CN110746616A

CN110746616A - Cellulose hydrogel containing phenylboronic acid and preparation method and application thereof

Info

Publication number: CN110746616A
Application number: CN201911025476.XA
Authority: CN
Inventors: 武伟; 张丹; 蒋锡群
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2019-10-25
Filing date: 2019-10-25
Publication date: 2020-02-04

Abstract

The invention discloses a cellulose-based three-dimensional hydrogel containing phenylboronic acid, a preparation method and application thereof. The hydrogel is simple and rapid in preparation process, and has good biocompatibility, self-repairability and cell adhesion.

Description

Cellulose hydrogel containing phenylboronic acid and preparation method and application thereof

Technical Field

The invention belongs to the field of high polymer materials, and particularly relates to a cellulose hydrogel containing phenylboronic acid and crosslinked by using boric acid ester, and a preparation method and application thereof.

Technical Field

Cellulose has ideal biocompatibility, good cell adhesion and high mechanical strength, and has been widely studied as a cell scaffold material in the fields of bone tissue engineering, cartilage tissue engineering, vascular tissue engineering and the like in recent years. The cellulose is widely distributed in natural resources and rich in content, and the content of the cellulose in various plants can almost account for 50 percent, so that the plant cellulose has wide sources and is cheap and easy to obtain. However, cellulose prepared from plant fiber resources does not have a three-dimensional network structure and cannot be directly used as a cell scaffold material, so that the application of the cellulose is greatly limited. If the plant cellulose molecules are prepared into the biocompatible three-dimensional hydrogel material through crosslinking, the biocompatible three-dimensional hydrogel material can be used as a scaffold material in the field of tissue engineering, and the high-added-value application of plant cellulose is realized, so that the development of the efficient preparation method of the cellulose-based three-dimensional hydrogel can greatly promote the application of cellulose in the field of tissue engineering, and has important significance for the recycling of forest residues. In addition, the cell adhesion is one of important indexes for evaluating the scaffold material, and the high cell adhesion is beneficial to the spreading and growth of cells along the scaffold material, so that the improvement of the cell adhesion of the cellulose three-dimensional hydrogel can obviously improve the service performance and the application potential of the cellulose three-dimensional hydrogel.

Disclosure of Invention

The invention provides a method for constructing cellulose three-dimensional hydrogel by using cellulose with flexible binary or polyhydric alcohol or cis-diol as a main polymer and using a poly-phenylboronic acid compound (such as polyethylene glycol, cyclodextrin, polyamino acid, protein, polysaccharide and the like which are covalently modified by phenylboronic acid) as a cross-linking agent. The hydrogel is simple and rapid in preparation process, and has good biocompatibility, self-repairability and cell adhesion.

The specific technical scheme of the invention is as follows:

a preparation method of cellulose hydrogel containing phenylboronic acid comprises the following steps:

(1) the method comprises the following steps of (1) carrying out covalent coupling on a compound with flexible dihydric or polyhydric alcohol or cis-diol and amino and a cellulose salt with carboxyl in an aqueous medium through amidation reaction in the presence of a condensing agent to obtain the cellulose with the flexible dihydric or polyhydric alcohol or cis-diol, wherein the compound with the flexible dihydric or polyhydric alcohol and the amino has the following structural general formula:

the compound with cis-diol and amino has the following structural general formula:

a represents saturated or unsaturated ring, two hydroxyl groups are ortho-or meta-cis-substituted, preferably selected from saturated five-membered ring, saturated six-membered ring or unsaturated cyclic group such as cyclopentene, cyclohexene, benzene ring, naphthalene, anthracene, quinoline, etc., in the above structure

The compound with flexible dihydric or polyhydric alcohol simultaneously has amino substituent groups, dihydric alcohol groups and amino groups can be connected through connecting groups of any number or types, and the position of the amino substituent group is any substitutable position;

(2) mixing a polyphenyl boric acid compound serving as a cross-linking agent with cellulose with flexible dihydric or polyhydric alcohol or cis-diol in an aqueous medium to obtain the cellulose hydrogel containing the phenyl boric acid, wherein the polyphenyl boric acid compound is polyethylene glycol, cyclodextrin, polyamino acid, protein or polysaccharide covalently modified by the phenyl boric acid.

Preferably, the cellulose salt having a carboxyl group in step (1) is selected from one or more of sodium carboxymethyl cellulose, sodium carboxyethyl cellulose and sodium carboxymethyl hydroxypropyl cellulose.

Preferably, the compound having a flexible di-or polyol or cis-diol and an amino group in the step (1) is selected from glucosamine or dopamine.

Preferably, the condensing agent in step (1) is selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or N-hydroxysuccinimide.

Preferably, the aqueous medium in step (1) is acidic or neutral, preferably having a pH of 4 to 7.

Preferably, the aqueous medium in step (2) is neutral or alkaline, preferably having a pH of 7 to 10.

Preferably, the reaction temperature of the step (1) is 10-50 ℃, and the reaction time is 1-24 hours. The preferred reaction temperature is 10-30 ℃ and the reaction time is 5 hours.

Preferably, the cellulose with the flexible dihydric or polyhydric alcohol or the cis-diol in the step (2) has a mass percentage concentration of 5-20%, and the polyphenyl boronic acid compound has a mass percentage concentration of 1-20%.

Preferably, the reaction temperature in the step (2) is 10-50 ℃, the reaction time is 10min to 1 hour, the reaction condition is standing, and further preferably, the reaction temperature is 10-30 ℃, and the reaction time is 10 min.

The polyphenylboronic acid compound in step (2) of the present invention is prepared by coupling a compound having two or more functional groups with phenylboronic acid or a phenylboronic acid derivative having amino groups or carboxyl groups, wherein the compound having two or more functional groups is one or more of polyethylene glycol, cyclodextrin, polyamino acid, protein or polysaccharide having carboxyl groups, amino groups, hydroxyl groups, halogens, alkynyl groups and azide, and preferably one or more of diaminopolyethylene glycol, dihydroxypolyethylene glycol, α -, β -, gamma-cyclodextrin, polyamino acid, protein and polysaccharide.

The invention also aims to provide the cellulose hydrogel containing the phenylboronic acid, which is prepared by adopting the method.

The cellulose hydrogel containing phenylboronic acid can be used for preparing tissue engineering materials.

The invention has the advantages that:

1) according to the preparation method, the phenylboronic acid group and the flexible diol or cis-diol group react to generate the cyclic borate, the reaction can be carried out in an aqueous medium and is quick and reversible, and any catalyst and auxiliary agent are not needed, so that the cellulose-based three-dimensional hydrogel can be quickly prepared by using the reaction in a simple mixing manner. 2) The reaction between the phenylboronic acid group and the diol group has the characteristic of quick reversibility, and the generated borate belongs to a typical dynamic covalent bond, so that the hydrogel prepared by utilizing the crosslinking reaction has a self-repairing function. 3) The hydrogel prepared by the method also has a large number of phenylboronic acid groups on the skeleton, and the phenylboronic acid groups can react with sialic acid residues on the surface of cells to generate boric acid esters, so that the cell adhesion of the hydrogel material can be remarkably improved. 4) The materials used by the hydrogel have good biocompatibility and have great application prospect in the field of tissue engineering.

Drawings

FIG. 1 shows NMR spectra of glucosamine-covalently modified cellulose.

FIG. 2 is the NMR spectrum of covalently modified poly (glutamic acid) with phenylboronic acid.

FIG. 3 is a scanning electron micrograph and a real photograph of the cellulose hydrogel prepared in example 10.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The present invention is further illustrated by the following examples, wherein carboxymethylcellulose and glucosamine or dopamine are coupled to prepare cellulose with cis-diol, and diaminopolyethylene glycol modified with phenylboronic acid, polyglutamic acid, and polylysine are used as polyphenylboronic acid cross-linking agents, but it should be noted that the scope of the present invention is not limited by these examples.

EXAMPLE 1 preparation of glucosamine modified cellulose

2g of carboxymethyl cellulose is dissolved in 200mL of deionized water, 10.8g (10mol) of EDC & HCl, 6.4g (10mol) of NHS and 6.04g (10mol) of glucosamine are added, the mixture is stirred at room temperature for 8 hours, the reaction mixture is dialyzed in a dialysis bag with the molecular weight cutoff of 14000Da for 2 days, and the product is obtained after freeze drying, and the nuclear magnetic resonance hydrogen spectrum is shown in figure 1. Glucosamine was shown to be covalently linked to cellulose with up to 30% substitution.

Example 2 preparation of dopamine-modified cellulose

2g of carboxymethyl cellulose is dissolved in 200mL of deionized water, 10.8g (10mol) of EDC & HCl, 6.4g (10mol) of NHS and 1.53g (10mol) of dopamine are added, the mixture is stirred for 8 hours at room temperature under a nitrogen atmosphere, the reaction mixture is dialyzed for 2 days in a dialysis bag with the molecular weight cutoff of 14000Da, and the product is obtained after freeze drying.

EXAMPLE 3 preparation of Phenylboronic acid-modified polyethylene glycols

2g (2mmol) of diaminopolyethylene glycol and 0.5mL of triethylamine were dissolved in 10mL of DMF, and 1.53g of EDC. HCl (8mmol), 0.92g of NHS (0.021mol) and 1.1g (8mmol) of 3-aminophenylboronic acid were added to the above solution, followed by stirring at room temperature for 8 hours. Concentrating the reaction mixture, separating and purifying by column chromatography, and drying in vacuum to obtain the product.

EXAMPLE 4 preparation of Phenylboronic acid modified β -Cyclodextrin

2g of β -cyclodextrin and 1mL of triethylamine were dissolved in 15mL of DMF, 3.811g of DCC (0.019mol) and 3.15g (0.019mol) of 4-carboxyphenylboronic acid were added to the above solution, the mixture was stirred at room temperature for 8 hours, the reaction mixture was concentrated, precipitated and washed in acetone for 6 times, and dried in vacuo to give the product.

EXAMPLE 5 preparation of Phenylboronic acid-modified polyglutamic acid

2g of polyglutamic acid (molecular weight: 5000) was dissolved in 20mL of deionized water, and 4.01g of EDC. HCl (0.021mol), 2.4g of NHS (0.021mol) and 2.88g (0.021mol) of 3-aminobenzeneboronic acid were added to the above solution, and stirred at room temperature for 8 hours. The reaction mixture is concentrated, precipitated and washed in acetone for 6 times, and dried in vacuum to obtain the product, and the nuclear magnetic resonance hydrogen spectrum is shown in figure 2. It was shown that phenylboronic acid was covalently linked to polyglutamic acid with a degree of substitution up to 25%.

EXAMPLE 6 preparation of Phenylboronic acid-modified polylysine

2g of polylysine (molecular weight: 5000) was dissolved in 20mL of deionized water, and 4.01g of EDC. HCl (0.021mol), 2.4g of NHS (0.021mol) and 3.49g (0.021mol) of 4-carboxyphenylboronic acid were added to the solution, and stirred at room temperature for 8 hours. And concentrating the reaction mixture, precipitating and washing in acetone for 6 times, and drying in vacuum to obtain the product.

Example 7 preparation of Phenylboronic acid-modified Bovine Serum Albumin (BSA)

0.58g (0.003mol) of EDC. HCl, 0.35g (0.003mol) of NHS and 0.5g (0.003mol) of 4-carboxyphenylboronic acid were dissolved in 2mL of DMF and stirred at room temperature for 1 hour. 2g BSA was dissolved in 20mL deionized water and added to the above solution, and stirred at room temperature for 8 hours. The reaction mixture was concentrated and dialyzed for 5 days, and lyophilized to obtain the product.

EXAMPLE 8 preparation of Phenylboronic acid-modified Chitosan

0.58g (0.003mol) of EDC. HCl, 0.35g (0.003mol) of NHS and 0.5g (0.003mol) of 4-carboxyphenylboronic acid were dissolved in 2mL of DMF and stirred at room temperature for 1 hour. 1g of chitosan was dissolved in 20mL of deionized water, and added to the above solution, and stirred at room temperature for 8 hours. And concentrating the reaction mixture, precipitating and washing in acetone for 6 times, and drying in vacuum to obtain the product.

Example 9 preparation of self-healing cellulose hydrogel

240mg of glucosamine modified cellulose is dissolved in 2mL of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, then 400mg of phenylboronic acid modified polyethylene glycol is added into the solution, and the solution is ultrasonically dispersed uniformly and stands for 10 minutes to form hydrogel. The hydrogel had an average pore size of 40 μm, a porosity of about 82%, and a Young's modulus of 1.5 kPa.

EXAMPLE 10 preparation of self-healing cellulose hydrogels

240mg of glucosamine modified cellulose is dissolved in 2mL of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, and then 120mg of phenylboronic acid modified polyglutamic acid is added into the solution, uniformly dispersed by ultrasonic waves and stood for 10 minutes to form hydrogel. The hydrogel had an average pore size of 25 μm, a porosity of about 80%, and a Young's modulus of 2.0 kPa. FIG. 3 shows scanning electron micrographs and physical photographs of the cellulose hydrogel. The scanning electron micrograph shows that the hydrogel has uniform pore diameter, and the average pore size is about 25 mu m.

EXAMPLE 11 preparation of self-healing cellulose hydrogels

240mg of glucosamine modified cellulose is dissolved in 2mL of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, and then 120mg of phenylboronic acid modified polylysine is added into the solution, is uniformly dispersed by ultrasonic waves and is stood for 10 minutes to form hydrogel. The hydrogel had an average pore size of 20 μm, a porosity of about 78%, and a Young's modulus of 2.8 kPa.

EXAMPLE 12 preparation of self-healing cellulose hydrogels

240mg of dopamine modified cellulose is dissolved in 2mL of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, then 150mg of phenylboronic acid modified β -cyclodextrin is added into the solution, the solution is uniformly dispersed by ultrasonic waves and stands for 10 minutes to form hydrogel, the average pore size of the hydrogel is 25 mu m, the porosity is about 78%, and the Young modulus is 2.3 kPa.

Example 13 preparation of self-healing cellulose hydrogel

240mg of dopamine-modified cellulose is dissolved in 2ml of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, and then 120mg of phenylboronic acid-modified BSA is added into the solution, uniformly dispersed by ultrasonic waves and stood for 10 minutes to form hydrogel. The hydrogel had an average pore size of 20 μm, a porosity of about 76%, and a Young's modulus of 2.5 kPa.

EXAMPLE 14 preparation of self-healing cellulose hydrogels

240mg of dopamine modified cellulose is dissolved in 2ml of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, then 120mg of phenylboronic acid modified chitosan is added into the solution, and the solution is ultrasonically dispersed uniformly and stands for 10 minutes to form hydrogel. The hydrogel had an average pore size of 20 μm, a porosity of about 78%, and a Young's modulus of 2.3 kPa.

Example 15 preparation of self-healing cellulose hydrogel

240mg of dopamine modified cellulose is dissolved in 2ml of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, then 120mg of phenylboronic acid modified polylysine is added into the solution, and the solution is ultrasonically dispersed uniformly and stands for 10 minutes to form hydrogel. The hydrogel had an average pore size of 16 μm, a porosity of about 76%, and a Young's modulus of 3.2 kPa.

EXAMPLE 16 preparation of covalently Cross-linked cellulose hydrogel

240mg of dopamine-modified cellulose is dissolved in 2mL of PBS buffer solution (0.01mol/L) with the pH value of 7.4 to prepare a solution with the mass concentration of 12%, and then 120mg of phenylboronic acid-modified polylysine, 77mg of EDC & HCL (0.4mmol) and 46mg of NHS (0.4mmol) are added into the solution, uniformly dispersed by ultrasonic waves and shaken for 2 hours to form the covalent cross-linked hydrogel. 3mL of deionized water was added, shaken and poured out, and washed three times to remove unreacted EDC & HCl and NHS, to obtain a pure hydrogel. The hydrogel has an average pore size of 15 μm, a porosity of about 75% and a Young's modulus of 3.8 MPa.

Claims

1. A preparation method of a cellulose hydrogel containing phenylboronic acid is characterized by comprising the following steps:

(1) the cellulose with the flexible dihydric or polyhydric alcohol or the cis-diol is prepared by the covalent coupling of a compound with the flexible dihydric or polyhydric alcohol or the cis-diol and an amino group and a cellulose salt with a carboxyl group in an aqueous medium through an amidation reaction in the presence of a condensing agent,

the compound with the flexible dihydric or polyhydric alcohol and the amino has the following structural general formula:

a represents a saturated or unsaturated ring, and two hydroxyl groups are in ortho-position or meta-position cis-substitution;

2. The preparation method according to claim 1, wherein the cellulose salt having a carboxyl group in the step (1) is one or more selected from the group consisting of sodium carboxymethyl cellulose, sodium carboxyethyl cellulose and sodium carboxymethylhydroxypropyl cellulose.

3. The method according to claim 1, wherein the compound having a flexible di-or polyol or cis-diol and an amino group in step (1) is selected from glucosamine and dopamine.

4. The method according to claim 1, wherein the condensing agent in the step (1) is selected from 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or N-hydroxysuccinimide.

5. The method according to claim 1, wherein the aqueous medium in the step (1) is acidic or neutral, and the aqueous medium in the step (2) is neutral or basic.

6. The method according to claim 1, wherein the step (1) is carried out in the presence of a catalyst

A represents a saturated five-membered ring, a saturated six-membered ring or an unsaturated cyclic group.

7. The method according to claim 6, wherein the step (1) is carried out in the presence of a catalyst

A represents cyclopentene, cyclohexene, a benzene ring, naphthalene, anthracene or quinoline.

8. The process according to claim 1, wherein the reaction temperature in the step (1) is 10 to 50 ℃ and the reaction time is 1 to 24 hours, the reaction temperature in the step (2) is 10 to 50 ℃ and the reaction time is 10min to 1 hour, and the reaction conditions are static.

9. The method according to claim 1, wherein the cellulose having a flexible diol or polyol or a cis-diol in the step (2) has a concentration of 5 to 20% by mass, and the polyphenylboronic acid compound has a concentration of 1 to 20% by mass.

10. The method according to claim 1, wherein the polyphenylboronic acid compound of step (2) is obtained by coupling a compound having two or more functional groups, which is polyethylene glycol, cyclodextrin, polyamino acid, protein or polysaccharide having one or more of carboxyl, amino, hydroxyl, halogen, alkynyl and azide, with phenylboronic acid or a phenylboronic acid derivative having an amino group or a carboxyl group.

11. The method according to claim 10, wherein the compound having two or more functional groups in step (2) is one or more selected from the group consisting of diaminopolyethylene glycol, dihydroxypolyethylene glycol, α -, β -, γ -cyclodextrin, polyamino acid, protein, and polysaccharide.

12. A phenylboronic acid-containing cellulose hydrogel prepared by the method of any one of claims 1 to 11.

13. Use of the phenylboronic acid-containing cellulose hydrogel of claim 12 in the preparation of a tissue engineering material.