WO2019192628A2

WO2019192628A2 - Thiolated chitosan derivative, chitosan hydrogel, and preparation methods therefor and applications thereof

Info

Publication number: WO2019192628A2
Application number: PCT/CN2019/089538
Authority: WO
Inventors: 戴建武; 陈艳艳; 黄雷; 储筠
Original assignee: 中国科学院苏州纳米技术与纳米仿生研究所
Priority date: 2018-04-03
Filing date: 2019-05-31
Publication date: 2019-10-10
Also published as: WO2019192628A3

Abstract

The present disclosure relates to a thiolated chitosan derivative, a chitosan hydrogel, a double crosslinked chitosan hydrogel, and preparation methods therefor and applications thereof. Alpha-beta unsaturated structures are respectively grafted onto chitosan molecule chains, thiol groups are grafted onto the chitosan molecule chains, and a chitosan hydrogel is obtained by means of a Michael addition reaction. A hydrogel obtained by reacting an alpha-beta unsaturated acylated chitosan with a thiolated chitosan is soaked in an ethanol solution to obtain a double crosslinked chitosan hydrogel. Chitosan is used as a raw material, and a sulfo compound having both sulfo groups and carboxyl groups is used as a modifying agent. Under the effect of a carboxyl group activating agent, and with the strong electron withdrawing effect of the sulfo groups, the sulfo groups are successfully introduced to the amino groups and the primary hydroxyl groups of the chitosan, and the sulfo groups are then converted into thiol groups via reduction to obtain the thiolated chitosan derivative of the present disclosure. The hydrogel of the present disclosure has a rapid curing speed, high mechanical strength, good biocompatibility, good stability in culture media, and a wide application range.

Description

Chitosan thiolated derivative, chitosan hydrogel, preparation method and application thereof

Cross-reference to related applications

This application claims the Chinese patent application entitled "Chitosan Hydrogel and Its Preparation Method and Application" submitted to the Chinese Patent Office on April 3, 2018, and submitted to China Patent on April 3, 2018. The application number of the bureau is 2018102910635, the Chinese patent application entitled "Chitosan thiolated derivative and its preparation method and application", and the application number of the Chinese Patent Office submitted on May 23, 2019 is 201910436288X, the name is " The priority of each of the entire contents of each of the entire contents of each of the entire contents of each of the entire contents of

Technical field

The present disclosure relates to the field of biomaterial technology, in particular, to chitosan thiolated derivatives, chitosan hydrogels, double crosslinked chitosan hydrogels, and preparation methods and applications thereof.

Background technique

3D printing, also known as additive manufacturing, has revolutionized many areas, such as engineering, manufacturing, education, and medicine. 3D bioprinting makes it possible to assemble biocompatible materials, cells and auxiliary components into human organs or tissues with three-dimensional functional activity. Compared to non-biological printing, 3D bioprinting is more complex, involving biocompatible material selection, cell type, growth, differentiation considerations, and tissue construction. The choice of materials is critical.

Hydrogel is a kind of three-dimensional network polymer with hydrophilic groups that can absorb a large amount of water and is insoluble in water. Because of its similar composition and structure to extracellular matrix, it is suitable for adhesion and growth of various cells. , proliferation and differentiation, become an important material for 3D bioprinting to construct human tissues and organs. At present, most 3D bio-printing hydrogels have problems such as slow curing speed, small mechanical strength and toughness of the colloid, or uncontrollable, which greatly reduces the printability and application range.

Chitosan is the only basic polysaccharide known in nature. It has good biocompatibility, biodegradability and non-cytotoxicity and is widely used in tissue engineering and regenerative medicine. The chitosan molecular chain is rich in amino groups and has good chemical activity.

Chitosan molecules play an important role in the body due to their unique molecular structure and physicochemical properties, and have been widely used in clinical practice. However, the existing chitosan molecules cannot be chemically reacted with other polymer derivatives containing maleimide, vinyl sulfone, α-β unsaturated aldehyde, ketone, acid, ester, etc. to prepare fast curing. Hydrogels.

The polymer hydrogel material is a low cross-linking material capable of quickly absorbing and retaining moisture without being soluble in water. It has polymer electrolyte properties and a three-dimensional network structure, and is a water-absorbing, water-retaining, and slow-absorbing material. A functional polymer material that functions in one.

Chitosan is a kind of natural bio-polysaccharide material widely used in the deacetylation of chitin. It has non-toxic, biocompatible, biodegradable, mucoadhesive and antibacterial properties. Characteristics, ideal for the preparation of hydrogels.

How to effectively crosslink the chitosan molecules to obtain the corresponding chitosan hydrogel materials is the hotspot of current research, and many researchers have made some explorations in the same or related fields. For example, in the prior art, a method of preparing a similar chitin hydrogel by using a combination of physical crosslinking and chemical crosslinking is disclosed, in which first, epichlorohydrin is used as a crosslinking agent. Chemical cross-linking is carried out, and then the product is placed in ethanol for cross-linking in the second step to obtain a high strength and strength-adjustable chitin hydrogel. Although the preparation method adopted in the prior art can achieve cross-linking between chitin molecules, the cross-linking agent epichlorohydrin used in the method is highly toxic, and the residual cross-linking agent is embedded after the cross-linking reaction occurs. It is difficult to remove in the colloid. At the same time, the chemical cross-linking reaction time in the first step of the method is long, and rapid prototyping cannot be achieved. Moreover, the maximum compressive modulus of the colloid prepared by the method is only as high as 260 KPa, and the maximum breaking strength is 3.98 MPa, which is difficult to meet the performance requirements as a high-strength hydrogel application.

Summary of the invention

The purpose of the present disclosure includes, for example, providing a method for preparing a chitosan hydrogel, which has a simple preparation process and can be effectively prepared to obtain a fast curing speed, good biocompatibility, adjustable mechanical strength, and stability in a medium. Good and chitosan hydrogel with adjustable biodegradability.

The purpose of the present disclosure includes, for example, providing a chitosan hydrogel having a fast curing speed, good biocompatibility, adjustable mechanical strength, good stability in a medium, an adjustable biodegradation rate, and a wide application range.

Objects of the present disclosure include, for example, the use of the above chitosan hydrogels for making biomedical materials or tissue engineering materials or 3D bioprinting materials.

The purpose of the present disclosure includes, for example, providing a method for preparing a double crosslinked chitosan hydrogel. In the present disclosure, by using chitosan with different functional groups to react and crosslink, not only the reaction crosslinking speed is fast, but also The use of chemical crosslinkers is avoided, and the resulting double crosslinked chitosan has good mechanical properties.

Objects of the present disclosure include, for example, providing a double crosslinked chitosan hydrogel obtained by the process of the present disclosure.

Objects of the present disclosure include, for example, the use of a double crosslinked chitosan of the present disclosure.

The object of the present disclosure includes, for example, providing a chitosan thiolated derivative obtained by modifying an amino group and a primary hydroxyl group in a chitosan molecule to be introduced into a thiol group, the chitosan thiolated derivative having a good The nucleophilic performance, antioxidant performance, and further derivatization by nucleophilic reaction, cross-linking reaction, etc., have a wide range of applications.

The object of the present disclosure includes, for example, providing a preparation method of the above chitosan thiolated derivative, which has a rational route design, a simple preparation method, low demand for equipment, and can rapidly and efficiently obtain a chitosan thiolated derivative.

Objects of the present disclosure include, for example, the use of the above chitosan thiolated derivatives in the preparation of hydrogels, such that the prepared hydrogels have properties of rapid cure, good biocompatibility, and adjustable mechanical strength.

The present disclosure provides a method for preparing a chitosan hydrogel, which comprises: Michael addition reaction of α-β unsaturated acylated chitosan with thiolated chitosan; α-β unsaturated acylation shell polymerization The formula of sugar is:

The general formula of thiolated chitosan is:

Wherein R ¹ is a residue portion of the chitosan polymer to remove an amino group; R ² is a hydrogen atom, an alkyl group or an alkylene group; and R ³ is a carbonyl group, a carboxyl group, an ester group, an amide group, an alkyl group or a substituted alkyl group; ⁴ is an alkylene group or a substituted alkylene group.

A chitosan hydrogel prepared by the method for preparing the above chitosan hydrogel, which has the formula:

The above chitosan hydrogel is used in the manufacture of biomedical materials or tissue engineering materials or 3D bioprinting materials.

The present disclosure also provides a method for preparing a high-toughness, high-strength and rapidly formable double-crosslinked chitosan hydrogel, the preparation method comprising: α-β-unsaturated acylated chitosan and thiolated shell The hydrogel obtained by the glycan reaction is immersed in an ethanol solution to obtain a double crosslinked chitosan hydrogel.

At the same time, the present disclosure also provides a double crosslinked chitosan hydrogel obtained by the process of the present disclosure.

The present disclosure also provides for the use of the present disclosed dual crosslinked chitosan hydrogel in a biomaterial; and/or a biomaterial comprising the double crosslinked chitosan hydrogel of the present disclosure.

The present disclosure provides a chitosan thiolated derivative which is produced by reacting and reacting chitosan with a sulfonic acid group compound, and has the formula:

Wherein R is an alkylene group or a substituted alkylene group.

A preparation method of the above chitosan thiolated derivative, wherein chitosan reacts with a sulfonic acid group compound in the presence of a carboxyl activator; and the sulfonic acid group is reduced to a mercapto group by a reducing agent; A compound having a carboxyl group and a sulfonic acid group.

The use of the above chitosan thiolated derivatives for the preparation of hydrogels.

The beneficial effects of the chitosan hydrogel of the embodiments of the present disclosure and the preparation method thereof include at least: by using a chitosan acylated with an acylating reagent and a thiolated chitosan as a raw material, the raw material can be realized by a Michael addition reaction. The rapid transition from liquid to solid greatly improves the printability of the material. By adjusting the concentration of the two and the graft ratio of the chitosan derivative, the elastic modulus of the chitosan hydrogel can be adjusted after curing. The range of applications of the material. The chitosan hydrogel prepared by the method has important applications in the fields of biomedicine and tissue engineering, and has the advantages of fast curing speed, good biocompatibility, adjustable mechanical strength, good stability in the medium, and adjustable biodegradation speed. , the scope of application is large.

In the present disclosure, chemical cross-linking is carried out by using chitosan reaction with different functional groups, and physical crosslinking is carried out by ethanol treatment, thereby obtaining a double containing both chemical cross-linking and physical cross-linking structure. The cross-linked chitosan hydrogel not only has a simple and rapid preparation method, but also has a fast preparation reaction speed, and does not need to use a chemical cross-linking agent with strong toxicity, and the preparation process is green, environmentally friendly and safe.

Meanwhile, the double crosslinked chitosan hydrogel obtained by the method of the present disclosure has a fast curing speed, high mechanical strength, good biocompatibility, good stability in a medium, and a large application range. Compared with the currently common ultraviolet-cured chitosan hydrogel, pH-responsive chitosan hydrogel, temperature-sensitive chitosan hydrogel, and ion-responsive chitosan hydrogel, the present invention double-crosslinked chitosan water The gel both increases the rate of cure and also improves the mechanical strength and elasticity of the chitosan hydrogel.

The beneficial effects of the chitosan thiolated derivative of the embodiment of the present disclosure and a preparation method thereof include at least: the preparation method comprises: using a compound having both a carboxyl group and a sulfonic acid group as a modifier, under the action of a carboxyl activator, in a shell The sulfonic acid group is successfully introduced into the amino group and the primary hydroxyl group of the glycan molecule, and the sulfonic acid group is reduced to a thiol group by the action of a reducing agent. The preparation method has reasonable route design, simple and feasible operation, low requirements on equipment, and high-yield chitosan thiolated derivatives. The chitosan thiolated derivative has good nucleophilic performance, antioxidant property and rich derivatization due to the action of the thiol side chain, and can be further derivatized by nucleophilic reaction, cross-linking reaction, etc., and the application range is very widely.

The beneficial effects of the chitosan thiolated derivatives of the embodiments of the present disclosure on the preparation of hydrogels include at least: the modified chitosan thiolated derivatives formed under alkaline conditions can be produced by leaving the protons Sulfur anion can be chemically reacted with other polymer derivatives containing a structure such as maleimide, vinyl sulfone, α-β unsaturated aldehyde, ketone, acid, ester, etc. to prepare a hydrogel and improve the hydrogel The rate of cure also improves the mechanical strength and elasticity of the hydrogel.

DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings to be used in the embodiments will be briefly described below. It should be understood that the following drawings show only certain embodiments of the present disclosure, and therefore It should be seen as a limitation on the scope, and those skilled in the art can obtain other related drawings according to these drawings without any creative work.

1 is a comparative analysis of Fourier transform infrared spectroscopy (FTIR) of chitosan, thiolated chitosan and α-β unsaturated acylated chitosan in Example 1 of the present disclosure;

2 is a scanning electron microscope (SEM) surface microstructure diagram of the chitosan hydrogel in Example 1 of the present disclosure;

3 is a surface aperture scanning electron microscope (SEM) image of a chitosan hydrogel in Example 1 of the present disclosure;

4 is a graph showing the mechanical test of the chitosan hydrogel in Example 1 of the present disclosure.

5 is a reaction formula of preparation of double crosslinked chitosan in Example 8 of the present disclosure;

6 is a reaction flow chart of preparation of double crosslinked chitosan in Example 8 of the present disclosure;

Figure 7 is a graph showing the mechanical properties of different hydrogel materials obtained according to the method of Example 8;

Figure 8 is a graph showing the mechanical properties of different hydrogel materials obtained according to the method of Example 8;

9 is a scanning electron micrograph of the double crosslinked chitosan biofiber in Experimental Example 3.

10 is a comparative analysis of chitosan and chitosan thiol derivative Fourier transform infrared spectroscopy (FTIR) in Example 15 of the present disclosure, wherein CS represents chitosan, and TCS represents chitosan thiolated derivative;

Figure 11 is a scanning electron microscope (SEM) surface microstructure diagram of the cured hydrogel in Example 19 of the present disclosure;

12 is a surface area scanning electron microscope (SEM) surface microstructure diagram of a cured hydrogel in Example 19 of the present disclosure;

Figure 13 is a graph showing the mechanical test of the cured hydrogel in Example 19 of the present disclosure.

detailed description

In order to make the objects, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.

The chitosan hydrogel of the examples of the present disclosure and a preparation method thereof will be specifically described below.

The present disclosure also provides a method for preparing a chitosan hydrogel, which comprises: Michael addition reaction of α-β unsaturated acylated chitosan with thiolated chitosan; α-β unsaturated acylate shell The general formula of glycans is:

The general formula of thiolated chitosan is:

Chitosan (CS), also known as chitosan, is a product of the deacetylation of chitin and is a widely used natural polysaccharide. Chitosan is the only basic polysaccharide in nature. It has a pair of unshared electron pairs on the free amino nitrogen atom in the molecular chain, which can bind a hydrogen proton, so that chitosan becomes a positively charged polyelectrolyte. Chitosan has good biocompatibility, biodegradability, bactericidal properties, etc. It is a safe natural high molecular polymer. The free amino group on the chitosan molecule has high chemical activity and is easily modified by a functional group having higher activity such as a carboxyl group. The chitosan can be modified by an acylating reagent to obtain a water-soluble chitosan, and at the same time in chitosan. The α-β unsaturated carbonyl structure is introduced into the molecular chain, and the addition reaction can be carried out by the attack of the nucleophilic reagent, and the crosslinking of the polymer is successfully achieved to achieve the effect of rapid curing.

In one or more embodiments, the alpha-beta unsaturated acylated chitosan (MCS) has the general formula:

R ³ is a carbonyl group, a carboxyl group, an ester group, an amide group, an alkyl group or a substituted alkyl group. Wherein the substituted alkyl group may be an alkyl group in which at least one hydrogen atom is substituted with at least one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group. That is, one hydrogen atom in the alkyl group may be substituted by one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group; or two hydrogen atoms in the alkyl group may be Substituting two groups of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group; or an alkyl group having two or more hydrogen atoms substituted by an alkyl group, a carboxyl group, an amino group, or an alkoxy group. Substituted with two or more groups in the group, an aryl group, an ester group, and a halogenated alkyl group; or a plurality of hydrogen atoms in the alkyl group may be an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group. A plurality of the same group in the group are substituted or substituted by a combination of a plurality of different groups.

The chitosan molecular chain has a free amino group, and the thiol-containing compound is used to modify the amino group, and the chitosan can be thiolated. The modified chitosan has the chemical properties of the sulfhydryl group, and the ruthenium matrix is removed under alkaline conditions. Sulfur anion can be produced, and chitosan acylated with an acylating reagent can be attacked as a nucleophilic reagent, and chitosan and MCS are rapidly grafted together in the form of a CS bond to form a network complex chitosan hydrogel.

In one or more embodiments, the thiolated chitosan has the formula:

Wherein R ⁴ is an alkylene group or a substituted alkylene group. The substituted alkylene group may be an alkylene group in which at least one hydrogen atom is substituted with at least one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group. That is, one of the alkylene groups may have one hydrogen atom substituted by one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group; or two hydrogen atoms in the alkylene group may be used. The atom is substituted by two groups of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group; or an alkylene group may have two or more hydrogen atoms selected from an alkyl group, a carboxyl group, or an amino group. Substituting two or more groups of an alkoxy group, an aryl group, an ester group, and a halogenated alkyl group; or a plurality of hydrogen atoms in the alkylene group may be an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group or an ester group. A plurality of the same group in the group and the haloalkyl group are substituted or substituted by a combination of a plurality of different groups.

Among them, R ⁴ may have 1 to 20 carbon atoms. That is, R ⁴ may be an alkylene group or a substitution of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20. Alkylene.

Since the free amino group is present in the molecular chain, the chitosan molecular chain can be modified and modified by reacting with the carboxyl group. The mercapto compound which reacts with chitosan is a compound having both a carboxyl group and a mercapto group; the above mercapto compound can be directly produced by a hydrolysis reaction, an aminolysis reaction, or the like, or can be obtained by reduction of a disulfide-containing compound. For example, the compound having both a carboxyl group and a thiol group may be: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioglycolic acid, 2-mercapto-3-pyridinecarboxylic acid, and the like.

Some embodiments of the present disclosure also provide a method for preparing a chitosan hydrogel comprising: performing a Michael addition reaction of an α-β unsaturated acylated chitosan solution with a thiolated chitosan solution.

In one or more embodiments, the α-β unsaturated acylated chitosan solution is prepared by dissolving α-β unsaturated acylated chitosan in an acid solution at a concentration of 10 to 100 mg/ml. a β-unsaturated acylated chitosan acid solution; the thiolated chitosan solution is a 10-100 mg/ml thiolated chitosan acid solution prepared by dissolving thiolated chitosan in an acid solution. By the above concentrations and the arrangement of the solvent thereof, the α-β unsaturated acylated chitosan and the thiolated chitosan can be well dissolved, and the Michael addition reaction between the two can be more favored.

In one or more embodiments, the preparation of the chitosan hydrogel is carried out by the following steps:

(1) Preparation of α-β unsaturated acylated chitosan (MCS).

Taking an α-β unsaturated acid as an acylating reagent as an example, the chemical reaction formula of α-β unsaturated acylated chitosan is:

In one or more embodiments, the chitosan is dissolved in an acid solution, and the acylating agent is dissolved in a polar solvent, and the molar ratio of the free amino group of the chitosan chain to the acylating agent is preferably 1: 1 to 3, the dissolved acylating reagent is slowly added to the chitosan acid solution on a magnetic stirrer, and thoroughly mixed at room temperature, and reacted at 10 to 90 ° C, preferably 40 to 90 ° C, and the reaction time is 2 to ～ 10h. After the completion of the reaction, the cells were dialyzed for 2 to 4 days, and lyophilized for 2 to 4 days to obtain a modified chitosan product.

In one or more embodiments, the reaction temperature of the chitosan-containing acid solution and the acylating agent-containing solution is 10 to 90 ° C, preferably 40 to 90 ° C, and the reaction time is 2 ~10h.

In one or more embodiments, the acid solution in which the chitosan is dissolved may be an organic acid solution, preferably an acetic acid solution, and more preferably, the acetic acid solution has a mass fraction of 0.01 to 30%. The polar solvent for dissolving the acylating agent includes acetone, methyl ethyl ketone, water, DMSO, DMF, etc., preferably acetone, and the amount of acetone is such that the acylating agent is completely dissolved. By setting the molar ratio of the free amino group on the chitosan chain to the carboxyl group of the acylating reagent to 1:1 to 3, the acid anhydride of the acylating reagent can react better with the free amino group on the chitosan chain, thereby The sugar is modified to give an α-β unsaturated acylated chitosan. At the same time, the dissolved acylating agent is stirred on a magnetic stirrer and slowly added to the chitosan acid solution, so that the two can be more fully contacted, thereby achieving a better reaction effect. After the reaction is completed, the reaction solution is subjected to dialysis and freeze-drying operation, and the small molecular impurities remaining in the α-β unsaturated acylated chitosan can be better removed to obtain a higher purity α-β unsaturated acylation. Chitosan, in turn, facilitates the subsequent Michael addition reaction to proceed better.

(2) Preparation of thiolated chitosan (CS-SH).

Thiolated chitosan (CS-SH) is produced by the following reaction formula:

In one or more embodiments, the thiolated chitosan (CS-SH) is prepared by reacting a solution containing chitosan with a solution containing a mercapto compound under the action of a carboxyl activator.

In one or more embodiments, when a thiol compound containing a carboxyl group is used as a raw material, thiolated chitosan (CS-SH) can be produced by the following method:

a. Preparation of a solution containing chitosan: The chitosan is dissolved in an acid solution having a mass fraction of 0.01 to 30%.

b. Preparation of a solution containing a mercapto compound: The mercapto compound is dissolved in a double distilled water or an alkali solution or an acid solution depending on its solubility.

c. The carboxyl activator is added to the above-mentioned solution containing a mercapto compound, and after mixing uniformly, the pH is adjusted to 4.5 to 6.5, and mixing and stirring are continued.

d. The activated solution containing a mercapto compound is mixed with a solution containing chitosan, and the molar ratio of the mercapto compound to the amino group on the chitosan is 1 to 10:1, and the mixture is sufficiently stirred, preferably transferred to a round bottom flask. , placed at a temperature of 50 ~ 60 ° C or less for constant temperature reaction.

e. The reaction solution obtained after the reaction is dialyzed for 2 to 5 days, and the obtained dialysis product is freeze-dried for 2 to 5 days to obtain a thiolated chitosan. The residual small molecule impurities can be removed by means of dialysis, and the purity of the obtained chitosan thiolated derivative is improved.

It should be noted that in the above process, steps b and c may also be performed first, and then step a is performed, which does not affect the formation of the final product.

In one or more embodiments, the acid solution during the preparation of the thiolated chitosan (CS-SH) can be an organic acid solution, preferably an acetic acid solution. That is, chitosan is preferably dissolved in an acetic acid solution having a mass fraction of 0.01 to 30%.

In one or more embodiments, the alkali solution may be a strong alkali solution, such as a sodium hydroxide solution, a potassium hydroxide solution, a calcium hydroxide solution, or the like, or may be a weak alkaline solution such as an aqueous ammonia solution or a sodium carbonate solution. A sodium hydrogen carbonate solution or the like is preferably a sodium hydroxide solution. The double distilled water can make the dissolved solution have less impurity content, higher purity, and the better the subsequent reaction effect. The acid solution in which the mercapto compound is dissolved may be an organic acid solution, preferably an acetic acid solution.

In one or more embodiments, the carboxyl activator comprises EDC and NHS, EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and EDC is soluble in water. A carbodiimide, used as an activating reagent for carboxyl groups in amide synthesis, also used to activate phosphate groups, cross-linking of proteins with nucleic acids, and preparation of immunoconjugates, often with N-hydroxysuccinimide (NHS) or N-hydroxy sulfosuccinimide is used in combination to increase the coupling efficiency. NHS, N-hydroxysuccinimide, activates the carboxyl group to facilitate the formation of an amide bond. For the synthesis of amino acid protective agents, semi-synthetic kanamycin and pharmaceutical intermediates. The carboxyl activator in the examples of the present disclosure can be prepared according to the ratio of the mass ratio of EDC to NHS of 1 to 10:1, which is advantageous for achieving better coupling efficiency and carboxy activation of the mercapto compound. better result.

In one or more embodiments, EDC and NHS can be added to the above-described thiol-containing compound solution under the action of a magnetic stirrer to achieve a better mixing effect.

In one or more embodiments, after mixing the carboxyl activating agent, the pH of the thiol compound solution to which the carboxyl activator is added is adjusted with a base or an acid solution to have a pH of 4.5 to 6.5. Preferably, the pH is adjusted with 1 M sodium hydroxide or 1 M hydrochloric acid solution. EDC and NHS can achieve the best activation effect on carboxyl groups at a pH of 4.5 to 6.5.

In one or more embodiments, the pH is adjusted and the time of mixing and stirring is 10 to 150 minutes, so that the carboxyl activator can sufficiently activate the carboxyl group of the mercapto compound to better modify the chitosan.

Among them, the reaction temperature is preferably 55 to 60 ° C, more preferably 55 ° C, and the reaction time may be 1 to 8 h, preferably 2 to 6 h, more preferably 4 to 5 h.

(3) A Michael addition reaction of the α-β unsaturated acylated chitosan solution with the thiolated chitosan solution.

The reaction formula of the Michael addition reaction is:

In one or more embodiments, the Michael addition reaction may be carried out by mixing an α-β unsaturated acylated chitosan solution with a thiolated chitosan solution and extruding into an alkali solution for reaction molding. The α-β unsaturated acylated chitosan solution and the thiolated chitosan solution are sufficiently mixed in a certain ratio, thereby further facilitating the progress of the reaction and adjusting the properties of the produced chitosan hydrogel.

In one or more embodiments, first, the α-β unsaturated acylated chitosan obtained in the step (1) is dissolved in an acid solution having a mass fraction of 0.1 to 30% to prepare a concentration of 10 to 50 mg/ A solution of ml; wherein the acid solution may be an organic acid solution, preferably an acetic acid solution.

Next, the thiolated chitosan obtained in the step (2) is dissolved in an acid solution to prepare a solution of 10 to 50 mg/ml, and the thiolated chitosan in the solution is treated with a reducing agent.

The reducing agent is metal Zn, dithiothreitol or hydroquinone. The time for carrying out the reduction treatment is preferably 5 to 50 minutes.

Then, the acylation reagent acylated chitosan solution and the thiolated chitosan solution are thoroughly mixed and extruded into an alkali solution for reaction molding to realize preparation of a novel 3D printed chitosan hydrogel.

The alkali solution may be a strong alkali solution, such as a sodium hydroxide solution, a potassium hydroxide solution, a calcium hydroxide solution, or the like, or may be a weak alkaline solution such as an aqueous ammonia solution, a sodium carbonate solution, a sodium hydrogencarbonate solution, or the like, preferably hydrogen. Sodium oxide solution.

It is worth noting that chitosan itself precipitates insoluble matter in an alkaline solution, but the precipitation of chitosan is an insoluble matter that is precipitated due to the change of the charge distribution of the alkali solution, and after neutralizing the alkali with acid Insolubles will redissolve. In contrast, the chitosan hydrogel prepared by the examples of the present disclosure is chemically crosslinked, has stable properties, and does not dissolve in a strong acid. This indirectly illustrates that the chitosan hydrogel provided by the embodiments of the present disclosure successfully undergoes an addition reaction during the formation process, rather than a simple physical change.

In the above preparation process, the α-β unsaturated acylation structure is grafted on the chitosan molecular chain, and the graft structure of the two biomaterials is modified by the grafting reaction of the two biomaterials in the chitosan molecule, and the Michael addition reaction is utilized. The chemical crosslinking mechanism is used to achieve rapid curing of the chitosan hydrogel to achieve 3D printability of the material, and the chitosan water is adjusted by changing the concentration of the modified material and the graft ratio of the chitosan derivative. The mechanical strength and elastic modulus of the gel. The 3D bioprinting chitosan hydrogel prepared by the embodiment of the present disclosure has a fast curing speed, good biocompatibility, adjustable mechanical strength, good stability in a medium, adjustable biodegradation speed, and large application range. Compared with the current UV-cured chitosan hydrogel, PH-responsive chitosan hydrogel, temperature-sensitive chitosan hydrogel, ion-responsive chitosan hydrogel, etc., it not only improves the curing speed, but also improves it. The mechanical strength and elasticity of the chitosan hydrogel make the 3D printed chitosan hydrogel have good support and fidelity, preventing the colloid from collapsing and deforming in a short time. The present disclosure also provides a novel grafted product of a sulfhydryl group with an alpha-beta unsaturated structure, which product has important applications in the fields of biomedical and tissue engineering.

Some embodiments of the present disclosure also provide the use of the chitosan hydrogel described above in the fabrication of biomedical materials or tissue engineering materials or 3D bioprinting materials.

The double crosslinked chitosan hydrogel provided by the present disclosure is based on a preliminary chitosan hydrogel study (refer to CN201810291744.1), and further adopts an ethanol treatment method, and the obtained chemical cross-linking and The physically crosslinked microstructure of the double crosslinked chitosan hydrogel material is not only faster than the prior art double crosslinked chitin hydrogel, but also requires no toxicity. The cross-linking agent and the obtained hydrogel material are more excellent in mechanical properties.

An aspect of the present disclosure provides a method for preparing a double crosslinked chitosan hydrogel, and the preparation method provided by the present disclosure mainly comprises:

The hydrogel obtained by reacting α-β unsaturated acylated chitosan with thiolated chitosan is immersed in an ethanol solution to obtain a double crosslinked chitosan hydrogel.

In one or more embodiments, the hydrogel obtained by reacting the alpha-beta unsaturated acylated chitosan with thiolated chitosan is the chitosan hydrogel of the foregoing disclosure.

In the above preparation method, a double crosslinked chitosan hydrogel is prepared by a chemical crosslinking and a physical crosslinking method. Different from the preparation method using epichlorohydrin as a crosslinking agent used in the prior art, the first step crosslinking in the present disclosure is carried out by a thiol click addition method, and maleic anhydride and sulfhydryl groups are used before the addition reaction. The succinic acid is chemically modified by chitosan, and the modified chitosan is used as a cross-linking agent to form a cross-linking agent, and an addition reaction occurs under the catalyst to achieve chemical cross-linking. The thiol-based click addition reaction has high stereoselectivity and fast reaction speed, which can realize rapid prototyping and facilitate the preparation of various three-dimensional structures. The second step of cross-linking with ethanol treatment can improve the intermolecular hydrogen bond of the hydrogel molecular side chain. The force and the hydrophobic interaction of the molecular backbone, thereby greatly increasing the mechanical strength of the chitosan hydrogel, by changing the concentration of ethanol (can be used as a treatment reagent with a concentration of 0% < vol% ≤ 100%) and shell polymerization The grafting ratio of the sugar derivative can effectively adjust the mechanical strength of the colloid. Further, the maximum breaking strength of the mechanical strength of the chitosan hydrogel obtained by the method of the present disclosure is up to 10.8 MPa, and the maximum elastic modulus is up to 1.32 MPa.

In one or more embodiments, the time (t) of the soaking treatment of the chemically crosslinked hydrogel in the ethanol solution is 0<t≤48h (for example, but not limited to 14, 16, 20, 24 , 30, 32, 36 or 42h, etc.);

In one or more embodiments, the soaking time is 24 hours.

In one or more embodiments, the reaction of the alpha-beta unsaturated acylated chitosan with the thiolated chitosan comprises:

Under the condition of the solution, the α-β unsaturated acylated chitosan is mixed with the thiolated chitosan, and then reacted with an alkali solution to obtain a hydrogel;

Preferably, in this step, the α-β unsaturated acylated chitosan is dissolved in an acid solution (preferably an organic acid solution, more preferably an acetic acid solution, particularly 0.1-30% (m/m) acetic acid solution. )in;

Preferably, in this step, the thiolated chitosan is dissolved in an acid solution (preferably an organic acid solution, more preferably an acetic acid solution, particularly an acetic acid solution of 0.1-30% (m/m));

Preferably, in this step, the step of reducing the thiolated chitosan is further included;

Preferably, the reducing treatment comprises: adding a reducing agent to the thiolated chitosan solution for reduction treatment;

More preferably, the reducing agent comprises: zinc (metal zinc), dithiothreitol, and at least one of hydroquinone;

More preferably, the reduction treatment time is 5-50 min (for example, but not limited to 10, 15, 20, 25, 30, 35, 40, or 45 min, etc.);

Preferably, in this step, after the α-β unsaturated acylated chitosan is mixed with the thiolated chitosan, the obtained product is extruded into an alkali solution to form a chemically crosslinked hydrogel;

In one or more embodiments, the alkali solution used for molding may be: a strong alkaline solution such as sodium hydroxide, potassium hydroxide, or a calcium hydroxide solution; or: ammonia water, sodium carbonate, carbonic acid a weakly alkaline solution such as a sodium hydrogen solution;

In one or more embodiments, the alkaline solution is a sodium hydroxide solution (preferably at a concentration of 0.01-10 M, such as, but not limited to, 0.05, 0.1, 1, 3, 5, 7, or 9 M, etc.).

In one or more embodiments, the preparation method further includes:

After reducing the thiolated chitosan, it is reacted with α-β unsaturated acylated chitosan;

Preferably, the thiolated chitosan reduction comprises the step of treating the thiolated chitosan with a reducing agent;

More preferably, the reducing agent comprises at least one of zinc, dithiothreitol, and hydroquinone.

In one or more embodiments, the alpha-beta unsaturated acylated chitosan as a raw material for hydrogel preparation comprises a compound of formula (i):

Wherein, in the formula (i), R ₁ is a residue portion of the chitosan to remove an amino group;

R ₂ , R ₃ , and R ₄ are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

In one or more embodiments, R ₂ , R ₃ , and R ₄ are each independently hydrogen, and a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms (preferably having a carbon number of 1 to 12) Or an unsubstituted alkyl group, more preferably a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, such as, but not limited to, methyl, ethyl, propyl, isopropyl, butyl of a substituted or unsubstituted alkane a substituted or unsubstituted alkoxy group having 1 to 20 carbon atoms (preferably a substituted or unsubstituted alkoxy group having 1 to 12 carbon atoms), a butyl group, a pentyl group, an isopentyl group, a hexyl group and the like. The group is more preferably a substituted or unsubstituted alkoxy group having 1 to 6 carbon atoms, such as, but not limited to, a substituted or unsubstituted methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, and a butyl group. a oxy group, a monobutoxy group, a pentyloxy group, an isopentyloxy group, a hexyloxy group or the like), a substituted or unsubstituted aryl group having 5 to 20 carbon atoms (preferably a substitution of 5 to 12 carbon atoms or Non-substituted aryl, such as, but not limited to, substituted or unsubstituted phenyl, naphthyl, biphenyl, etc., and substituted or unsubstituted heteroaryl having 5 to 20 carbon atoms (preferably a carbon atom) Number 5-12 Generation or substituted heteroaryl, for example, but not limited to: substituted or unsubstituted pyrrole, indole, pyrazole, indazole, imidazole, phenylpropyl pyrazole, triazole, benzotriazole, etc.);

In one or more embodiments, when any R group of R ₂ , R ₃ , R ₄ is a substituted alkyl group, a substituted alkoxy group, a substituted aryl group or a substituted heteroaryl group, the substitution At least one hydrogen atom of the alkyl group, substituted alkoxy group, substituted aryl group or substituted heteroaryl group may be an alkyl group (preferably an alkyl group having 1 to 20 carbon atoms, more preferably 1 to 1 carbon atom). The alkyl group of 12 is further preferably an alkyl group having 1 to 6 carbon atoms, such as, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, isopentyl. , hexyl or the like), a carboxyl group, an amino group, an alkoxy group (preferably an alkoxy group having 1 to 20 carbon atoms, more preferably an alkoxy group having 1 to 12 carbon atoms, still more preferably 1 to 1 carbon atom) Alkoxy group of 6, for example, but not limited to: methoxy, ethoxy, propoxy, isopropoxy, butoxy, tertoxy, pentyloxy, isopentyloxy, hexyloxy And the like, an aryl group (preferably an aryl group having 5 to 20 carbon atoms, more preferably an aryl group having 5 to 12 carbon atoms, such as, but not limited to, a phenyl group, a naphthyl group, a biphenyl group, etc.), a heteroaryl group Base (preferably 5-2 carbon atoms) a heteroaryl group of 0, preferably a substituted or unsubstituted heteroaryl group having 5 to 12 carbon atoms, such as, but not limited to, substituted or unsubstituted pyrrole, indole, pyrazole, oxazole, imidazole, phenylpyrrolid Oxazole, triazole, benzotriazole, etc.), ester or halogen (fluorine, chlorine, bromine or iodine);

Wherein, when the number of substituents is greater than 1, different substituents may be optionally the same or different;

R ₅ is carbonyl, carboxy, ester, amide, substituted or unsubstituted alkyl (preferably C1-C12 substituted or unsubstituted alkyl, more preferably C1-C6 substituted or unsubstituted alkyl), substituted or unsubstituted Alkoxy (preferably a C1-C12 substituted or unsubstituted oxyalkyl group, more preferably a C1-C6 substituted or unsubstituted oxyalkyl group), a substituted or unsubstituted aryl group (preferably a C5-C20 substituted or unsubstituted) An aryl group, more preferably a C5-C12 substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group (preferably a C1-C12 substituted or unsubstituted heteroaryl group, more preferably a C1-C6 substituted or unsubstituted) Heteroaryl)

In one or more embodiments, when R ₅ is a carbonyl group, the carbonyl structure is:

Wherein R may be a substituted or unsubstituted alkyl group (preferably a C1-C12 substituted or unsubstituted alkyl group, more preferably a C1-C6 substituted or unsubstituted alkyl group), a substituted or unsubstituted alkoxy group (preferably C1- a C12 substituted or unsubstituted oxyalkyl group, more preferably a C1-C6 substituted or unsubstituted oxyalkyl group, a substituted or unsubstituted aryl group (preferably a C5-C20 substituted or unsubstituted aryl group, more preferably a C5-C12) a substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group (preferably a C1-C12 substituted or unsubstituted heteroaryl group, more preferably a C1-C6 substituted or unsubstituted heteroaryl group);

In one or more embodiments, when R ₅ is an ester group, the structure of the ester group is:

or

Wherein R' may be a substituted or unsubstituted alkyl group (preferably a C1-C12 substituted or unsubstituted alkyl group, more preferably a C1-C6 substituted or unsubstituted alkyl group), a substituted or unsubstituted alkoxy group (preferably C1) a -C12 substituted or unsubstituted oxyalkyl group, more preferably a C1-C6 substituted or unsubstituted oxyalkyl group), a substituted or unsubstituted aryl group (preferably a C5-C20 substituted or unsubstituted aryl group, more preferably C5-) a C12-substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group (preferably a C1-C12 substituted or unsubstituted heteroaryl group, more preferably a C1-C6 substituted or unsubstituted heteroaryl group);

In one or more embodiments, when R ₅ is an amide group, the structure of the amide group is:

Wherein R" and R"" are each independently hydrogen, substituted or unsubstituted alkyl (preferably C1-C12 substituted or unsubstituted alkyl, more preferably C1-C6 substituted or unsubstituted alkyl), substituted or not a substituted alkoxy group (preferably a C1-C12 substituted or unsubstituted oxyalkyl group, more preferably a C1-C6 substituted or unsubstituted oxyalkyl group), a substituted or unsubstituted aryl group (preferably a C5-C20 substituted or non-substituted) a substituted aryl group, more preferably a C5-C12 substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group (preferably a C1-C12 substituted or unsubstituted heteroaryl group, more preferably a C1-C6 substituted or non-substituted group) Substituted heteroaryl).

In one or more embodiments, the method for synthesizing the α-β unsaturated acylated chitosan comprises:

The chitosan is reacted with an acylating reagent to obtain an α-β unsaturated acylated chitosan;

Wherein the acylating agent comprises: at least one of an α-β unsaturated acid, an α-β unsaturated acid anhydride, an α-β unsaturated acid halide, and an α-β unsaturated ester;

Preferably, the chitosan has a molecular weight of 0.1 to 10 million, such as, but not limited to, 1, 5, 10, 50, 100, 300, 500, 700 or 9 million;

More preferably, the chitosan has a molecular weight of 50,000 and a degree of deacetylation of 80%. The structure can be referred to as follows:

Preferably, the α-β unsaturated acid comprises: β-methacrylic acid, and at least one of β-isopropylacrylic acid;

The α-β unsaturated acid anhydride includes: maleic anhydride;

The α-β unsaturated acid halide includes at least one of acryloyl chloride and methacryloyl chloride;

The α-β unsaturated ester includes at least one of methyl methacrylate and ethyl methacrylate;

Preferably, the synthesis method further comprises the step of purifying the obtained α-β unsaturated acylated chitosan;

More preferably, the purification comprises: dialysis, by using dialysis purification, the residual modifier (acylating agent) can also be removed, thereby avoiding the effect of the modifier on subsequent reactions.

Preferably, the synthesis method further comprises the step of purifying the obtained α-β unsaturated acylated chitosan.

To the chitosan dissolved in the acid solution, an acylating agent (which may be added as a solution) is added, and after stirring at room temperature, at 10-90 ° C (for example, but not limited to 20, 30, 40, 50, 60-70 or 80 ° C, etc., preferably 40-90 ° C) reaction 2-10h (such as, but not limited to 3, 4, 5, 6, 7, 8, or 9h, etc.);

Then, the product is subjected to dialysis (preferably dialysis 2-4d), dried (preferably by freeze-drying) to obtain α-β unsaturated acylated chitosan;

Preferably, the concentration of the chitosan solution is 10 to 100 mg/ml;

Preferably, the acid solution for dissolving chitosan comprises an organic acid solution, preferably an acetic acid solution (particularly a solution of 0.01-30% acetic acid);

Preferably, the solution for dissolving the acylating agent comprises at least one of a polar solvent such as acetone, methyl ethyl ketone, water, DMSO and DMF (particularly acetone);

Preferably, the molar ratio of chitosan to acylating agent is from 1:1 to 1:3.

In one or more embodiments, the thiolated chitosan as a raw material for the preparation of the hydrogel comprises a compound of the following formula (ii):

Wherein, in the formula (ii), R ₁ is a residue portion of the chitosan to remove an amino group;

R ₆ is a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, and a substituted or unsubstituted heteroarylene group;

Preferably, R ₆ is a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms (preferably a substituted or unsubstituted alkyl group having 1 to 12 carbon atoms, more preferably a substituent having 1 to 6 carbon atoms) Or an unsubstituted alkyl group, such as, but not limited to, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. of a substituted or unsubstituted alkene, carbon a substituted or unsubstituted alkoxy group having 1 to 20 atomic atoms (preferably a substituted or unsubstituted alkoxy group having 1 to 12 carbon atoms, more preferably a substituted or unsubstituted alkane having 1 to 6 carbon atoms) Oxyl group, for example, but not limited to, substituted or unsubstituted methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyl Oxyl or the like), a substituted or unsubstituted aryl group having 5 to 20 carbon atoms (preferably a substituted or unsubstituted aryl group having 5 to 12 carbon atoms, for example, but not limited to, a substituted or unsubstituted phenyl group , naphthyl, biphenyl, etc.), and a substituted or unsubstituted heteroaryl group having 5 to 20 carbon atoms (preferably a substituted or unsubstituted heteroaryl group having 5 to 12 carbon atoms), for example, but not limited to :take Or unsubstituted pyrrole, indole, pyrazole, indazole, imidazole, phenylpropyl pyrazole, triazole, benzotriazole, etc.);

Preferably, when R ₆ is a substituted alkyl, substituted alkoxy, substituted aryl or substituted heteroaryl, the substituted alkyl, substituted alkoxy, substituted aryl or substituted hetero At least one hydrogen atom in the aryl group may be an alkyl group (preferably an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 1 to 12 carbon atoms, still more preferably an alkyl group having 1 to 6 carbon atoms). For example, but not limited to: methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, isopentyl, hexyl, etc.), carboxyl, amino, alkoxy (preferably having a carbon number of The alkoxy group of 1-20 is more preferably an alkoxy group having 1 to 12 carbon atoms, more preferably an alkoxy group having 1 to 6 carbon atoms, such as, but not limited to, a methoxy group and an ethoxy group. Base, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy, etc.), aryl (preferably an aryl group having 5 to 20 carbon atoms, more Preferred is an aryl group having 5 to 12 carbon atoms, such as, but not limited to, a phenyl group, a naphthyl group, a biphenyl group, etc., a heteroaryl group (preferably a heteroaryl group having 5 to 20 carbon atoms, preferably a carbon atom) Number 5-12 substitution Non-substituted heteroaryl, such as, but not limited to, substituted or unsubstituted pyrrole, indole, pyrazole, oxazole, imidazole, phenylpropyrazole, triazole, benzotriazole, etc.), ester or halogen ( Replaced by fluorine, chlorine, bromine or iodine;

Wherein, when the number of the substituents is more than 1, the different substituents may be optionally the same or different.

In one or more embodiments, the method for synthesizing the thiolated chitosan comprises:

The chitosan is reacted with a thiolation reagent to obtain a thiolated chitosan;

Preferably, the thiolation reagent comprises: a compound having a thiol group and a carboxyl group;

More preferably, the thiolation reagent comprises at least one of: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioacetic acid, and 2-mercapto-3-pyridinecarboxylic acid;

Preferably, the synthetic method further comprises the step of purifying the obtained thiolated chitosan.

More preferably, the thiolation reagent comprises at least one of: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioglycolic acid, and 2-mercapto-3-pyridinecarboxylic acid;

More preferably, the chitosan has a molecular weight of 50,000 and a degree of deacetylation of 80%;

Preferably, the synthetic method further comprises the step of purifying the obtained thiolated chitosan;

More preferably, the purification comprises: dialysis, and similarly, the purification of the thiolated chitosan by dialysis can also avoid the residue of the modifier (thiolizing agent) in the product thiolated chitosan;

Preferably, the thiolated chitosan is reacted in the presence of a carboxyl activator;

More preferably, the carboxyl activator comprises: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHC (N-hydroxysuccinimide);

Preferably, the thiolation reagent comprises: a compound having both a carboxyl group and a thiol group;

More preferably, the thiolation reagent comprises at least one of: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioglycolic acid, and 2-mercapto-3-pyridinecarboxylic acid; and the like;

Preferably, the molar ratio of chitosan to thiolation reagent is from 1:1 to 10:1.

(a) dissolving the chitosan in an acid solution (preferably an organic acid solution, more preferably an acetic acid solution, particularly an acetic acid solution having a concentration of 0.01-30%);

Preferably, the concentration of the obtained chitosan solution is 10 to 100 mg/ml;

(b) dissolving the thiolation reagent (depending on solubility) in water (preferably double distilled water), an alkali solution (such as a strong alkaline solution such as sodium hydroxide, potassium hydroxide, or calcium hydroxide solution, or ammonia water, a weakly alkaline solution such as sodium carbonate or sodium hydrogencarbonate solution, preferably sodium hydroxide solution or an acid solution (preferably an organic acid solution, more preferably an acetic acid solution, particularly an acetic acid solution having a concentration of 0.01 to 30%);

(c) adding a carboxyl activator to the thiolation reagent solution, mixing uniformly, and preferably continuing to mix after adjusting the pH to 4.5-6.5;

(d) mixing the steps (a) and (c), and then preferably performing the reaction for 1-8 h at 50-60 ° C (for example, but not limited to 52, 55, 58 ° C, etc.) (for example, but It is not limited to 2, 3, 4, 5, 6, or 7 h, preferably 2-6 h, and more preferably 4-5 h).

(e) Step (d) The reaction solution is dialyzed (preferably dialyzed 2-4 d) and dried (preferably by freeze-drying) to obtain a thiolated chitosan.

The present disclosure also provides a double crosslinked chitosan hydrogel obtained by the above preparation method of the present disclosure.

The micro-chemical structure of the double crosslinked chitosan hydrogel prepared by the present disclosure has both chemical bond cross-linking and physical cross-linking, so that the double-crosslinked chitosan hydrogel of the present disclosure has good mechanical properties. Compared with the chitin hydrogel in the prior art or the hydrogel which has not been physically cross-linked, there is a significant improvement in mechanical properties.

In still another aspect, the present disclosure also provides an application of the disclosed double crosslinked chitosan hydrogel in the preparation of biological materials;

Further, the present disclosure can also provide a biomaterial comprising the double crosslinked chitosan hydrogel of the present disclosure;

Biomaterials as described above preferably include biofibers.

A chitosan thiolated derivative having the formula:

Wherein R is an alkylene group or a substituted alkylene group.

In one or more embodiments, when R is a substituted alkylene group, the substituted alkylene group may be at least one hydrogen atom selected from an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, a hydroxyl group, and an alkyl halide. An alkylene group substituted with at least one group in the group. That is, one of the alkylene groups may have one hydrogen atom substituted by one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, a hydroxyl group, and a halogenated alkyl group; or two of the alkylene groups may be used. One hydrogen atom is substituted by two groups of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, a hydroxyl group and a halogenated alkyl group; or two or more hydrogen atoms in the alkylene group may be alkyl groups. Substituting two or more groups of a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, a hydroxyl group and a halogenated alkyl group; or a plurality of hydrogen atoms in the alkylene group may be an alkyl group, a carboxyl group, an amino group or an alkoxy group. A plurality of the same group in the group, an aryl group, an ester group, a hydroxyl group, and a halogenated alkyl group are substituted or substituted by a combination of a plurality of different groups.

In one or more embodiments, R may have 1 to 20 carbon atoms, preferably 1 to 15, more preferably 1 to 10 carbon atoms. That is, R may be an alkylene group or a substituted subunit of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20. alkyl.

In one or more embodiments, the substituted alkylene group is substituted with at least one hydrogen atom of at least one of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aryl group, an ester group, a carboxyl group, and a halogenated alkyl group. Alkylene.

In one or more embodiments, R has from 1 to 20 carbon atoms.

Due to the presence of free amino groups and hydroxyl groups on the chitosan molecular chain, it is possible to modify and modify the chitosan molecular chain by reacting with a carboxyl group. The most straightforward way to introduce a thiol group into a chitosan molecule is to react a compound having both a carboxyl group and a thiol group with chitosan. However, in the above reaction, since the electrophilicity of the carboxyl group is insufficient, it can only react with the amino group having a strong nucleophilicity in the chitosan molecule without reacting with the hydroxyl group. In the embodiment of the present disclosure, the sulfonic acid group compound having both a carboxyl group and a sulfonic acid group is reacted with chitosan. Due to the strong electron-withdrawing property of the sulfonic acid group, the carboxyl group of the sulfonic acid group compound has strong electrophilicity and can be Simultaneously reacting with the amino group and the primary hydroxyl group in the chitosan, introducing a sulfonic acid group into the chitosan molecular chain, and then reacting with the reducing agent to reduce the sulfonic acid group to a sulfhydryl group, thereby obtaining an amino group and a primary hydroxyl group. Chitosan thiolated derivatives after thiolation. The compound having a carboxyl group and a sulfonic acid group can be directly produced by a hydrolysis reaction, an aminolysis reaction, or the like, or can be directly obtained by reduction after containing a disulfide bond and then vigorously oxidizing. For example, the compound having both a carboxyl group and a sulfonic acid group may be: disulfonic acid succinic acid, sulfonic acid acetic acid, 3-sulfonic acid propionic acid, sulfonic acid succinic acid, or the like.

In one or more embodiments, the chitosan thiolated derivative is produced by the following reaction formula:

Some embodiments of the present disclosure relate to a process for the preparation of a chitosan thiolated derivative comprising reacting a chitosan with a sulfonic acid based compound in the presence of a carboxyl activator and then reducing the sulfonic acid group to a thiol group.

In one or more embodiments, the method for preparing a chitosan thiolated derivative comprises: reacting a solution containing the chitosan with a solution containing the sulfonic acid group compound under the action of a carboxyl activator .

In one or more embodiments, the chitosan thiolated derivatives of the presently disclosed embodiments can be prepared by the following methods:

(1) Preparation of a solution containing chitosan: The chitosan is dissolved in an acid solution of 0.01 to 30%.

(2) Preparation of a solution containing a modifier compound: The sulfonic acid group compound is dissolved in a double distilled water or an alkali solution or an acid solution according to its solubility, and the modifier itself is a solution for the reaction operation to be diluted with a corresponding solvent.

(3) The carboxyl activating agent is added to the above solution containing the sulfonic acid group compound, and after mixing uniformly, the pH is adjusted to 4.5 to 6.5, and mixing and stirring are continued.

(4) The activated sulfonic acid group-containing compound is mixed with the chitosan-containing solution, and sufficiently stirred, and preferably transferred to a round bottom flask, and placed at a temperature of 50 to 60 ° C or lower for constant temperature reaction.

(5) An appropriate amount of a reducing agent was added to the reaction mixture after the reaction, and the mixture was stirred at room temperature for 24 hours.

(6) The reaction solution obtained after the reaction is dialyzed for 2 to 5 days, and the obtained dialysis product is freeze-dried for 2 to 5 days to obtain a chitosan thiolated derivative. The residual small molecule impurities can be removed by means of dialysis to obtain a chitosan thiolated derivative of higher purity.

In one or more embodiments, the solution containing the chitosan is an acid solution. According to some embodiments, the acid solution may be an organic acid solution, preferably an acetic acid solution. That is, it is preferred to dissolve the chitosan in a 0.01 to 30% acetic acid solution.

In one or more embodiments, the solution containing the sulfonic acid group compound is a double distilled water or an alkali solution or an acid solution. According to some embodiments, the alkali solution may be a strong alkali solution, such as a sodium hydroxide solution, a potassium hydroxide solution, a calcium hydroxide solution, or the like, or may be a weak alkaline solution such as an aqueous ammonia solution, a sodium carbonate solution, a sodium hydrogencarbonate solution, or the like. Preferably, a sodium hydroxide solution is used. The double distilled water will be subjected to a once distilled water, and the obtained water is again distilled, which makes the solution after dissolution higher in purity, and the subsequent reaction effect is better. The acid solution in which the sulfonic acid group compound is dissolved may be an organic acid solution, preferably an acetic acid solution.

In one or more embodiments, the carboxyl activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) ). EDC is a water-soluble carbodiimide used as an activation reagent for carboxyl groups in amide synthesis. It is also used to activate phosphate groups, cross-linking of proteins and nucleic acids, and preparation of immunoconjugates, often with NHS. Or N-hydroxy sulfosuccinimide is used in combination to increase the coupling efficiency. NHS, N-hydroxysuccinimide, activates the carboxyl group for the formation of an amide bond. For the synthesis of amino acid protective agents, semi-synthetic kanamycin and pharmaceutical intermediates. The carboxyl activator in the embodiment of the present disclosure may be formulated in a ratio of EDC to NHS of 10:1 to 1:1, which ratio is advantageous for achieving better coupling efficiency, so that the sulfonic acid group is The carboxyl group activation effect of the compound is better.

In one or more embodiments, EDC and NHS may be added to the above sulfonic acid group-containing compound solution under the action of a magnetic stirrer to achieve a better mixing effect.

In one or more embodiments, after mixing the carboxyl activating agent, the pH of the sulfonic acid based compound solution to which the carboxyl activator is added is adjusted with a base or an acid solution to have a pH of 4.5 to 6.5. Preferably, the pH is adjusted with 1 M sodium hydroxide or 1 M hydrochloric acid solution. EDC and NHS can achieve the best activation effect on carboxyl groups at a pH of 4.5 to 6.5.

In one or more embodiments, the pH is adjusted and the mixing time is 10 to 150 minutes, so that the carboxyl activator can fully activate the carboxyl group of the sulfonic acid compound to better modify the chitosan. .

In one or more embodiments, the reaction temperature is preferably 55 to 60 ° C, further preferably 55 ° C. The reaction time may be from 1 to 8 h, preferably from 2 to 6 h, more preferably from 4 to 5 h.

It should be noted that, in the above process, steps (2) and (3) may be performed first, and then step (1) may be performed without affecting the formation of the final product.

In one or more embodiments, the solution containing the chitosan is mixed with a solution containing the sulfonic acid group compound and reacted in the presence of a carboxyl activator; and the sulfonic acid group is reduced by a reducing agent. It is a mercapto group; wherein the solution containing the chitosan is preferably an acid solution; and the solution containing the sulfonic acid group compound is preferably a double distilled water or an alkali solution or an acid solution.

In one or more embodiments, the carboxyl activator comprises EDC and NHS, and the mass ratio of the EDC to the NHS is from 10:1 to 1:1.

In one or more embodiments, the carboxyl group to be reacted in the solution containing the sulfonic acid group compound is activated by the carboxyl activator, and then the solution containing the sulfonic acid group compound is mixed and the shell is contained. The solution of the polysaccharide is subjected to a reaction, and the reaction time is preferably from 1 to 8 hours.

In one or more embodiments, the sulfonic acid based compound is a compound having both a carboxyl group and a sulfonic acid group.

The method for preparing chitosan thiolated derivatives in the embodiments of the present disclosure has a simple operation process and mild reaction conditions. It provides a simple and feasible new method for preparing a novel chitosan thiolated derivative and hydrogel. The resulting chitosan thiolated derivatives are useful in the fields of regenerative medicine, tissue engineering scaffolds, and medical and health applications.

It should be noted that chitosan thiolated derivatives can also be reacted with small molecules or nanoparticles to prepare other chemical materials.

The chitosan thiolated derivative obtained by the above preparation method can be applied in the preparation of a hydrogel, and the hydrogel can be prepared by mixing the chitosan thiolated derivative with the maleated chitosan.

The features and capabilities of the present disclosure are further described in detail below in conjunction with the embodiments.

Example 1

The preparation method of the chitosan hydrogel provided by the embodiment includes:

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 0.01%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 30 min. The acylating reagent (maleic anhydride) 1.8 g was accurately weighed, and 5 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for about 20 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 40 ° C, and heated at a constant temperature for 2 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The MCS product was obtained by freeze drying for three days.

(2) Accurately weigh 1.5 g of thiosuccinic acid and add it to a certain amount of double distilled water for dissolution. 1.2 g of EDC and 300 mg of NHS were weighed into the above solution, and after thorough mixing, the pH of the mixed solution was adjusted to 6.5 with a sodium hydroxide solution, and stirred on a magnetic stirrer for 30 minutes. Accurately weigh 1.5 g of chitosan, dissolve it in an acetic acid solution with a mass fraction of 0.01%, slowly and evenly pour the chitosan solution into the above mixture, and stir to complete the mixing. The mixture was transferred to a 250 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 55 ° C, and heated at a constant temperature for 5 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The chitosan thiol product was obtained by freeze drying for three days.

(3) Accurately weigh 60 mg of MCS, dissolve it with a 1% acetic acid solution, and perform ultrasonic vibration and nitrogen degassing in sequence to prepare a solution with a concentration of 10 mg/ml. Then accurately weigh 60 mg of CS-SH, dissolve it with 1% acetic acid solution, then perform ultrasonic vibration and remove air bubbles by nitrogen, prepare a solution with a concentration of 10 mg/ml, treat with Zn for 10 min, and mix well by shaking. After the prepared MCS solution and the CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 2

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 0.01%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 30 min. 1.8 g of the acylating reagent (β-methacrylic acid) was accurately weighed, and 5 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for about 20 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 40 ° C, and heated at a constant temperature for 2 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The MCS product was obtained by freeze drying for three days.

(3) Accurately weigh 120 mg of MCS, dissolve it with 10% acetic acid solution, and perform ultrasonic vibration and nitrogen degassing in sequence to prepare a solution with a concentration of 50 mg/ml. Then accurately weigh 120 mg of CS-SH, dissolve it with 10% acetic acid solution, then perform ultrasonic vibration and remove air bubbles by nitrogen, prepare a solution with a concentration of 50 mg/ml, treat with Zn for 10 min, and mix well by shaking. After the prepared MCS solution and the CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 3

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 30%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 35 min. The acylating reagent (maleic anhydride) 4.5 g was accurately weighed, and 15 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for 30 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 90 ° C, and heated at a constant temperature for 10 hours. The reaction was stopped and dialyzed for two days, and the dialysate was changed every 4 hours. The MCS product was obtained by freeze drying for two days.

(2) Accurately weigh 1.5 g of 2-mercaptonicotinic acid and add it to a certain amount of double distilled water for dissolution. 1.2 g of EDC and 300 mg of NHS were weighed into the above solution, and after thorough mixing, the pH of the mixed solution was adjusted to about 5.5 with a sodium hydroxide solution, and stirred on a magnetic stirrer for 30 minutes. Accurately weigh 1.5 g of chitosan, dissolve it in an acetic acid solution with a mass fraction of 10%, slowly and uniformly pour the chitosan solution into the above mixture, and stir to complete the mixing. The mixture was transferred to a 250 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 50 ° C, and heated at a constant temperature for 8 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The chitosan thiol product was obtained by freeze drying for three days.

(3) Accurately weigh 60 mg of MCS, dissolve it with acetic acid solution with a mass fraction of 10%, perform ultrasonic vibration in sequence, remove bubbles by nitrogen, and prepare a solution with a concentration of 10 mg/ml. Then accurately weigh 60 mg of CS-SH, dissolve it with 10% acetic acid solution, then perform ultrasonic vibration, remove air bubbles by nitrogen, prepare a solution with a concentration of 10 mg/ml, treat with Zn for 15 min, and mix well by shaking. After the prepared MCS solution and the CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 4

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 30%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 35 min. The acylating reagent (β-isopropylacrylic acid) 4.5 g was accurately weighed, and 15 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for 30 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 90 ° C, and heated at a constant temperature for 10 hours. The reaction was stopped and dialyzed for two days, and the dialysate was changed every 4 hours. The MCS product was obtained by freeze drying for two days.

(3) Accurately weigh 120 mg of MCS, dissolve it with acetic acid solution with a mass fraction of 10%, perform ultrasonic vibration in sequence, remove bubbles by nitrogen, and prepare a solution with a concentration of 50 mg/ml. Then we accurately weighed 120 mg of CS-SH, dissolved it with 10% acetic acid solution, and then ultrasonically oscillated and degassed with nitrogen to prepare a solution with a concentration of 50 mg/ml, treated with Zn for 15 min, and shaken evenly. After the prepared MCS solution and the CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 5

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 30%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 15 min. 5.4 g of the acylating reagent (maleic anhydride) was accurately weighed, and 20 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for 150 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 90 ° C, and heated at a constant temperature for 10 hours. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The MCS product was obtained by freeze drying for three days.

(2) Accurately weigh 1.5g of 3-mercaptopropionic acid solution and add a certain amount of double distilled water for dilution. 1.2 g of EDC and 400 mg of NHS were weighed into the above solution, and after thorough mixing, the pH of the mixed solution was adjusted to about 5 with a sodium hydroxide solution, and stirred on a magnetic stirrer for 60 minutes. Accurately weigh 1.5 g of chitosan, dissolve it in 0.1% by mass of acetic acid solution, slowly and uniformly pour the chitosan solution into the above mixture, and stir to complete the mixing. The mixture was transferred to a 250 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 60 ° C, and heated at a constant temperature for 3 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The CS-SH product was obtained by freeze drying for three days.

(3) Accurately weigh 240 mg of MCS, dissolve it with acetic acid solution with a mass fraction of 1%, ultrasonically shake, remove bubbles by nitrogen, and prepare a solution with a concentration of 100 mg/ml. Accurately weigh 240 mg of CS-SH, dissolve it with 1% acetic acid solution, ultrasonically shake, remove bubbles by nitrogen, prepare a solution with a concentration of 100 mg/ml, treat with the reducing agent dithiothreitol for 100 min, and mix well by shaking. After the prepared MCS and CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 6

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 30%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 15 min. 5.4 g of the acylating reagent (β-methacrylic acid) was accurately weighed, and 20 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for 150 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 90 ° C, and heated at a constant temperature for 10 hours. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The MCS product was obtained by freeze drying for three days.

(2) Accurately weigh 1.5g of 3-mercaptopropionic acid solution and add a certain amount of double distilled water for dilution. Weigh 700mg EDC and 700mg NHS into the above solution at the same time, mix well, adjust the pH of the mixed solution to about 5 with sodium hydroxide solution, stir on a magnetic stirrer for 60min. Accurately weigh 1.5 g of chitosan, dissolve it in 0.1% by mass of acetic acid solution, slowly and evenly pour the chitosan solution into the above mixture, and stir to complete the mixing. The mixture was transferred to a 250 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 60 ° C, and heated at a constant temperature for 3 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The CS-SH product was obtained by freeze drying for three days.

(3) Accurately weigh 60 mg of MCS, dissolve it with acetic acid solution with a mass fraction of 1%, ultrasonically shake, remove bubbles by nitrogen, and prepare a solution with a concentration of 10 mg/ml. Accurately weigh 60 mg of CS-SH, dissolve it with 1% acetic acid solution, ultrasonically shake, remove bubbles by nitrogen, prepare a solution with a concentration of 10 mg/ml, treat with the reducing agent dithiothreitol for 100 min, shake and mix evenly. After the prepared MCS and CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Example 7

(1) Accurately weigh 1.5 g of chitosan and dissolve it in acetic acid with a mass fraction of 15%. After being substantially dissolved uniformly under the action of a magnetic stirrer, it was placed in an ultrasonic oscillator for 15 min. 1.2 g of the acylating reagent (maleic anhydride) was accurately weighed, and 6 ml of acetone was added to completely dissolve it. The dissolved acylating reagent was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for 40 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 60 ° C, and heated at a constant temperature for 6 hours. After the reaction was stopped, the solution was dialyzed for three days, and the dialysate was changed every 4 hours. The MCS product was obtained by freeze drying for three days.

(2) Accurately weigh 1.5g thioglycolic acid solution and add a certain amount of double distilled water for dilution. 1.5 g of EDC and 150 mg of NHS were weighed into the above solution, and after thorough mixing, the pH of the mixed solution was adjusted to 6.5 with a sodium hydroxide solution, and stirred on a magnetic stirrer for 8 minutes. Accurately weigh 1.5 g of chitosan, dissolve in 2% by mass of acetic acid solution, slowly and evenly pour the chitosan solution into the above mixture, and stir to complete the mixing. The mixture was transferred to a 250 ml round bottom flask, placed in a collecting magnetic stirrer, set at a temperature of 55 ° C, and heated at a constant temperature for 5 h. After the reaction was stopped, the solution was dialyzed for three days, and the dialysate was changed every 4 hours. The CS-SH product was obtained by freeze drying for three days.

(3) Accurately weigh 240 mg of MCS, dissolve it with acetic acid solution with a mass fraction of 1%, ultrasonically shake, remove bubbles by nitrogen, and prepare a solution with a concentration of 100 mg/ml. Accurately weigh 240 mg of CS-SH, dissolve it with 1% acetic acid solution, ultrasonically shake, remove bubbles by nitrogen, prepare a solution with a concentration of 100 mg/ml, treat with hydroquinone with reducing agent for 100 min, and mix well by shaking. After the prepared MCS and CS-SH solution are thoroughly mixed, they are extruded into a NaOH solution to form a gel, thereby obtaining a desired chitosan hydrogel.

Chemical reaction formula:

Experimental example 1

Fourier infrared analysis of the chitosan, thiolated chitosan and α-β unsaturated acylated chitosan in Example 1 gave Fig. 1, chitosan and thiolated chitosan in Fig. 1. the IR analysis shows comparison, whether or MCS CS-SH in the vicinity of a wave number of 3400cm ^-1, -OH stretching can be seen the introduction of a carboxyl group, generated; in the vicinity of 2900cm ^-1, represents the CH stretching branched Vibration; amide I band and amide II band near 1500cm ^-1 -1650cm ^-1 , it can be seen that both the MCS and CS-SH amide bands are strengthened, especially the MCS, due to the introduction of C=C double bond The generated C=C stretching vibration peak overlaps with the amide band, and the peak intensity in the band is obviously increased; in the vicinity of 1100 cm ^-1 , both MCS and CS-SH show obvious CO at this position due to the introduction of carboxyl groups. Stretching vibration peak; in the absorption band of -NH ₃ ⁺ near 2100 cm ^-1 , the free amino group of chitosan is modified, the content of protonated amino group is decreased, and the peak intensity of absorption band of -NH ₃ ⁺ is significantly reduced. The mercapto compound and the acylating agent were successfully introduced onto the chitosan chain. At the same time, the surface structure and surface pore size of the chitosan hydrogel of Example 1 were subjected to scanning electron microscopy (SEM) analysis by scanning electron microscopy. The results are shown in Figure 2 and Figure 3. SEM analysis showed that many cross-linked pores appeared on the surface of the chitosan hydrogel after curing, indicating that the cross-linking reaction proceeded completely, further indicating that the prepared thiolated chitosan could successfully prepare a fast-curing chitosan hydrogel.

Further, the chitosan hydrogel obtained in Example 1 was tested by using an instron mechanical testing machine, and a 10N sensor was placed on the sensor compression substrate, and the test parameters were set according to the size of the sample. Stop compression and the system automatically derives the elastic modulus value.

The test parameters are: length 10 mm, width 8 mm, height 5 mm, compression rate 0.8 mm/min. The test results are shown in Fig. 4. The fracture occurred at a compression ratio of 75% and a compressive stress of about 700 kPa. Therefore, it can be seen that the hydrogel has good mechanical strength and elastic properties.

In summary, by using the acylation reagent acylated chitosan and thiolated chitosan as raw materials, the rapid addition of raw materials from liquid to solid can be achieved by Michael addition reaction, which greatly improves the printability of the material. By adjusting the concentration of the two, the elastic modulus of the cured chitosan hydrogel can be adjusted, and the application range of the material can be expanded. The chitosan hydrogel prepared by the method has important applications in the fields of biomedicine and tissue engineering, The curing speed is fast, the biocompatibility is good, the mechanical strength is adjustable, the stability in the medium is good, the biodegradation speed is adjustable, and the application range is large.

Example 8

1) 1.5g of chitosan (molecular weight of about 50,000, deacylation degree of about 80%) was added to 0.01% (m / m) of acetic acid solution, stirred evenly and then ultrasonic for 30min;

Add 1.8g maleic anhydride to 10ml of acetone and stir to dissolve, then add the obtained solution to the chitosan solution, stir and mix well, and react at 40 ° C for 2h;

After completion of the reaction, the reaction solution was dialyzed for 3 days, and dialyzate was replaced every 5 hours, and the obtained product was freeze-dried to obtain a product α-β unsaturated acylated chitosan (abbreviated as MCS).

Step 1) The reaction formula can be referred to as follows:

2) 1.5g of mercapto succinic acid was added to the double distilled water to stir and dissolve, and then 1.2g of EDC and 300mg of NHS were added. After thorough mixing, the pH of the solution was adjusted to 6.5 with 1M NaOH, and stirring was continued;

1.5 g of chitosan was added to a 0.01% (m / m) acetic acid solution and stirred to dissolve, and then the resulting chitosan solution was added to a mixed solution containing mercapto succinic acid, and reacted at 55 ° C for 5 h;

The reaction solution was dialyzed for 3 days, and dialyzate was replaced every 5 hours, and the obtained product was freeze-dried to obtain a product thiolated chitosan (abbreviated as CS-SH).

Step 2) The reaction formula can be referred to as follows:

3) 60 mg of MCS was added to a 1% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 10 mg/L MCS solution;

60 mg of CS-SH was added to a 1% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 10 mg/L CS-SH solution;

Adding metal zinc to the obtained CS-SH solution, shaking the reaction, and then filtering to obtain a CS-SH solution after reduction treatment;

The MCS solution was mixed with the reduced-treated CS-SH solution, and then extruded into a NaOH solution to obtain a chemically crosslinked hydrogel.

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 8.

The reaction formula of Example 8 is shown in Fig. 5, and the reaction scheme is shown in Fig. 6.

Example 9

Add 1.8g β-methacrylic acid to 5ml of acetone and stir to dissolve, then add the obtained solution to the chitosan solution, stir and mix well, and react at 40 ° C for 2h;

3) 120 mg of MCS was added to a 10% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 50 mg/L MCS solution;

120 mg of CS-SH was added to a 10% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 50 mg/L CS-SH solution;

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 9.

Example 10

1) 1.5 g of chitosan (having a molecular weight of about 50,000 and a deacylation degree of about 80%) is added to a 0.30% (m/m) acetic acid solution, stirred uniformly and then ultrasonicated for 35 min;

Adding 4.5 g of maleic anhydride to 15 ml of acetone, stirring and dissolving, then adding the obtained solution to the chitosan solution, stirring and mixing uniformly, and reacting at 40 ° C for 2 h;

2) 1.5 g of 2-mercaptonicotinic acid was added to double distilled water to stir and dissolve, then 1.2 g of EDC and 300 mg of NHS were added, and after thorough mixing, the pH of the solution was adjusted to 5.5 with 1 M NaOH, and stirring was continued;

1.5 g of chitosan was added to a 10% (m/m) acetic acid solution to stir and dissolve, and then the obtained chitosan solution was added to a mixed solution containing 2-mercaptonicotinic acid, and reacted at 50 ° C for 8 hours;

3) 60 mg of MCS was added to a 10% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 10 mg/L MCS solution;

60 mg of CS-SH was added to a 10% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 10 mg/L CS-SH solution;

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 10.

Example 11

1) 1.5 g of chitosan (having a molecular weight of about 50,000 and a deacylated degree of about 80%) is added to a 30% (m/m) acetic acid solution, stirred uniformly and then ultrasonicated for 35 min;

4.5g β-isopropylacrylic acid was added to 15ml of acetone and stirred to dissolve, and then the obtained solution was added to the chitosan solution, stirred and mixed uniformly, and reacted at 90 ° C for 10 hours;

After completion of the reaction, the reaction solution was dialyzed for 2 d, and dialyzate was replaced every 4 hours, and the obtained product was freeze-dried to obtain a product α-β unsaturated acylated chitosan (abbreviated as MCS).

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 11.

Example 12

1) 1.5 g of chitosan (having a molecular weight of about 50,000 and a deacylation degree of about 80%) is added to a 30% (m/m) acetic acid solution, stirred uniformly and then ultrasonicated for 150 min;

Adding 5.4 g of maleic anhydride to 20 ml of acetone, stirring and dissolving, then adding the obtained solution to the chitosan solution, stirring and mixing uniformly, and reacting at 90 ° C for 10 h;

2) 1.5 g of 3-mercaptopropionic acid was added to double distilled water to stir and dissolve, then 1.2 g of EDC and 400 mg of NHS were added, and after thorough mixing, the pH of the solution was adjusted to 5 with 1 M NaOH, and stirring was continued;

1.5 g of chitosan was added to a 0.1% (m/m) acetic acid solution to stir and dissolve, and then the obtained chitosan solution was added to a mixed solution containing 3-mercaptopropionic acid, and reacted at 60 ° C for 3 h;

3) 240 mg of MCS was added to a 1% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 100 mg/L MCS solution;

Adding dithiothreitol to the obtained CS-SH solution, shaking the reaction for 100 min, and then filtering to obtain a CS-SH solution after reduction;

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 12.

Example 13

Add 5.4g of β-methacrylic acid to 20ml of acetone and stir to dissolve, then add the obtained solution to the chitosan solution, stir and mix well, and react at 90 ° C for 10h;

2) 1.5 g of 3-mercaptopropionic acid was added to double distilled water to stir and dissolve, then 700 mg of EDC and 700 mg of NHS were added, and after thorough mixing, the pH of the solution was adjusted to 5 with 1 M NaOH, and stirring was continued;

Adding dithiothreitol to the obtained CS-SH solution, shaking the reaction, and then filtering to obtain a CS-SH solution after reduction treatment;

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 13.

Example 14

1) 1.5 g of chitosan (molecular weight of about 50,000, deacylation degree of about 80%) was added to a 15% (m / m) acetic acid solution, stirred evenly and then ultrasonic for 150 min;

1.2g β-methacrylic acid was added to 6ml of acetone and stirred to dissolve, and then the obtained solution was added to the chitosan solution, stirred and mixed uniformly, and reacted at 60 ° C for 6 hours;

After completion of the reaction, the reaction solution was dialyzed for 3 days, and dialyzate was replaced every 4 hours, and the obtained product was freeze-dried to obtain a product α-β unsaturated acylated chitosan (abbreviated as MCS).

2) 1.5 g of thioglycolic acid was added to double distilled water to stir and dissolve, then 1.5 g of EDC and 700 mg of NHS were added, and after thorough mixing, the pH of the solution was adjusted to 6.5 with 1 M NaOH, and stirring was continued;

1.5g of chitosan was added to a 2% (m / m) acetic acid solution and stirred to dissolve, and then the resulting chitosan solution was added to a mixed solution containing thioglycolic acid, and reacted at 60 ° C for 3 h;

The reaction solution was dialyzed for 3 days, and dialyzate was replaced every 4 hours, and the obtained product was freeze-dried to obtain a product thiolated chitosan (abbreviated as CS-SH).

240 mg of CS-SH was added to a 1% (m/m) acetic acid solution, stirred and dissolved, and then ultrasonically shaken and deaerated with nitrogen to obtain a 100 mg/L CS-SH solution;

Adding hydroquinone to the obtained CS-SH solution, shaking the reaction, and then filtering to obtain a CS-SH solution after reduction treatment;

The obtained chemically crosslinked hydrogel was added to absolute ethanol and immersed for 24 hours to obtain a double crosslinked chitosan hydrogel of Example 14.

Experimental example 2

The unmodified chitosan was immersed in absolute ethanol for 24 hours to obtain a physically crosslinked hydrogel, which was recorded as CSH-E10;

Referring to the method in Example 8, a chemically crosslinked hydrogel was obtained according to a molar ratio of maleic anhydride to chitosan of 1.2:1 and a molar ratio of mercapto succinic acid to chitosan of 1.2:1. For M4S4-E0;

Meanwhile, referring to the method in Example 8, a chemically crosslinked hydrogel was obtained according to a molar ratio of maleic anhydride to chitosan of 1.2:1 and a molar ratio of mercapto succinic acid to chitosan of 1.2:1. Then, the obtained chemically crosslinked hydrogel was respectively placed in a 20%, 40%, 60%, 80% ethanol solution and absolute ethanol, and immersed for 24 hours to obtain a corresponding double crosslinked hydrogel. The hydrogels were recorded as M4S4-E2, M4S4-E4, M4S4-E6, M4S4-E8, M4S4-E10, respectively;

Referring to the method of Example 8, a chemically crosslinked hydrogel was obtained according to a molar ratio of maleic anhydride to chitosan of 0.2:1 and a molar ratio of mercapto succinic acid to chitosan of 0.2:1, and then, The obtained chemically crosslinked hydrogel was placed in absolute ethanol and immersed for 24 hours to obtain a corresponding double crosslinked hydrogel, which was recorded as M1S1-E10;

Referring to the method of Example 8, a chemically crosslinked hydrogel is obtained according to a molar ratio of maleic anhydride to chitosan of 0.5:1 and a molar ratio of mercapto succinic acid to chitosan of 0.5:1, and then, The obtained chemically crosslinked hydrogel was placed in absolute ethanol and immersed for 24 hours to obtain a corresponding double crosslinked hydrogel, which was recorded as M2S2-E10;

Referring to the method of Example 8, a molar ratio of maleic anhydride to chitosan was 1:1, and a molar ratio of mercapto succinic acid to chitosan was 1:1, thereby obtaining a chemically crosslinked hydrogel, and then, The obtained chemically crosslinked hydrogel was placed in absolute ethanol and immersed for 24 hours to obtain a corresponding double crosslinked hydrogel, which was recorded as M3S3-E10;

The different hydrogel test data are shown in the following table:

From the results of the tests shown in FIG. 7 and FIG. 8 and the above table data, the double crosslinked hydrogel of the present disclosure has remarkable mechanical properties compared to the single physical crosslinked or chemically crosslinked hydrogel. Increase, both compression modulus and fracture strength, are significantly improved;

At the same time, the performance of the double crosslinked hydrogels of M4S4-E2, M4S4-E4, M4S4-E6, M4S4-E8, and M4S4-E10 groups shows that when the ratio of raw materials is the same, the water is physically crosslinked with absolute ethanol. The gel has the best physical properties;

Further, the performance comparison of the M4S4-E10, M1S1-E10, M2S2-E10, and M3S3-E10 double crosslinked hydrogels shows that the raw material acylating agent and the thiolation reagent are combined with chitosan to double crosslink the final product. The mechanical properties of chitosan also have a significant effect. After the reaction of acylated chitosan and thiolated chitosan prepared in a molar ratio of 1.2:1, double-crosslinked chitosan with better mechanical properties can be obtained.

Experimental example 3

According to the method of Example 8, the MCS solution and the CS-SH solution after the reduction treatment were respectively obtained, and the two were uniformly mixed, then sucked into a syringe, and sampled by a syringe pump, and the mixed solution was rapidly formed in a receiving dish containing NaOH. Obtaining chitosan biofibers;

Then, the obtained chitosan biofibers were immersed in absolute ethanol for 24 hours to obtain double crosslinked chitosan biofibers, and the scanning electron micrograph thereof is shown in FIG.

Example 15

The reaction equation of the chitosan thiolated derivative in this example is:

The specific reaction steps are as follows:

(1) 0.3 g of chitosan was dissolved in a 0.01% by mass acetic acid solution to obtain a chitosan acetic acid solution.

(2) Take 5 ml of sulfonic acid succinic acid solution and dilute to 50 ml with double distilled water.

(3) Take 0.8g EDC and 0.2g NHS under the action of magnetic stirrer, add the above sulfonic acid succinic acid solution once, mix well, and adjust the pH of the mixed solution to 6.5 with 1M diluted hydrochloric acid, and mix the solution. Stir for 30 min to fully activate the carboxyl groups.

(4) Mixing the sulfonic acid succinic acid solution after activation of the carboxyl group with the chitosan acetic acid solution, stirring for 10 minutes, stirring well, transferring the mixed solution to a round bottom flask, and reacting the temperature at a temperature of 55 ° C. 5h.

(5) Metal zinc particles having surface zinc oxide treated with hydrochloric acid were added to the above reaction liquid, and stirred at room temperature for 24 hours.

(6) After the reaction solution was subjected to dialysis for 3 days and freeze-dried for 3 days, a chitosan thiolated derivative was obtained.

Example 16

The reaction equation of the chitosan thiolated derivative in this example is:

The specific reaction steps are as follows:

(1) 4.5 g of chitosan was dissolved in a 30% by mass acetic acid solution to obtain a chitosan acetic acid solution.

(2) Take 2.8 g of 2-sulfonic acid nicotinic acid and dilute to 50 ml with double distilled water.

(3) Take 1.6g EDC and 0.4g NHS in the above 2-sulfonic acid nicotinic acid solution under the action of magnetic stirrer, mix well, and adjust the pH of the mixed solution to 6.5 with 1M diluted hydrochloric acid, and mix The solution was stirred for 30 min to fully activate the carboxyl groups.

(4) Mixing the 2-sulfonic acid nicotinic acid solution activated by the carboxyl group with the chitosan acetic acid solution, stirring for 10 minutes, stirring well, transferring the mixed solution to a round bottom flask, and reacting at a constant temperature of 60 ° C. Number 3h.

Example 17

The reaction equation of the chitosan thiolated derivative in this example is:

The specific reaction steps are as follows:

(1) 2.1 g of chitosan was dissolved in a 2% by mass acetic acid solution to obtain a chitosan acetic acid solution.

(2) Take 4.5 g of 3-sulfonic acid propionic acid and dilute to 50 ml with double distilled water.

(3) Take 1.0g EDC and 1.0g NHS in the above 3-sulfonic acid propionic acid solution under the action of magnetic stirrer, mix well, and adjust the pH of the mixed solution to 5.5 with 1M diluted hydrochloric acid, and mix The solution was stirred for 60 min to fully activate the carboxyl group.

(4) After mixing the carboxyl group-activated 3-sulfonic acid propionic acid solution with the chitosan acetic acid solution, stirring for 10 minutes, stirring well, transferring the mixed solution to a round bottom flask, and maintaining the temperature at a temperature of 50 ° C. Number 8h.

Example 18

The reaction equation of the chitosan thiolated derivative in this example is:

The specific reaction steps are as follows:

(1) 0.15 g of chitosan was dissolved in a 0.01% by mass acetic acid solution to obtain a chitosan acetic acid solution.

(2) Take 0.09 g of sulfonic acid acetic acid and dilute to 50 ml with double distilled water.

(3) Take 0.090g EDC and 0.009g NHS in the above sulfonic acid acetic acid solution under the action of magnetic stirrer, mix well, and adjust the pH of the mixed solution with 1M diluted hydrochloric acid to make the pH 6.5, then stir. The carboxyl group was fully activated in 30 min.

(4) Add the activated sulfonic acid acetic acid solution to the chitosan acetic acid solution for 150 min, stir well, and transfer the mixture to a round bottom flask, and react at a constant temperature of 55 ° C for 5 h.

(6) The reacted reaction solution was dialyzed for 3 days and lyophilized for 3 days to obtain a chitosan thiolated derivative.

Example 19

Accurately weigh 1.5g of chitosan dissolved in acetic acid solution with a mass fraction of 0.01% by mass. After being dissolved uniformly under the action of magnetic stirrer, it was placed in an ultrasonic oscillator for about 30 minutes. 1.8 g of maleic anhydride was accurately weighed, and 5 ml of acetone was added to completely dissolve it. The dissolved maleic anhydride was slowly and slowly added to the chitosan acetic acid solution, and stirred under a magnetic stirrer for about 20 minutes to be thoroughly mixed. The uniformly mixed liquid was transferred into a 150 ml round bottom flask, placed in a hot magnetic stirrer, set at a temperature of 40 ° C, and heated at a constant temperature for 2 h. After the reaction was stopped, it was dialyzed for three days, and the dialysate was changed every 5 hours. The MCS product was obtained by freeze drying for about three days.

Chemical reaction formula:

Accurately weigh 60 mg of MCS, dissolve it with a certain mass fraction of acetic acid solution, ultrasonically shake it, remove the bubbles by nitrogen, and prepare a solution with a concentration of 10 mg/ml. Accurately weigh 60 mg of the chitosan thiolated derivative (TCS) in Example 15, dissolved in sodium hydroxide solution, ultrasonically shaken, and deaerated with nitrogen to prepare a solution having a concentration of 10 mg/ml, and treated with a reducing agent for about 10 minutes. , shake and mix evenly. The prepared MCS and TCS solutions were injected from the injection system on a 3D printer, and the computer program controlled the sample speed and print mode to uniformly mix and prepare a 3D bio-printing hydrogel.

The Fourier transform infrared spectrum of chitosan and chitosan thiolated derivatives in Example 15 were respectively obtained in Figure 10. The comparison of the infrared spectra of chitosan and thiolated chitosan in Figure 10 shows that the thiol group can be seen. chitosan in the vicinity of a wave number of 3400cm ^-1, -OH stretching vibration can be seen at the carboxy group; in the vicinity of 2900cm ^-1, represents the CH stretching vibration introduced branched; in the vicinity of 1500cm ^-1 ~ 1650cm amide ⅰ ^-1 The band and the amide II band, the modified chitosanamide band is strengthened; in the vicinity of 1100 cm ^-1 , the modified chitosan exhibits a distinct CO stretching vibration peak at this position due to the introduction of the carboxyl group; near 2100 cm ^-1 In the absorption band of -NH ₃ ⁺ , the free amino group of chitosan was reduced, the content of protonated amino group was decreased, and the peak intensity of absorption band of -NH ₃ ⁺ was significantly reduced. The above phenomenon indicates that the sulfhydryl group was successfully introduced into the chitosan chain. At the same time, the surface structure and surface pore diameter of the cured hydrogel were analyzed by scanning electron microscopy (SEM). The results are shown in Figure 11 and Figure 12 in sequence. SEM analysis showed that many cross-linked pores appeared on the surface of the hydrogel after curing, indicating that the cross-linking reaction proceeded completely, further indicating that the prepared chitosan thiolated derivative can successfully prepare a fast-curing hydrogel.

Further, the hydrogel obtained in Example 19 was tested by using an instron mechanical testing machine, and a 10N sensor was placed on the sensor compression substrate, and the test parameters were set according to the size of the sample. After the test curve was abrupt, the compression was stopped. The system automatically derives the elastic modulus value.

The test parameters are: length 10 mm, width 8 mm, height 5 mm, compression rate 0.8 mm/min. The test results are shown in Fig. 13. The fracture occurred at a compression ratio of 75% and a compressive stress of about 700 kPa. Therefore, it can be seen that the hydrogel has good mechanical strength and elastic properties.

In summary, in the embodiments of the present disclosure, by using a sulfonic acid group compound containing both a sulfonic acid group and a carboxyl group as a modifying agent, the amino group and the primary hydroxyl group in the chitosan molecular chain are successfully succeeded by the carboxyl activator. A sulfonic acid group is introduced, and further a thiol group is obtained by reduction. The preparation method has reasonable route design, simple and feasible operation, low requirements on equipment, and high-yield chitosan thiolated derivatives. The chitosan thiolated derivative has good nucleophilic performance, antioxidant property and rich derivatization due to the action of the thiol side chain, and can be further derivatized by nucleophilic reaction, crosslinking reaction, etc., and its application range Very extensive. For example, the chitosan thiolated derivative is chemically reacted with other polymer derivatives containing a structure such as maleimide, vinyl sulfone, α-β unsaturated aldehyde, ketone, acid, ester, etc. to prepare rapid curing. Hydrogels have important applications in the fields of regenerative medicine and tissue engineering. For example, due to the better nucleophilic performance and biocompatibility of the chitosan thiolated derivative, the chitosan thiolated derivative can be used for preparing a pharmaceutical carrier, and its good nucleophilic performance is beneficial to The binding of target cells can achieve the purpose of increasing the efficacy; or, by using the chitosan thiolated derivatives, the antioxidant properties can be made into a polymer film with natural polymers, and play a role in the field of storage and preservation. .

The embodiments described above are a part of the embodiments of the present disclosure, and not all of them. The detailed description of the embodiments of the present disclosure is not intended to limit the scope of the disclosure The chitosan thiolated derivatives have a wide variety of variations and derivatizations, which are widely applicable, and only some of their possible uses are listed herein, and are not exhaustive. Based on the examples in the present disclosure, those skilled in the art will All other embodiments or other possible applications obtained without creative efforts are within the scope of the present disclosure.

Industrial applicability

(1) The chitosan hydrogel prepared by the method of the present disclosure has important applications in the fields of biomedicine and tissue engineering, and has good printability, fast curing speed, good biocompatibility, adjustable mechanical strength, and medium. Good stability, adjustable biodegradation speed and wide application range.

(2) The double cross-linked chitosan hydrogel containing both chemical cross-linking and physical cross-linking structure in the present disclosure not only has a simple and rapid preparation method, but also has a fast preparation reaction speed and does not require the use of highly toxic chemicals. Cross-linking agent, the preparation process is green, environmentally friendly and safe. The double crosslinked chitosan hydrogel obtained by the method of the present invention has a fast curing speed, high mechanical strength, good biocompatibility, good stability in a medium, and a large application range. Compared with the currently common ultraviolet-cured chitosan hydrogel, pH-responsive chitosan hydrogel, temperature-sensitive chitosan hydrogel, and ion-responsive chitosan hydrogel, the present invention double-crosslinked chitosan water The gel both increases the rate of cure and also improves the mechanical strength and elasticity of the chitosan hydrogel.

(3) The preparation method of the chitosan thiolated derivative of the embodiment of the present disclosure has a rational route design, simple and feasible operation, low requirements on equipment, and high-yield chitosan thiolated derivatives. The chitosan thiolated derivative has good nucleophilic performance, antioxidant property and rich derivatization due to the action of the thiol side chain, and can be further derivatized by nucleophilic reaction, cross-linking reaction, etc., and the application range is very widely. The modified chitosan thiolated derivative formed under the alkaline condition can form a sulfur anion under alkaline conditions, and can be combined with maleimide, vinyl sulfone, α-β unsaturated aldehyde, ketone, acid, Other polymer derivatives of esters and other structures undergo a chemical reaction to prepare a hydrogel, which improves the curing speed of the hydrogel and also improves the mechanical strength and elasticity of the hydrogel.

Claims

A method for preparing a chitosan hydrogel comprises: performing a Michael addition reaction of α-β unsaturated acylated chitosan with thiolated chitosan; and the α-β unsaturated acylated chitosan The formula is:
The general formula of the thiolated chitosan is:

Wherein R 1 is a residue portion of the chitosan polymer to remove an amino group; R 2 is a hydrogen atom, an alkyl group or an alkylene group; and R 3 is a carbonyl group, a carboxyl group, an ester group, an amide group, an alkyl group or a substituted alkyl group; 4 is an alkylene group or a substituted alkylene group.
The method for preparing a chitosan hydrogel according to claim 1, wherein the thiolated chitosan is treated with a reducing agent before the Michael addition reaction, preferably, the reducing agent is metal Zn, two Thiositol or hydroquinone.
The method for producing a chitosan hydrogel according to claim 1 or 2, wherein the α-β unsaturated acylated chitosan solution and the thiolated chitosan solution are subjected to a Michael addition reaction, preferably, The concentration of the α-β unsaturated acylated chitosan solution is 10 to 100 mg/ml, and the pH is less than 7; preferably, the concentration of the thiolated chitosan solution is 10 to 100 mg/ml, and the pH is less than 7.
The method for producing a chitosan hydrogel according to any one of claims 1 to 3, wherein the Michael addition reaction is carried out by using the α-β unsaturated acylated chitosan solution and the thiol group After the chitosan solution is mixed, it is reacted with an alkali solution to form a gel.
The method for producing a chitosan hydrogel according to any one of claims 1 to 4, wherein the preparation of the α-β unsaturated acylated chitosan comprises the steps of: an acid solution containing chitosan After mixing with a solution containing an acylating reagent, the reaction is carried out, followed by dialysis and freeze-drying; wherein the acylating agent comprises an α-β unsaturated acid, an α-β unsaturated acid anhydride, an α-β unsaturated acid chloride, and α- At least one of the β-unsaturated esters; preferably, the molar ratio of the free amino groups on the chitosan chain to the acylating agent is from 1:1 to 10.
The method for producing a chitosan hydrogel according to any one of claims 1 to 5, wherein the preparation of the thiolated chitosan comprises the steps of: a solution containing chitosan and a solution containing a mercapto compound The reaction is carried out under the action of a carboxyl activator; preferably, the chitosan-containing solution dissolves chitosan in an acid solution having a mass fraction of 0.01 to 30%, and the solution containing the mercapto compound is the mercapto group. a compound which, according to its solubility, is dissolved in a double distilled water or an alkali solution or an acid solution, the carboxyl activator comprising EDC and NHS, the mass ratio of the EDC to the NHS being from 1 to 10:1; further preferably The molar ratio of the mercapto compound to the amino group on the chitosan is from 1 to 10:1; preferably, the reaction temperature is from 50 to 60 ° C, further preferably from 15 to 20 ° C, and the reaction time is from 4 to 12 h, and will pass The mixed solution after the reaction is subjected to dialysis and freeze-drying; preferably, the mercapto compound is a compound containing both a carboxyl group and a mercapto group, and more preferably, the mercapto compound includes acetylcysteine, cysteine, mercaptobutane acid, DMSA, any one of 2-mercapto-3-pyridinecarboxylic acid in.
The method for producing a chitosan hydrogel according to any one of claims 1 to 6, wherein the thiolated chitosan is prepared by first using the carboxyl activator in a solution containing the mercapto compound. The carboxyl group is activated, and then a solution containing the mercapto compound and a solution containing chitosan are mixed to carry out a reaction.
A chitosan hydrogel prepared by the method for preparing a chitosan hydrogel according to any one of claims 1 to 7, wherein the chitosan hydrogel has the formula:

Wherein R 1 is a residue portion of the chitosan polymer to remove an amino group; R 2 is a hydrogen atom, an alkyl group or an alkylene group; and R 3 is a carbonyl group, a carboxyl group, an ester group, an amide group, an alkyl group or a substituted alkyl group; 4 is an alkylene group or a substituted alkylene group.
Use of the chitosan hydrogel of claim 8 in the manufacture of biomedical materials or tissue engineering materials or 3D bioprinting materials.
A method for preparing a double crosslinked chitosan hydrogel, wherein the preparation method comprises:

The hydrogel obtained by reacting α-β unsaturated acylated chitosan with thiolated chitosan is immersed in an ethanol solution to obtain a double crosslinked chitosan hydrogel.
The preparation method according to claim 10, wherein the immersion treatment time is 0-48h, but does not include 0h;

Preferably, the time of the soaking treatment is 24 hours.
The production method according to claim 10 or 11, wherein the α-β unsaturated acylated chitosan comprises a compound of the following formula (i):

Wherein, in the formula (i), R 1 is a residue portion of the chitosan to remove an amino group;

R 2 , R 3 , and R 4 are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl;

R 5 is carbonyl, carboxyl, ester, amide, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
The production method according to any one of claims 10 to 12, wherein the thiolated chitosan comprises a compound represented by the following formula (ii):

Wherein, in the formula (ii), R 1 is a residue portion of the chitosan to remove an amino group;

R 6 is a substituted or unsubstituted alkylene group, a substituted or unsubstituted arylene group, and a substituted or unsubstituted heteroarylene group.
The production method according to any one of claims 10 to 13, comprising: mixing α-β unsaturated acylated chitosan with thiolated chitosan under solution conditions, and then reacting with an alkali solution , get a hydrogel.
The production method according to claim 10 or 11, wherein the hydrogel obtained by reacting the α-β unsaturated acylated chitosan with thiolated chitosan is the chitosan hydrogel according to claim 8. .
A double crosslinked chitosan hydrogel obtained by the production method according to any one of claims 10-15.
The use of the double crosslinked chitosan hydrogel of claim 16 in the preparation of a biological material; preferably, the biological material comprises: a biofiber;

And/or a biomaterial comprising the double crosslinked chitosan hydrogel of claim 16;

Preferably, the biological material comprises: a biofiber.
A chitosan thiolated derivative having the formula:

Wherein R is an alkylene group or a substituted alkylene group.
A method for producing a chitosan thiolated derivative according to claim 18, wherein the chitosan is reacted with a sulfonic acid group compound in the presence of a carboxyl group activator; and the sulfonic acid group is reduced to a reducing agent by a reducing agent Sulfhydryl compound; the sulfonic acid group compound is a compound having both a carboxyl group and a sulfonic acid group.
Use of the chitosan thiolated derivative of claim 18 for the preparation of a hydrogel.