WO2019192628A2 - Dérivé de chitosane thiolé, hydrogel de chitosane, et procédés de préparation associés et applications associées - Google Patents

Dérivé de chitosane thiolé, hydrogel de chitosane, et procédés de préparation associés et applications associées Download PDF

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WO2019192628A2
WO2019192628A2 PCT/CN2019/089538 CN2019089538W WO2019192628A2 WO 2019192628 A2 WO2019192628 A2 WO 2019192628A2 CN 2019089538 W CN2019089538 W CN 2019089538W WO 2019192628 A2 WO2019192628 A2 WO 2019192628A2
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chitosan
group
solution
hydrogel
substituted
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PCT/CN2019/089538
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Chinese (zh)
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WO2019192628A3 (fr
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戴建武
陈艳艳
黄雷
储筠
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中国科学院苏州纳米技术与纳米仿生研究所
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Priority claimed from CN201810291744.1A external-priority patent/CN110343264B/zh
Priority claimed from CN201810291063.5A external-priority patent/CN110343194B/zh
Priority claimed from CN201910436288.XA external-priority patent/CN111333878B/zh
Application filed by 中国科学院苏州纳米技术与纳米仿生研究所 filed Critical 中国科学院苏州纳米技术与纳米仿生研究所
Publication of WO2019192628A2 publication Critical patent/WO2019192628A2/fr
Publication of WO2019192628A3 publication Critical patent/WO2019192628A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • 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.
  • 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.
  • the existing chitosan molecules cannot be chemically reacted with other polymer derivatives containing maleimide, vinyl sulfone, ⁇ - ⁇ 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.
  • 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.
  • the chemical cross-linking reaction time in the first step of the method is long, and rapid prototyping cannot be achieved.
  • 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.
  • 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.
  • a method for preparing a double crosslinked chitosan hydrogel 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 ⁇ - ⁇ unsaturated acylated chitosan with thiolated chitosan; ⁇ - ⁇ unsaturated acylation shell polymerization
  • Michael addition reaction of ⁇ - ⁇ unsaturated acylated chitosan with thiolated chitosan ⁇ - ⁇ unsaturated acylation shell polymerization
  • the formula of sugar is:
  • the general formula of thiolated chitosan is:
  • 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
  • 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:
  • 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
  • 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 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: ⁇ - ⁇ -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.
  • 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:
  • R is an alkylene group or a substituted alkylene group.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ - ⁇ 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.
  • FTIR Fourier transform infrared spectroscopy
  • Example 2 is a scanning electron microscope (SEM) surface microstructure diagram of the chitosan hydrogel in Example 1 of the present disclosure
  • Example 3 is a surface aperture scanning electron microscope (SEM) image of a chitosan hydrogel in Example 1 of the present disclosure
  • Example 4 is a graph showing the mechanical test of the chitosan hydrogel in Example 1 of the present disclosure.
  • Example 5 is a reaction formula of preparation of double crosslinked chitosan in Example 8 of the present disclosure.
  • Example 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.
  • Example 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;
  • FTIR Fourier transform infrared spectroscopy
  • Figure 11 is a scanning electron microscope (SEM) surface microstructure diagram of the cured hydrogel in Example 19 of the present disclosure
  • Example 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.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan with thiolated chitosan; ⁇ - ⁇ unsaturated acylate shell
  • Michael addition reaction of ⁇ - ⁇ unsaturated acylated chitosan with thiolated chitosan ⁇ - ⁇ unsaturated acylate shell
  • the general formula of glycans is:
  • the general formula of thiolated chitosan is:
  • 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
  • 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.
  • 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 ⁇ - ⁇ 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.
  • the alpha-beta unsaturated acylated chitosan 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.
  • 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.
  • 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.
  • the thiolated chitosan has the formula:
  • 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.
  • 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.
  • 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.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan solution with a thiolated chitosan solution.
  • the ⁇ - ⁇ unsaturated acylated chitosan solution is prepared by dissolving ⁇ - ⁇ unsaturated acylated chitosan in an acid solution at a concentration of 10 to 100 mg/ml.
  • a ⁇ -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.
  • the preparation of the chitosan hydrogel is carried out by the following steps:
  • 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.
  • 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.
  • 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.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan.
  • 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.
  • the reaction solution is subjected to dialysis and freeze-drying operation, and the small molecular impurities remaining in the ⁇ - ⁇ unsaturated acylated chitosan can be better removed to obtain a higher purity ⁇ - ⁇ unsaturated acylation.
  • Chitosan facilitates the subsequent Michael addition reaction to proceed better.
  • CS-SH Thiolated chitosan
  • 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.
  • thiolated chitosan (CS-SH) can be produced by the following method:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the acid solution during the preparation of the thiolated chitosan 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%.
  • 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.
  • the carboxyl activator comprises EDC and NHS
  • EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Michael addition reaction may be carried out by mixing an ⁇ - ⁇ unsaturated acylated chitosan solution with a thiolated chitosan solution and extruding into an alkali solution for reaction molding.
  • the ⁇ - ⁇ 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.
  • the ⁇ - ⁇ 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.
  • 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.
  • 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 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 such as 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
  • 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.
  • 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.
  • the ⁇ - ⁇ 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 is used to achieve rapid curing of the chitosan hydrogel to achieve 3D printability of the material.
  • 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.
  • PH-responsive chitosan hydrogel 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 ⁇ - ⁇ unsaturated acylated chitosan with thiolated chitosan is immersed in an ethanol solution to obtain a double crosslinked chitosan hydrogel.
  • the hydrogel obtained by reacting the alpha-beta unsaturated acylated chitosan with thiolated chitosan is the chitosan hydrogel of the foregoing disclosure.
  • a double crosslinked chitosan hydrogel is prepared by a chemical crosslinking and a physical crosslinking method.
  • 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 grafting ratio of the sugar derivative can effectively adjust the mechanical strength of the colloid.
  • 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.
  • 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.);
  • the soaking time is 24 hours.
  • reaction of the alpha-beta unsaturated acylated chitosan with the thiolated chitosan comprises:
  • the ⁇ - ⁇ unsaturated acylated chitosan is mixed with the thiolated chitosan, and then reacted with an alkali solution to obtain a hydrogel;
  • the ⁇ - ⁇ 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;
  • an acid solution preferably an organic acid solution, more preferably an acetic acid solution, particularly 0.1-30% (m/m) acetic acid solution.
  • 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));
  • 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)
  • the step of reducing the thiolated chitosan is further included;
  • the reducing treatment comprises: adding a reducing agent to the thiolated chitosan solution for reduction treatment;
  • the reducing agent comprises: zinc (metal zinc), dithiothreitol, and at least one of hydroquinone;
  • 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.);
  • the obtained product is extruded into an alkali solution to form a chemically crosslinked hydrogel;
  • 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;
  • 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.).
  • the preparation method further includes:
  • the thiolated chitosan reduction comprises the step of treating the thiolated chitosan with a reducing agent
  • the reducing agent comprises at least one of zinc, dithiothreitol, and hydroquinone.
  • the alpha-beta unsaturated acylated chitosan as a raw material for hydrogel preparation comprises a compound of 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 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.
  • a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms preferably having a carbon number of
  • 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.);
  • 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 atom
  • 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
  • R 5 when R 5 is a carbonyl group, the carbonyl structure is:
  • 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 alky
  • R 5 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
  • R 5 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 or unsub
  • the method for synthesizing the ⁇ - ⁇ unsaturated acylated chitosan comprises:
  • the chitosan is reacted with an acylating reagent to obtain an ⁇ - ⁇ unsaturated acylated chitosan;
  • the acylating agent comprises: at least one of an ⁇ - ⁇ unsaturated acid, an ⁇ - ⁇ unsaturated acid anhydride, an ⁇ - ⁇ unsaturated acid halide, and an ⁇ - ⁇ unsaturated ester;
  • 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;
  • the chitosan has a molecular weight of 50,000 and a degree of deacetylation of 80%.
  • the structure can be referred to as follows:
  • the ⁇ - ⁇ unsaturated acid comprises: ⁇ -methacrylic acid, and at least one of ⁇ -isopropylacrylic acid;
  • the ⁇ - ⁇ unsaturated acid anhydride includes: maleic anhydride;
  • the ⁇ - ⁇ unsaturated acid halide includes at least one of acryloyl chloride and methacryloyl chloride;
  • the ⁇ - ⁇ unsaturated ester includes at least one of methyl methacrylate and ethyl methacrylate;
  • the synthesis method further comprises the step of purifying the obtained ⁇ - ⁇ unsaturated acylated chitosan;
  • 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.
  • the method for synthesizing the ⁇ - ⁇ unsaturated acylated chitosan comprises:
  • the chitosan is reacted with an acylating reagent to obtain an ⁇ - ⁇ unsaturated acylated chitosan;
  • the acylating agent comprises: at least one of an ⁇ - ⁇ unsaturated acid, an ⁇ - ⁇ unsaturated acid anhydride, an ⁇ - ⁇ unsaturated acid halide, and an ⁇ - ⁇ unsaturated ester;
  • the synthesis method further comprises the step of purifying the obtained ⁇ - ⁇ unsaturated acylated chitosan.
  • the method for synthesizing the ⁇ - ⁇ unsaturated acylated chitosan comprises:
  • 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.);
  • the product is subjected to dialysis (preferably dialysis 2-4d), dried (preferably by freeze-drying) to obtain ⁇ - ⁇ unsaturated acylated chitosan;
  • the concentration of the chitosan solution is 10 to 100 mg/ml
  • 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);
  • 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);
  • a polar solvent such as acetone, methyl ethyl ketone, water, DMSO and DMF (particularly acetone);
  • the molar ratio of chitosan to acylating agent is from 1:1 to 1:3.
  • the thiolated chitosan as a raw material for the preparation of the hydrogel comprises a compound of the following 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;
  • 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.
  • 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
  • R 6 is a substituted alkyl, substituted alkoxy, substituted aryl or substituted heteroaryl
  • 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).
  • 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.
  • 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;
  • the different substituents may be optionally the same or different.
  • the method for synthesizing the thiolated chitosan comprises:
  • the chitosan is reacted with a thiolation reagent to obtain a thiolated chitosan
  • the thiolation reagent comprises: a compound having a thiol group and a carboxyl group;
  • the thiolation reagent comprises at least one of: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioacetic acid, and 2-mercapto-3-pyridinecarboxylic acid;
  • the synthetic method further comprises the step of purifying the obtained thiolated chitosan.
  • the method for synthesizing the thiolated chitosan comprises:
  • the chitosan is reacted with a thiolation reagent to obtain a thiolated chitosan
  • the thiolation reagent comprises: a compound having a thiol group and a carboxyl group;
  • the thiolation reagent comprises at least one of: dimercaptosuccinic acid, mercapto succinic acid, mercaptopropionic acid, thioglycolic acid, and 2-mercapto-3-pyridinecarboxylic acid;
  • 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;
  • the chitosan has a molecular weight of 50,000 and a degree of deacetylation of 80%;
  • the synthetic method further comprises the step of purifying the obtained thiolated chitosan
  • 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;
  • the thiolated chitosan is reacted in the presence of a carboxyl activator
  • the carboxyl activator comprises: EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHC (N-hydroxysuccinimide);
  • the thiolation reagent comprises: a compound having both a carboxyl group and a thiol group;
  • 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;
  • the molar ratio of chitosan to thiolation reagent is from 1:1 to 10:1.
  • the method for synthesizing the thiolated chitosan comprises:
  • 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%;
  • the concentration of the obtained chitosan solution is 10 to 100 mg/ml
  • 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%);
  • 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
  • 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%
  • 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.
  • the present disclosure also provides an application of the disclosed double crosslinked chitosan hydrogel in the preparation of biological materials
  • present disclosure can also provide a biomaterial comprising the double crosslinked chitosan hydrogel of the present disclosure
  • Biomaterials as described above preferably include biofibers.
  • R is an alkylene group or a substituted alkylene group.
  • the substituted alkylene group when R is a 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.
  • 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.
  • 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.
  • 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 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.
  • R has from 1 to 20 carbon atoms.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • the chitosan thiolated derivatives of the presently disclosed embodiments can be prepared by the following methods:
  • 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.
  • 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.
  • 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.
  • the solution containing the chitosan is an acid solution.
  • 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.
  • the solution containing the sulfonic acid group compound is a double distilled water or an alkali solution or an acid solution.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • steps (2) and (3) may be performed first, and then step (1) may be performed without affecting the formation of the final product.
  • 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.
  • the carboxyl activator comprises EDC and NHS, and the mass ratio of the EDC to the NHS is from 10:1 to 1:1.
  • 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.
  • the chitosan thiolated derivative is produced by the following reaction formula:
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 1 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.
  • 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.
  • concentration of the two 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.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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).
  • CS-SH product thiolated chitosan
  • CS-SH 60 mg 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;
  • 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.
  • Example 8 The reaction formula of Example 8 is shown in Fig. 5, and the reaction scheme is shown in Fig. 6.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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).
  • CS-SH product thiolated chitosan
  • CS-SH 120 mg 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 MCS solution was mixed with the reduced-treated CS-SH solution, and then extruded into a NaOH solution to obtain a chemically crosslinked hydrogel.
  • chitosan having a molecular weight of about 50,000 and a deacylation degree of about 80%
  • a 0.30% (m/m) acetic acid solution stirred uniformly and then ultrasonicated for 35 min;
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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;
  • 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).
  • CS-SH product thiolated chitosan
  • CS-SH 60 mg 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 MCS solution was mixed with the reduced-treated CS-SH solution, and then extruded into a NaOH solution to obtain a chemically crosslinked hydrogel.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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;
  • 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).
  • CS-SH product thiolated chitosan
  • CS-SH 120 mg 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 MCS solution was mixed with the reduced-treated CS-SH solution, and then extruded into a NaOH solution to obtain a chemically crosslinked hydrogel.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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;
  • 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).
  • CS-SH product thiolated chitosan
  • CS-SH 60 mg 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 MCS solution was mixed with the reduced-treated CS-SH solution, and then extruded into a NaOH solution to obtain a chemically crosslinked hydrogel.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5 g 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;
  • 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).
  • CS-SH product thiolated chitosan
  • CS-SH 60 mg 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;
  • 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.
  • 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 ⁇ - ⁇ unsaturated acylated chitosan (abbreviated as MCS).
  • MCS product ⁇ - ⁇ unsaturated acylated chitosan
  • chitosan 1.5g 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).
  • CS-SH product thiolated chitosan
  • 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 unmodified chitosan was immersed in absolute ethanol for 24 hours to obtain a physically crosslinked hydrogel, which was recorded as CSH-E10;
  • 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.
  • 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;
  • 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;
  • 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;
  • 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 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;
  • 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;
  • 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.
  • 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.
  • reaction equation of the chitosan thiolated derivative in this example is:
  • reaction equation of the chitosan thiolated derivative in this example is:
  • reaction equation of the chitosan thiolated derivative in this example is:
  • reaction equation of the chitosan thiolated derivative in this example is:
  • 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.
  • 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
  • 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.
  • Example 19 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.
  • 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.
  • the chitosan thiolated derivative is chemically reacted with other polymer derivatives containing a structure such as maleimide, vinyl sulfone, ⁇ - ⁇ unsaturated aldehyde, ketone, acid, ester, etc. to prepare rapid curing.
  • Hydrogels have important applications in the fields of regenerative medicine and tissue engineering.
  • 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 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.
  • 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.
  • 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 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, ⁇ - ⁇ 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.

Abstract

La présente invention concerne un dérivé de chitosane thiolé, un hydrogel de chitosane, un hydrogel de chitosane réticulé double, et des procédés de préparation associés et des applications associées. Des structures alpha-bêta insaturées sont respectivement greffées sur des chaînes de molécule de chitosane, des groupes thiol sont greffés sur les chaînes de molécule de chitosane, et un hydrogel de chitosane est obtenu au moyen d'une réaction d'addition de Michael. Un hydrogel obtenu par réaction d'un chitosane acylé alpha-bêta insaturé avec un chitosane thiolé est immergé dans une solution d'éthanol pour obtenir un hydrogel de chitosane réticulé double. Le chitosane est utilisé comme matière première, et un composé sulfo ayant à la fois des groupes sulfo et des groupes carboxyle est utilisé en tant qu'agent de modification. Sous l'effet d'un agent d'activation de groupe carboxyle, et avec l'effet de retrait d'électrons forts des groupes sulfo, les groupes sulfo sont introduits avec succès dans les groupes amino et les groupes hydroxyle primaires du chitosane, et les groupes sulfo sont ensuite convertis en groupes thiol par réduction pour obtenir le dérivé de chitosane thiolé de la présente invention. L'hydrogel de la présente invention présente une vitesse de durcissement rapide, une résistance mécanique élevée, une bonne biocompatibilité, une bonne stabilité dans des milieux de culture, et une large gamme d'applications.
PCT/CN2019/089538 2018-04-03 2019-05-31 Dérivé de chitosane thiolé, hydrogel de chitosane, et procédés de préparation associés et applications associées WO2019192628A2 (fr)

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CN112940291A (zh) * 2021-02-05 2021-06-11 中国科学技术大学 一种基于壳聚糖的透明水凝胶及其制备方法
CN114507916A (zh) * 2022-04-18 2022-05-17 中国科学院苏州纳米技术与纳米仿生研究所 具有沟槽拓扑结构的壳聚糖微纤维及其制备方法与应用

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KR100849185B1 (ko) * 2006-01-19 2008-07-30 서울산업대학교 산학협력단 키토산 또는 히알루론산-폴리에틸렌옥사이드 및키토산-히알루론산-폴리에틸렌옥사이드를 기저로 하는하이드로젤과　 이의 제조방법
CN104958783B (zh) * 2015-06-19 2017-08-08 暨南大学 一种天然多糖基水凝胶及制备和在眼结膜修复中的应用
CN105536046B (zh) * 2016-02-26 2018-08-07 闫策 一种含双硫键的可注射骨水泥及制备方法
CN105754016B (zh) * 2016-03-09 2019-07-16 沈阳药科大学 一种生物粘附性巯基化壳聚糖的合成方法
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CN112940291A (zh) * 2021-02-05 2021-06-11 中国科学技术大学 一种基于壳聚糖的透明水凝胶及其制备方法
CN112940291B (zh) * 2021-02-05 2024-02-09 中国科学技术大学 一种基于壳聚糖的透明水凝胶及其制备方法
CN114507916A (zh) * 2022-04-18 2022-05-17 中国科学院苏州纳米技术与纳米仿生研究所 具有沟槽拓扑结构的壳聚糖微纤维及其制备方法与应用

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