.

3.6 Dissolve 1g of the poly(L-glutamic acid) obtained in 3.5 in 50ml of dimethylsulfoxide, then add 0.45g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.27g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 2.32g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 3.2 and 0.2g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

3.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 3.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 3.6 is analyzed by nuclear magnetic resonance, and the results obtained are: the grafting rate of polyethylene glycol monomethyl ether is 16%, and the grafting rate of tyramine is 14%, and the yield was 89%.

Example 4

4.1 Azeotropically remove water from 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene at 140°C, then remove the toluene under reduced pressure, add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

4.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 4.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

4.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

4.4 Dissolve 2 g of γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 4.3 in 20 ml of N,N-dimethylformamide from which water has been removed, and add 6 μL of n-hexylamine as an initiator Under the condition of nitrogen protection, the reaction was stirred at room temperature for 72 hours. After the reaction, it was settled with ether, and the solid was dried in vacuum for 24 hours to obtain poly(γ-benzyl-L-glutamate).

4.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 4.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Calculated by NMR, its molecular weight is 21000.

4.6 Dissolve 1g of the poly(L-glutamic acid) obtained in 4.5 in 50ml of dimethyl sulfoxide, then add 0.60g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.36g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 3.1g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 4.2 and 0.27g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

4.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 4.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 4.6 is analyzed by nuclear magnetic resonance, and the results obtained are: the grafting rate of polyethylene glycol monomethyl ether is 21%, and the grafting rate of tyramine is 19%, and the yield was 82%.

Example 5

5.1 Azeotropically remove 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene at 140° C., then remove the toluene under reduced pressure, add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

5.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 5.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72h. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

5.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

5.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 5.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

5.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 5.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

5.6 Dissolve 1g of the poly(L-glutamic acid) obtained in 5.5 in 50ml of dimethyl sulfoxide, then add 0.45g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.27g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 2.32g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 5.2 and 0.20g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

5.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 5.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in formula (I) obtained in 5.6 is analyzed by nuclear magnetic resonance, and its hydrogen nuclear magnetic resonance spectrum is obtained, as shown in Figure 1; the result obtained is: polyethylene glycol monomethyl ether The grafting rate was 14%, the grafting rate of tyramine was 15%, and the yield was 79%.

Example 6

6.1 Azeotropically remove water from 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene at 140 ° C, then remove the toluene under reduced pressure, add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

6.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 6.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72h. After the reaction, saturate the reaction solution with sodium chloride, and then use two The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

6.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

6.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 6.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

6.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 6.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

6.6 Dissolve 1g of the poly(L-glutamic acid) obtained in 6.5 in 50ml of dimethylsulfoxide, then add 0.60g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.36g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 3.1g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 6.2 and 0.27g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

6.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 6.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 6.6 is analyzed by nuclear magnetic resonance, and its hydrogen nuclear magnetic resonance spectrum is obtained, as shown in Figure 2; the result obtained is: polyethylene glycol monomethyl ether The grafting rate was 21%, the grafting rate of tyramine was 20%, and the yield was 85%.

Example 7

7.1 Azeotropically remove water from 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene at 140° C., then remove the toluene under reduced pressure, and add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

7.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 7.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use di The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

7.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

7.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 7.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

7.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 7.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

7.6 Dissolve 1 g of the poly(L-glutamic acid) obtained in 7.5 in 50 ml of dimethyl sulfoxide, then add 0.74 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.45g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 3.9g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 7.2 and 0.34g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

7.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 7.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

When the mass concentration of polyamino acid graft copolymer is 5%, and the concentration of hydrogen peroxide is 4.9mmol/L, the gel time of injectable hydrogel varies with the concentration of horseradish peroxidase, as shown in Figure 5 . It can be seen from Figure 5 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gelation time can be adjusted.

When the mass concentration of polyamino acid graft copolymer is 10%, and the concentration of hydrogen peroxide is 4.9mmol/L, the gel time of injectable hydrogel varies with the concentration of horseradish peroxidase, as shown in Figure 6. . It can be seen from Figure 6 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gelation time can be adjusted.

When the mass concentration of polyamino acid graft copolymer is 15%, and the concentration of hydrogen peroxide is 4.9mmol/L, the gel time of injectable hydrogel varies with the concentration of horseradish peroxidase, as shown in Figure 7. . It can be seen from Figure 7 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gelation time can be adjusted.

The polyamino acid graft copolymer shown in formula (I) obtained in 7.6 is analyzed by nuclear magnetic resonance, and its hydrogen nuclear magnetic resonance spectrum is obtained, as shown in Figure 3; the result obtained is: polyethylene glycol monomethyl ether The grafting rate was 26%, the grafting rate of tyramine was 24%, and the yield was 86%.

Example 8

8.1 Azeotropically remove water from 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and 100 ml of toluene at 140° C., then remove the toluene under reduced pressure, and add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 4ml triethylamine and 8ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, filter and remove after the reaction Precipitate, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

8.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 8.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use di The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

8.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

8.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 8.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

8.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 8.4 in 10ml of dichloroacetic acid, after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, and keep at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

8.6 Dissolve 1 g of the poly(L-glutamic acid) obtained in 8.5 in 50 ml of dimethyl sulfoxide, then add 0.89 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.53g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 4.6g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 8.2 and 0.4g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

8.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 8.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

When the mass concentration of polyamino acid graft copolymer is 10%, and the concentration of hydrogen peroxide is 24.5mmol/L, the gel time of injectable hydrogel varies with the concentration of horseradish peroxidase, as shown in Figure 8 . It can be seen from Figure 8 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gelation time can be adjusted.

When the mass concentration of the polyamino acid graft copolymer is 10%, and the concentration of horseradish peroxidase is 0.5 mg/ml, the gel time of the injectable hydrogel varies with the concentration of hydrogen peroxide, as shown in Figure 9 . It can be seen from Figure 9 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gelation time can be adjusted.

When the mass concentration of polyamino acid graft copolymer is 10%, the concentration of horseradish peroxidase is 0.5mg/ml, and the concentration of hydrogen peroxide is 24.5mmol/L, the three-dimensional energy storage of injectable hydrogel is measured by rheometer The change of modulus with time is obtained from the dynamic mechanical test diagram, as shown in Figure 10. It can be seen from Figure 10 that the polyamino acid graft copolymer can quickly form a hydrogel, and the gel strength can be adjusted.

The polyamino acid graft copolymer shown in formula (I) obtained in 8.6 is analyzed by nuclear magnetic resonance, and its hydrogen nuclear magnetic resonance spectrum is obtained, as shown in Figure 4; the result obtained is: polyethylene glycol monomethyl ether The grafting rate was 31%, the grafting rate of tyramine was 29%, and the yield was 76%.

Example 9

9.1 Azeotropically remove 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 5000 and 100 ml of toluene at 140° C., then remove the toluene under reduced pressure, and add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 1.6ml triethylamine and 3.2ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, after the reaction The precipitate was removed by filtration, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

9.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 9.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use di The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

9.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

9.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 9.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

9.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 9.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

9.6 Dissolve 1 g of the poly(L-glutamic acid) obtained in 9.5 in 50 ml of dimethyl sulfoxide, then add 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.36g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 7.75g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 9.2 and 0.27g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

9.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 9.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 9.6 is analyzed by nuclear magnetic resonance, and the results obtained are: the grafting rate of polyethylene glycol monomethyl ether is 19%, and the grafting rate of tyramine is 20%, the yield is 81%.

Example 10

10.1 Azeotropically remove 10 g of polyethylene glycol monomethyl ether with a number average molecular weight of 5000 and 100 ml of toluene at 140°C to remove water, then remove the toluene under reduced pressure, add 50 ml of dichloromethane to dissolve to obtain a polyethylene glycol monomethyl ether solution. Under the condition of 0°C and anhydrous, slowly add 1.6ml triethylamine and 3.2ml methanesulfonyl chloride to the polyethylene glycol monomethyl ether solution, react at 0°C for 2h, rise to room temperature and stir for 24h, after the reaction The precipitate was removed by filtration, the filtrate was settled with ether, and the solid was obtained by filtration. After vacuum drying at room temperature for 24 hours, polyethylene glycol monomethyl ether methanesulfonate was obtained.

10.2 Dissolve 5g of polyethylene glycol monomethyl ether methanesulfonate obtained in 10.1 and 5g of ammonium chloride in 250ml of ammonia water, and react at room temperature for 72 hours. After the reaction, saturate the reaction solution with sodium chloride, and then use di The reaction product was extracted with methyl chloride, and the obtained organic phase was washed with a 5% sodium chloride aqueous solution, the organic phase was collected, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated and settled with ether, filtered, and the obtained solid was vacuum Dry for 24 hours to obtain polyethylene glycol monomethyl ether with the structure of formula (IV).

10.3 Add 150ml of dried tetrahydrofuran to a dry reaction flask, add 10g of γ-benzyl-L-glutamate and 6g of triphosgene under a nitrogen atmosphere, and react at 55°C for 2 to 3 hours under the protection of nitrogen. After the reaction solution was clarified, it was stirred at room temperature for 30 min, then precipitated with cold petroleum ether, filtered and drained, the solid was dissolved in ethyl acetate, washed with cold water three times, and the organic phase was dried overnight with anhydrous magnesium sulfate. After removing magnesium sulfate by filtration, transfer the filtrate to a dry reaction bottle, recrystallize three times with ethyl acetate and n-hexane, and dry the solid in vacuum for 24 hours to obtain γ-benzyl-L-glutamate-N-carboxylic acid anhydride.

10.4 Dissolve 2 g of the γ-benzyl-L-glutamate-N-carboxylic acid internal anhydride obtained in 10.3 in 20 ml of dehydrated dioxane, add 4 μL of triethylamine as an initiator, and Under protected conditions, the reaction was stirred at room temperature for 72 hours. After the reaction was completed, it was settled with ether, and the solid was vacuum-dried for 24 hours to obtain poly(γ-benzyl-L-glutamic acid ester).

10.5 Dissolve 1g of the poly(γ-benzyl-L-glutamate) obtained in 10.4 in 10ml of dichloroacetic acid, and after it is completely dissolved, add 3ml of acetic acid solution with a volume fraction of 33% hydrobromic acid, at room temperature The deprotection reaction was carried out for 2 hours. After the reaction was completed, it was settled with ether, and the filtered product was dissolved in dimethyl sulfoxide, then transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain poly(L-glutamic acid). Tested by viscosity method, its molecular weight is 110,000.

10.6 Dissolve 1 g of the poly(L-glutamic acid) obtained in 10.5 in 50 ml of dimethyl sulfoxide, then add 1.0 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC·HCl) and 0.55g N-hydroxysuccinimide (NHS) to activate the carboxyl group for 24h, then add 11.6g of polyethylene glycol monomethyl ether with the structure of formula (IV) obtained in 10.2 and 0.4g of phenol After reacting with amine at room temperature for 24 hours, the reaction liquid was transferred into a dialysis bag, dialyzed for three days, and freeze-dried to obtain a polyamino acid graft copolymer represented by formula (I).

10.7 Prepare the polyamino acid graft copolymer represented by formula (I) obtained in 10.6 into a phosphate buffer solution with a mass concentration of 6% to 20% as the first solution; prepare hydrogen peroxide to 2.45 to 49mmol/L Phosphate buffered saline solution is the second solution; Horseradish peroxidase is prepared into 0.0156～0.5mg/ml phosphate buffered saline solution is the third solution; 200 μL of the first solution, 50 μL of the second solution, 50 μL of the third solution Mix well, and use the small tube inversion method to observe the gelation situation. When the small tube is inverted, gelation does not occur within 30 seconds.

The polyamino acid graft copolymer shown in the formula (I) obtained in 10.6 is analyzed by nuclear magnetic resonance, and the results obtained are: the grafting rate of polyethylene glycol monomethyl ether is 31%, and the grafting rate of tyramine is 32%, and the yield was 87%.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a polyamino acid graft copolymer, as shown in the formula (I):

Described-R ₁there is formula (II) structure:

Described-R ₂there is formula (III) structure:

Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.

2. polyamino acid graft copolymer according to claim 1, is characterized in that, the scope of described m is 10≤m≤300.

3. a preparation method for polyamino acid graft copolymer, is characterized in that, comprises the following steps:

Tyrasamine, the poly glycol monomethyl ether with formula (IV) structure, the PLGA with formula V structure are mixed with organic solvent, under the effect of coupling agent, condensation reaction occurs, obtain the polyamino acid graft copolymer shown in formula (I); Described-R ₁there is formula (II) structure; Described-R ₂there is formula (III) structure;

Wherein, x, y, z, m and n are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000; N=x+y+z.

4. preparation method according to claim 3, is characterized in that, described tyrasamine with the mol ratio of carboxyl of PLGA with formula V structure for (0.1～0.4): 1.

5. preparation method according to claim 3, is characterized in that, described in the poly glycol monomethyl ether with formula (IV) structure and the mol ratio of carboxyl with the PLGA of formula V structure be (0.1～0.4): 1.

6. preparation method according to claim 3, is characterized in that, described coupling agent is EDC hydrochloride, N, one or more in N '-dicyclohexylcarbodiimide and N-hydroxy-succinamide.

7. a syringeability hydrogel, is characterized in that, comprises polyamino acid graft copolymer, aqueous solvent, horseradish peroxidase and the hydrogen peroxide shown in formula (I);

Described-R ₁there is formula (II) structure:

Described-R ₂there is formula (III) structure:

Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.

8. syringeability hydrogel according to claim 7, is characterized in that, the polyamino acid graft copolymer shown in described formula (I) and aqueous solvent are mixed into the first mixing solutions of 2～30wt%.

9. syringeability hydrogel according to claim 7, is characterized in that, described horseradish peroxidase and aqueous solvent are mixed into the second mixing solutions of 0.01mg/mL～2.0mg/mL.

10. syringeability hydrogel according to claim 7, is characterized in that, described hydrogen peroxide and aqueous solvent are mixed into the 3rd mixing solutions of 0.1mmol/L～200mmol/L.

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