阅读说明：本技术 一种聚乙二醇衍生物、其制备方法及可快速发生交联反应的聚乙二醇水凝胶 (Polyethylene glycol derivative, preparation method thereof and polyethylene glycol hydrogel capable of rapidly generating crosslinking reaction ) 是由 贺超良 张震 任慧 陈学思 于 2020-05-26 设计创作，主要内容包括：本发明提供了一种聚乙二醇衍生物,包含具有式(I)结构的重复单元和具有式(II)结构的端基。本发明提供的聚乙二醇衍生物由于具有式(I)结构的重复单元,具有良好的生物相容性。由于具有式(II)结构的邻苯二醛端基,能够和氨基、(酰)肼基、胺氧基等多种基团反应,反应速率快、反应条件温和。将本发明提供的聚乙二醇衍生物与端基含氨基的聚乙二醇在水性介质中混合,可快速形成化学交联水凝胶材料,其制备条件温和,成胶速度快,机械强度高,稳定性好。这种聚乙二醇水凝胶可作为药物缓释载体或组织工程支架等应用于生物医用材料领域。(The invention provides a polyethylene glycol derivative which comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II). The polyethylene glycol derivative provided by the invention has good biocompatibility due to the repeating unit with the structure of the formula (I). The o-phthalaldehyde end group with the structure of the formula (II) can react with various groups such as amino, (acyl) hydrazino, aminoxy and the like, and has the advantages of high reaction rate and mild reaction conditions. The polyethylene glycol derivative provided by the invention and the polyethylene glycol with amino-containing terminal group are mixed in an aqueous medium, so that a chemical crosslinking hydrogel material can be quickly formed. The polyethylene glycol hydrogel can be used as a drug sustained-release carrier or a tissue engineering scaffold and the like to be applied to the field of biomedical materials.)

1. A polyethylene glycol derivative, which is characterized by comprising a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II);

2. the polyethylene glycol derivative according to claim 1, wherein the polyethylene glycol derivative has any one of the structures of formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), and formula (IIIe);

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, and b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is a terminal group with a structure of formula (II).

3. A method for preparing the polyethylene glycol derivative according to claim 1 or 2, comprising the steps of:

A) reacting 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester with polyethylene glycol with an amino group as a terminal group to obtain a reaction product;

B) and deprotecting the reaction product to obtain the polyethylene glycol derivative.

4. The preparation method according to claim 3, characterized in that the succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate is prepared by the following method:

1) carrying out bromination reaction on 3, 4-dimethylbenzoic acid to prepare 3, 4-di (dibromomethyl) benzoic acid;

2) hydrolyzing 3, 4-di (dibromomethyl) benzoic acid to obtain 3, 4-dibenzoyl benzoic acid;

3) reacting 3, 4-dibenzoyl benzoic acid with methanol to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid;

4) 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid reacts with N-hydroxysuccinimide and a condensation reagent to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester.

5. A polyethylene glycol hydrogel capable of rapidly generating a crosslinking reaction, which is characterized in that the polyethylene glycol hydrogel is formed by connecting a polyethylene glycol derivative and polyethylene glycol of which the end group contains amino through a chemical bond, wherein the polyethylene glycol derivative is the polyethylene glycol derivative according to claim 1 or 2.

6. The polyethylene glycol hydrogel according to claim 5, wherein the terminal amino group-containing polyethylene glycol comprises a repeating unit having a structure of formula (IV) and a terminal group having a structure of any one of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve);

-NH₂(V_a)

7. the polyethylene glycol hydrogel of claim 5, wherein the terminal amino group-containing polyethylene glycol has any one of the structures of formula (VIa), formula (VIb), formula (VIc), formula (VId), and formula (VIe):

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is an end group having any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve).

8. A preparation method of the polyethylene glycol hydrogel according to claims 5 to 7, wherein the polyethylene glycol hydrogel is prepared from a polyethylene glycol derivative, polyethylene glycol with an amino group at the end group and a solvent.

9. The method according to claim 8, wherein the solvent is water, physiological saline or a buffer solution;

the mass-volume concentration of the polyethylene glycol derivative is 1-1000 mg/m L;

the mass-volume concentration of the amino-containing polyethylene glycol at the end group is 1-1000 mg/m L.

10. The preparation method according to claim 8, wherein the mass ratio of the polyethylene glycol derivative to the terminal amino group-containing polyethylene glycol is 1: 0.01 to 100.

Technical Field

The invention belongs to the technical field of new biomedical materials, and particularly relates to a polyethylene glycol derivative, a preparation method thereof and polyethylene glycol hydrogel capable of rapidly generating a crosslinking reaction.

Background

Hydrogels are materials with a three-dimensional network structure formed by some cross-linking. Hydrogels are widely used in the biomedical field due to their physicochemical properties similar to those of extracellular matrices and good biocompatibility.

Polyethylene glycols are common starting materials for preparing hydrogels, and include linear polyethylene glycols and multi-arm polyethylene glycols. Because the reactivity of the terminal hydroxyl of polyethylene glycol is low, the polyethylene glycol is required to be derivatized to generate terminal groups with different functions, such as carboxyl, amino, sulfhydryl and aldehyde. Through the chemical reaction of the end group, the chemical crosslinking hydrogel can be conveniently prepared, and other molecules can be coupled to functionalize the hydrogel. The prior art discloses various polyethylene glycol hydrogels, such as polyethylene glycol hydrogels reacted via aldehyde groups with amino groups, polyethylene glycol hydrogels reacted via enzymatic cross-linking. However, the polyethylene glycol hydrogel prepared by the prior art has the defects of low gelling speed, poor stability, low mechanical strength, need of adding a toxic catalyst and the like, and the application of the polyethylene glycol hydrogel in the biomedical field is limited.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a polyethylene glycol derivative, a preparation method thereof, and a polyethylene glycol hydrogel capable of rapidly generating a crosslinking reaction.

The invention provides a polyethylene glycol derivative, which comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II);

preferably, the polyethylene glycol derivative has any one of the structures of formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), and formula (IIIe);

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, and b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is a terminal group with a structure of formula (II).

The invention also provides a preparation method of the polyethylene glycol derivative, which comprises the following steps:

A) reacting 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester with polyethylene glycol with an amino group as a terminal group to obtain a reaction product;

B) and deprotecting the reaction product to obtain the polyethylene glycol derivative.

Preferably, the succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate is prepared by the following method:

1) carrying out bromination reaction on 3, 4-dimethylbenzoic acid to prepare 3, 4-di (dibromomethyl) benzoic acid;

2) hydrolyzing 3, 4-di (dibromomethyl) benzoic acid to obtain 3, 4-dibenzoyl benzoic acid;

3) reacting 3, 4-dibenzoyl benzoic acid with methanol to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid;

4) 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid reacts with N-hydroxysuccinimide and a condensation reagent to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester.

The invention also provides polyethylene glycol hydrogel capable of rapidly generating crosslinking reaction, which is formed by connecting polyethylene glycol derivatives and polyethylene glycol with amino groups at the end groups through chemical bonds, wherein the polyethylene glycol derivatives are the polyethylene glycol derivatives.

Preferably, the amino group-containing polyethylene glycol comprises a repeating unit having a structure of formula (IV) and a terminal group having any one of a structure of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve);

-NH₂(Va)

preferably, the terminal amino group-containing polyethylene glycol has any one of the structures of formula (VIa), formula (VIb), formula (VIc), formula (VId), and formula (VIe):

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is an end group having any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve).

The invention also provides a preparation method of the polyethylene glycol hydrogel, which is characterized by comprising a polyethylene glycol derivative, polyethylene glycol with an amino-containing end group and a solvent.

Preferably, the solvent is water, physiological saline or a buffer solution;

the mass-volume concentration of the polyethylene glycol derivative is 1-1000 mg/m L;

the mass-volume concentration of the amino-containing polyethylene glycol at the end group is 1-1000 mg/m L.

Preferably, the mass ratio of the polyethylene glycol derivative to the amino-containing polyethylene glycol at the end group is 1: 0.01 to 100.

Compared with the prior art, the invention provides a polyethylene glycol derivative which comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II). The polyethylene glycol derivative provided by the invention has good biocompatibility due to the repeating unit with the structure of the formula (I). The o-phthalaldehyde end group with the structure of the formula (II) can react with various groups such as amino, (acyl) hydrazino, aminoxy and the like, and has the advantages of high reaction rate and mild reaction conditions. The polyethylene glycol derivative provided by the invention and the polyethylene glycol with amino-containing terminal group are mixed in an aqueous medium, so that a chemical crosslinking hydrogel material can be quickly formed. The polyethylene glycol hydrogel can be used as a drug sustained-release carrier or a tissue engineering scaffold and the like to be applied to the field of biomedical materials.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate prepared in example 4 of the present invention;

FIG. 2 is a NMR spectrum of a terminal-protected four-arm PEG derivative prepared in example 7 of the present invention;

FIG. 3 is a NMR chart of a four-arm PEG derivative prepared in example 7 of the present invention;

FIG. 4 is a graph showing the change with time of storage modulus and loss modulus of a mixed solution prepared in example 11 of the present invention;

FIG. 5 is a graph showing the change in storage modulus and loss modulus with time of a mixed solution prepared in example 12 of the present invention;

FIG. 6 is a graph showing the change with time of storage modulus and loss modulus of a mixed solution prepared in example 13 of the present invention;

FIG. 7 shows the application of the polyethylene glycol hydrogel prepared in example 14 of the present invention in rat skin wound closure;

FIG. 8 shows that the polyethylene glycol hydrogel prepared in example 15 of the present invention is used for hemostasis of rat liver defect closure;

FIG. 9 shows that the PEG hydrogel prepared in example 16 of the present invention is used for rabbit abdominal aorta defect sealing hemostasis.

Detailed Description

The invention provides a polyethylene glycol derivative, which comprises a repeating unit with a structure shown in a formula (I) and a terminal group with a structure shown in a formula (II);

in the present invention, the polyethylene glycol derivative may be a linear polyethylene glycol derivative or a multi-arm polyethylene glycol derivative.

In some embodiments of the invention, the polyethylene glycol derivative has any one of the structures of formula (IIIa), formula (IIIb), formula (IIIc), formula (IIId), and formula (IIIe);

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is a terminal group with a structure of formula (II).

The invention also provides a preparation method of the polyethylene glycol derivative, which comprises the following steps:

A) reacting 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester with polyethylene glycol with an amino group as a terminal group to obtain a reaction product;

B) and deprotecting the reaction product to obtain the polyethylene glycol derivative.

Wherein the reaction process is as follows:

the invention dissolves 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester and polyethylene glycol with an amino group as a terminal group in an organic solvent, and the reaction product is obtained by reaction and sedimentation in the presence of an acid-binding agent.

The molar equivalent of the 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester is 1.2-5 times, preferably 2 times of that of amino in polyethylene glycol with an end group of amino; the organic solvent is preferably anhydrous dichloromethane; the acid scavenger is preferably anhydrous pyridine.

The reaction time is 24-72 hours, preferably 48 hours; the reaction temperature is 10-40 ℃, and preferably 25 ℃.

The invention preferably adopts anhydrous ether for sedimentation; and filtering the settled solid, and drying in vacuum to obtain a reaction product.

After a reaction product is obtained, the invention carries out deprotection, dialysis and freeze drying on the reaction product to obtain the polyethylene glycol derivative.

The deprotection can be carried out using techniques well known to those skilled in the art. The invention preferably adopts a mixed solvent of trifluoroacetic acid and water; the volume of the mixed solvent is 5-20 times, preferably 5 times of the mass of the reaction product; the deprotection time is 0.5-3 h, preferably 1 h; the deprotection temperature is 10-40 ℃, and preferably 25 ℃.

The dialysis and freeze-drying may be carried out using techniques well known to those skilled in the art.

In the present invention, the succinimidyl 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylate is prepared according to the following method:

1) carrying out bromination reaction on 3, 4-dimethylbenzoic acid to prepare 3, 4-di (dibromomethyl) benzoic acid;

2) hydrolyzing 3, 4-di (dibromomethyl) benzoic acid to obtain 3, 4-dibenzoyl benzoic acid;

3) reacting 3, 4-dibenzoyl benzoic acid with methanol to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid;

4) 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid reacts with N-hydroxysuccinimide and a condensation reagent to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester.

Wherein the reaction process is as follows:

specifically, the 3, 4-di (dibromomethyl) benzoic acid is prepared by bromination reaction of 3, 4-dimethylbenzoic acid.

The bromination reaction may be carried out using techniques well known to those skilled in the art. The invention adopts N-bromosuccinimide as a brominating reagent, any one of benzoyl peroxide and azodiisobutyronitrile as a free radical initiator, and carbon tetrachloride as a solvent to carry out bromination reaction.

The molar equivalent of the N-bromosuccinimide is 3-5 times, preferably 4 times of that of 3, 4-dimethylbenzoic acid; the molar equivalent of the benzoyl peroxide is 0.05-0.5 time of that of the 3, 4-dimethylbenzoic acid, and preferably 0.1 time; the volume (ml) of the carbon tetrachloride is 10-50 times, preferably 20 times of the mass (g) of the 3, 4-dimethylbenzoic acid.

The temperature of the bromination reaction is 70-90 ℃, and the optimal temperature is 81 ℃; the bromination reaction time is 10-20 h, preferably 15 h.

After the bromination reaction is finished, filtering the reaction mixture; washing the filter cake with benzene and diethyl ether; all the filtrates are combined, concentrated and then vacuum-dried; the solid product was recrystallized from acetonitrile to give 3, 4-bis (dibromomethyl) benzoic acid.

The invention preferably adopts a rotary evaporator for concentration; preferably, the concentration temperature is 30 ℃; preferably to 10% of the liquid volume.

The recrystallization may be carried out by a technique known to those skilled in the art.

The invention carries out hydrolysis reaction on 3, 4-di (dibromomethyl) benzoic acid to obtain 3, 4-diformylbenzoic acid.

The hydrolysis reaction may be carried out using techniques well known to those skilled in the art. In the present invention, it is preferable to carry out the hydrolysis reaction of 3, 4-bis (dibromomethyl) benzoic acid in an aqueous solution of sodium carbonate.

The mass-volume concentration of the sodium carbonate aqueous solution is 10 percent; the volume of the sodium carbonate aqueous solution is 5 to 20 times, preferably 10 times of the mass of the 3, 4-bis (dibromomethyl) benzoic acid.

The temperature of the hydrolysis reaction is 60-80 ℃, and preferably 70 ℃; the time of the hydrolysis reaction is 3-5h, and preferably 4 h.

After the hydrolysis reaction is finished, selecting concentrated hydrochloric acid to adjust the pH of the reaction solution to 0-3, preferably 1; extracting with ethyl acetate; concentrating, and vacuum-pumping to obtain 3, 4-diformylbenzoic acid.

The invention makes 3, 4-diformylbenzoic acid react with methanol and catalyst to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid.

The catalyst is preferably scandium trifluoromethanesulfonate; the reaction temperature is 10-40 ℃, and preferably 25 ℃; the reaction time is 6-24 h, and preferably 12 h.

The invention makes 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid react with N-hydroxysuccinimide and a condensation reagent in an organic solvent to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester.

The condensation reagent is any one of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and diisopropylcarbodiimide, and preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the organic solvent is any one of dimethylformamide, dimethyl sulfoxide and acetonitrile, and acetonitrile is preferred.

The reaction temperature is 10-40 ℃, and preferably 25 ℃; the reaction time is 6-24 h, and preferably 12 h.

After the reaction is finished, the crude product is purified by silica gel column chromatography to obtain 1, 3-dimethoxy-1, 3-dihydroisobenzofuran-5-carboxylic acid succinimide ester. The mobile phase of the column chromatography is preferably n-hexane and ethyl acetate, and the volume ratio is 1: 5-1: 1, preferably 1: 3.

the invention also provides polyethylene glycol hydrogel capable of rapidly generating crosslinking reaction, which is formed by connecting polyethylene glycol derivatives and polyethylene glycol with amino groups at the end groups through chemical bonds, wherein the polyethylene glycol derivatives are the polyethylene glycol derivatives.

Wherein the polyethylene glycol with the amino-containing terminal group comprises a repeating unit with a structure of a formula (IV) and a terminal group with any one structure of a formula (Va), a formula (Vb), a formula (Vc), a formula (Vd) and a formula (Ve);

-NH₂(Va)

the polyethylene glycol with the amino group at the end group can be linear polyethylene glycol with the amino group at the end group or multi-arm polyethylene glycol with the amino group at the end group.

In some embodiments of the invention, the terminal amino group-containing polyethylene glycol has any one of the structures of formula (VIa), formula (VIb), formula (VIc), formula (VId), and formula (VIe):

wherein a is the degree of polymerization, and a is more than or equal to 1 and less than or equal to 1000;

b is polymerization degree, b is more than or equal to 1 and less than or equal to 333;

c is polymerization degree, and c is more than or equal to 1 and less than or equal to 250;

d is polymerization degree, d is more than or equal to 1 and less than or equal to 166;

e is polymerization degree, and e is more than or equal to 1 and less than or equal to 125;

r is an end group having any one of the structures of formula (Va), formula (Vb), formula (Vc), formula (Vd) and formula (Ve).

The invention also provides a preparation method of the polyethylene glycol hydrogel, which is prepared from polyethylene glycol derivatives, polyethylene glycol with amino groups at the end groups and a solvent.

In the present invention, the preparation process of the polyethylene glycol hydrogel comprises:

(1) preparing a solution 1 of the polyethylene glycol derivative;

(2) preparing a solution 2 of the polyethylene glycol with the amino-containing terminal group;

(3) and mixing the solution 1 and the solution 2, and carrying out chemical reaction to obtain the polyethylene glycol hydrogel.

In the solution 1, the mass-volume concentration of the polyethylene glycol derivative is 1-1000 mg/m L, preferably 10-200 mg/m L.

In the solution 2, the mass-volume concentration of the amino-containing polyethylene glycol at the end group is 1-1000 mg/m L, preferably 10-200 mg/m L.

The solvent used for preparing the solution 1 and the solution 2 is selected from water, physiological saline or a buffer solution, and the buffer solution is selected from phosphate buffer solution.

The mass ratio of the polyethylene glycol derivative to the polyethylene glycol with the amino group at the end group is 1: 0.01 to 100, preferably 1: 0.1 to 10.

The polyethylene glycol derivative provided by the invention has good water solubility and biocompatibility, and phthalic aldehyde at the tail end of the polyethylene glycol derivative can be chemically reacted with various groups.

The polyethylene glycol derivative provided by the invention comprises a main chain of polyethylene glycol and a terminal group of o-phthalaldehyde. The polyethylene glycol hydrogel is formed by connecting the polyethylene glycol derivative and polyethylene glycol with amino groups at the end groups through chemical bonds. The polyethylene glycol hydrogel has the advantages of high gelling speed, high mechanical strength, excellent tissue adhesion capability and good biocompatibility. The polyethylene glycol hydrogel of the invention has potential applications in the following fields: drug slow release carrier, tissue engineering bracket and hemostatic sealing coating.

In order to further understand the present invention, the following examples are provided to illustrate the polyethylene glycol derivatives, the preparation method thereof, and the polyethylene glycol hydrogel capable of rapidly generating a crosslinking reaction, and the scope of the present invention is not limited by the following examples.