WO2019192628A2 - 壳聚糖巯基化衍生物、壳聚糖水凝胶及其制备方法与应用 - Google Patents
壳聚糖巯基化衍生物、壳聚糖水凝胶及其制备方法与应用 Download PDFInfo
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- 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.
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Abstract
本公开涉及壳聚糖巯基化衍生物、壳聚糖水凝胶、双交联壳聚糖水凝胶及其制备方法与应用。分别在壳聚糖分子链上接枝α-β不饱和结构,在壳聚糖分子链接枝巯基基团,然后利用Michael加成反应得到壳聚糖水凝胶。将α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶在乙醇溶液中浸泡处理,得到双交联壳聚糖水凝胶。以壳聚糖为原料,以同时带有磺酸基和羧基的磺酸基化合物作修饰剂,在羧基活化剂的作用下,依靠磺酸基的强吸电子作用,在壳聚糖分子链的氨基和伯羟基上成功引入磺酸基,并进一步通过还原将磺酸基转化为巯基得到本公开的壳聚糖巯基化衍生物。本公开的水凝胶固化速度快,力学强度高,生物相容性好,在培养基中的稳定性好,应用范围大。
Description
相关申请的交叉引用
本申请要求于2018年04月3日提交中国专利局的申请号为2018102917441、名称为“壳聚糖水凝胶及其制备方法与应用”的中国专利申请,于2018年04月3日提交中国专利局的申请号为2018102910635、名称为“壳聚糖巯基化衍生物及其制备方法和应用”的中国专利申请,以及于2019年05月23日提交中国专利局的申请号为201910436288X、名称为“一种双交联壳聚糖水凝胶及其制备方法和应用”的中国专利申请的优先权,其每个文件的全部内容通过引用结合在本申请中。
本公开涉及生物材料技术领域,具体而言,涉及壳聚糖巯基化衍生物、壳聚糖水凝胶、双交联壳聚糖水凝胶及其制备方法与应用。
3D打印,也称增材制造,在很多领域引起了巨大变革,如工程学、制造工业、教育、医学。3D生物打印使将生物相容性材料、细胞以及辅助性成分组装成具有三维功能活性的人体器官或组织成为可能。与非生物打印相比,3D生物打印更为复杂,涉及生物相容性材料的选择、细胞类型、生长、分化因素的考虑以及组织构建等问题,其中材料的选择至关重要。
水凝胶是一类具有亲水基团,能吸收大量水分溶胀但不溶于水的三维网状聚合物,因其与细胞外基质类似的组成和结构,适合各种不同细胞的粘附、生长、增殖和分化,成为3D生物打印构建人体组织和器官的重要材料。目前大部分3D生物打印水凝胶存在固化速度较慢、胶体机械强度和韧性较小或不可调控等问题,大大降低了其可打印性和应用范围。
壳聚糖是自然界中已知的唯一碱性多糖,具备良好的生物相容性、生物降解性、无细胞毒性,被广泛应用于组织工程和再生医学领域。壳聚糖分子链上含有丰富的氨基基团,具备较好的化学活性。
壳聚糖分子以其独特的分子结构和理化性质在机体内发挥着重要的作用,临床上也得到了广泛应用。但现有的壳聚糖分子不能够与含马来酰亚胺、乙烯砜、α-β不饱和醛、酮、酸、酯等结构的其他高分子衍生物发生化学反应,来制备快速固化的水凝胶。
高分子水凝胶材料是一种能够迅速吸收并保持水分,同时又不会溶于水的低交联度材料,其具有高分子电解质特性以及三维网络结构,是一种集吸水、保水、缓释等功能于一体的功能高分子材料。
壳聚糖是由甲壳素脱乙酰基所得到的一种具有广泛应用价值的天然生物多糖材料,其具有无毒,生物相容性好,生物可降解,黏膜黏附性和抗菌性等优异的生物特性,是制备水凝胶的理想材料。
如何将壳聚糖分子间进行有效的交联以得到对应的壳聚糖水凝胶材料,则是目前研究的热点所在,也有很多研究人员在相同或者相关领域做出了一些探索。例如,现有技术中就公开了一种采用物理交联和化学交联结合的方法以制备类似的几丁质水凝胶的方法,该方法中,首先以环氧氯丙烷作为交联剂,进行化学交联,然后将产物置于乙醇中进行第二步交联,得到一种高强度且强度可调的几丁质水凝胶。如上现有技术采用的制备方法虽然能够实现几丁质分子间的交联,然而,该方法中所用交联剂环氧氯丙烷有较大毒性,且交联反应发生后残留的交联剂嵌入在胶体中难以清除掉。同时,该方法中第一步的化学交联反应时间较长,无法实现快速成型。而且,该方法所制得的胶体最大压缩模量仅高达260KPa,最大断裂强度达3.98MPa,难以满足作为高强度水凝胶应用的性能要求。
发明内容
本公开的目的包括例如提供一种壳聚糖水凝胶的制备方法,其制备工艺简单,能够有效地制备得到固化速度快、生物相容性好、机械强度可调、在培养基中的稳定性好以及生物降解速度可调的壳聚糖水凝胶。
本公开的目的包括例如提供一种壳聚糖水凝胶,其固化速度快,生物相容性好、机械强度可调、在培养基中的稳定性好,生物降解速度可调,应用范围大。
本公开的目的包括例如提供上述壳聚糖水凝胶在制作生物医用材料或组织工程材料或3D生物打印 材料上的应用。
本公开的目的包括例如提供一种双交联壳聚糖水凝胶的制备方法,本公开中,利用带有不同官能基团的壳聚糖反应交联,不仅反应交联速度较快,而且能够避免化学交联剂的使用,同时所得到的双交联壳聚糖具有良好的力学性能。
本公开的目的包括例如提供一种由本公开制备方法所得到的双交联壳聚糖水凝胶。
本公开的目的包括例如提供一种本公开双交联壳聚糖的应用。
本公开的目的包括例如提供一种壳聚糖巯基化衍生物,其通过将壳聚糖分子中的氨基和伯羟基进行衍生引入巯基进行改性得到,该壳聚糖巯基化衍生物具有很好的亲核性能、抗氧化性能,并且可以进一步通过亲核反应、交联反应等进行衍生,应用范围十分广泛。
本公开的目的包括例如提供上述壳聚糖巯基化衍生物的制备方法,其路线设计合理,制作方法简单,对设备需求低,能够快速高效地得到壳聚糖巯基化衍生物。
本公开的目的包括例如提供上述壳聚糖巯基化衍生物在制备水凝胶中的应用,使得制备的水凝胶具有快速固化、生物相容性好以及机械强度可调的性能。
其中,R
1为壳聚糖高分子去除氨基的残基部分;R
2为氢原子、烷基或亚烷基;R
3为羰基、羧基、酯基、酰胺、烷基或取代烷基;R
4为亚烷基或取代亚烷基。
一种壳聚糖水凝胶,其由上述壳聚糖水凝胶的制备方法制备得到,其通式为:
其中,R
1为壳聚糖高分子去除氨基的残基部分;R
2为氢原子、烷基或亚烷基;R
3为羰基、羧基、酯基、酰胺、烷基或取代烷基;R
4为亚烷基或取代亚烷基。
上述壳聚糖水凝胶在制作生物医用材料或组织工程材料或3D生物打印材料上的应用。
本公开也提供了一种高韧性、高强度以及可快速成型的双交联壳聚糖水凝胶的制备方法,所述制备方法包括:将α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶在乙醇溶液中浸泡处理,得到双交联壳聚糖水凝胶。
同时,本公开还提供了由本公开方法所得到的双交联壳聚糖水凝胶。
本公开也提供了本公开双交联壳聚糖水凝胶在生物材料中的应用;以及/或者,包含本公开双交联壳聚糖水凝胶的生物材料。
本公开提供了一种壳聚糖巯基化衍生物,其由壳聚糖与磺酸基化合物反应再还原生成,其通式为:
其中,R为亚烷基或取代亚烷基。
一种上述壳聚糖巯基化衍生物的制备方法,壳聚糖与磺酸基化合物在羧基活化剂的存在下进行反应;再通过还原剂将磺酸基还原为巯基;磺酸基化合物为同时具有羧基和磺酸基的化合物。
上述壳聚糖巯基化衍生物在制备水凝胶中的应用。
本公开实施例的壳聚糖水凝胶及其制备方法的有益效果至少包括:通过将酰化试剂酰化的壳聚糖与巯基化的壳聚糖作为原材料,通过Michael加成反应可实现原材料由液态到固态的快速转变,大大提高了材料的可打印性,通过调节二者的浓度及壳聚糖衍生物的接枝率,可实现固化后壳聚糖水凝胶弹性模量的调节,可扩大材料的应用范围。该方法制备的壳聚糖水凝胶在生物医学及组织工程领域具有重要用途,其固化速度快,生物相容性好、机械强度可调、在培养基中的稳定性好,生物降解速度可调,应用范围大。
本公开中,利用带有不同官能基团的壳聚糖反应进行化学交联,同时通过乙醇处理以进行物理交联,从而得到了一种既包含化学交联、又包含物理交联结构的双交联壳聚糖水凝胶,不仅制备方法简便、快捷,而且制备反应速度较快,同时无需使用毒性较强的化学交联剂,制备过程绿色、环保、安全。
同时,由本公开方法所得到的双交联壳聚糖水凝胶的固化速度快,力学强度高、生物相容性好、在培养基中的稳定性好,应用范围大。与目前常见的紫外光固化壳聚糖水凝胶、pH响应壳聚糖水凝胶、温敏型壳聚糖水凝胶、以及离子响应壳聚糖水凝胶等相比,本公开双交联壳聚糖水凝胶既提高了固化速度,也改善了壳聚糖水凝胶的机械强度和弹性。
本公开实施方式的壳聚糖巯基化衍生物及其制备方法的有益效果至少包括:该制备方法通过将同时含有羧基和磺酸基的化合物作为修饰剂,在羧基活化剂的作用下,在壳聚糖分子的氨基和伯羟基上成功引入磺酸基,再在还原剂的作用下将磺酸基还原成巯基。该制备方法的路线设计合理,操作简单可行,对设备要求低,能够高效高产率地得到壳聚糖巯基化衍生物。该壳聚糖巯基化衍生物由于巯基侧链的作用,而具有很好的亲核性能、抗氧化性能以及丰富的衍生性,其可以进一步通过亲核反应、交联反应等进行衍生,应用范围十分广泛。
本公开实施方式的壳聚糖巯基化衍生物在制备水凝胶上的应用的有益效果至少包括:改性后的形成的壳聚糖巯基化衍生物在碱性条件下巯基质子离去可产生硫负离子,可与含马来酰亚胺、乙烯砜、α-β不饱和醛、酮、酸、酯等结构的其他高分子衍生物发生化学反应来制备得到水凝胶,提高了水凝胶的固化速度,也改善了水凝胶的机械强度和弹性。
为了更清楚地说明本公开实施方式的技术方案,下面将对实施方式中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本公开的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本公开实施例1中的壳聚糖、巯基化壳聚糖与α-β不饱和酰基化壳聚糖的傅里叶红外图谱(FTIR)对比分析图;
图2为本公开实施例1中的壳聚糖水凝胶的扫描电子显微镜(SEM)表面微观结构图;
图3为本公开实施例1中的壳聚糖水凝胶的表面孔径扫描电子显微镜(SEM)图;
图4为本公开实施例1中的壳聚糖水凝胶力学测试曲线图。
图5为本公开实施例8中双交联壳聚糖制备的反应式;
图6为本公开实施例8中双交联壳聚糖制备的反应流程图;
图7为根据实施例8的方法,得到的不同水凝胶材料的力学性能测试图;
图8为根据实施例8的方法,得到的不同水凝胶材料的力学性能测试图;
图9为实验例3中双交联壳聚糖生物纤维的扫描电镜图。
图10为本公开实施例15中的壳聚糖与壳聚糖巯基衍生物傅里叶红外图谱(FTIR)对比分析图,其中,CS表示壳聚糖,TCS表示壳聚糖巯基化衍生物;
图11为本公开实施例19中的固化后水凝胶的扫描电子显微镜(SEM)表面微观结构图;
图12为本公开实施例19中的固化后水凝胶的表面孔径扫描电子显微镜(SEM)表面微观结构图;
图13为本公开实施例19中的固化后水凝胶的力学测试曲线图。
为使本公开实施例的目的、技术方案和优点更加清楚,下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
下面对本公开实施例的壳聚糖水凝胶及其制备方法进行具体说明。
其中,R
1为壳聚糖高分子去除氨基的残基部分;R
2为氢原子、烷基或亚烷基;R
3为羰基、羧基、酯基、酰胺、烷基或取代烷基;R
4为亚烷基或取代亚烷基。
壳聚糖(CS),又称脱乙酰壳多糖,是甲壳素脱乙酰化的产物,是一种应用广泛的天然多糖。壳聚糖是自然界唯一的碱性多糖,其分子链上的游离氨基氮原子上具有一对未共用电子对,能结合一个氢质子,从而使壳聚糖成为带正电荷的聚电解质。壳聚糖具有良好的生物相容性、生物降解性、杀菌性等,是一种安全的天然高分子聚合物。壳聚糖分子上的游离氨基具有较高的化学活性,易于被羧基等活性较高的官能团修饰,利用酰化试剂对壳聚糖进行修饰,可得到水溶性壳聚糖,同时在壳聚糖分子链上引入了α-β不饱和羰基结构,可以被亲核试剂进攻发生加成反应,成功实现聚合物的交联,达到快速固化的效果。
R
3为羰基、羧基、酯基、酰胺、烷基或取代烷基。其中,取代烷基可以为至少一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中至少一种基团取代的烷基。即可以是烷基中有一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的一种基团取代;也可以是烷基中有两个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的两种基团取代;也可以是烷基中 有两个以上的氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的两种以上的基团取代;也可以是烷基中多个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的多个同一种基团取代或被其中多个不同基团的组合对应进行取代。
壳聚糖分子链上存在游离氨基基团,用带巯基的化合物去修饰氨基,可将壳聚糖巯基化,修饰后的壳聚糖具备巯基的化学性质,在碱性条件下巯基质子离去可产生硫负离子,作为亲核试剂进攻用酰化试剂酰化的壳聚糖,以C-S键的形式将壳聚糖和MCS快速接枝在一起,生成网状复合壳聚糖水凝胶。
其中,R
4为亚烷基或取代亚烷基。取代亚烷基可以为至少一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中至少一种基团取代的亚烷基。即可以是亚烷基中有一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的一种基团取代;也可以是亚烷基中有两个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的两种基团取代;也可以是亚烷基中有两个以上的氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的两种以上的基团取代;也可以是亚烷基中多个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基和卤代烷基中的多个同一种基团取代或被其中多个不同基团的组合对应进行取代。
其中,R
4的碳原子数均可为1~20个。即R
4可以为C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20的亚烷基或取代亚烷基。
由于分子链上存在游离的氨基,从而可以通过与羧基进行反应,达到对壳聚糖分子链进行修饰改性的目的。与壳聚糖发生反应的巯基化合物为同时具有羧基和巯基的化合物;上述巯基化合物可以通过经水解反应、氨解反应等直接生成,或者可通过含二硫键的化合物经还原后得到。例如,同时具有羧基和巯基的化合物可以为:二巯基丁二酸、巯基丁二酸、巯基丙酸、硫代乙醇酸、2-巯基-3-吡啶甲酸等。
本公开的一些实施方式还提供一类壳聚糖水凝胶的制备方法,包括:将α-β不饱和酰基化壳聚糖溶液与巯基化壳聚糖溶液进行Michael加成反应。
在一种或多种实施方式中,α-β不饱和酰基化壳聚糖溶液是将α-β不饱和酰基化壳聚糖溶解在酸溶液而配成的浓度为10~100mg/ml的α-β不饱和酰基化壳聚糖酸溶液;巯基化壳聚糖溶液是将巯基化壳聚糖溶解在酸溶液中而配成的10~100mg/ml的巯基化壳聚糖酸溶液。通过上述浓度以及其溶剂的设置,使得α-β不饱和酰基化壳聚糖以及巯基化壳聚糖能够很好的进行溶解,并且能够更加有利于二者之间的Michael加成反应的进行。
在一种或多种实施方式中,壳聚糖水凝胶的制备通过以下步骤进行:
(1)制备α-β不饱和酰基化壳聚糖(MCS)。
以α-β不饱和酸作为酰化试剂为例,α-β不饱和酰基化壳聚糖的化学反应式为:
在一种或多种实施方式中,将壳聚糖溶解在酸溶液中,将酰化试剂溶解于极性溶剂中,壳聚糖链上游离氨基与酰化试剂的羧基摩尔比优选为1:1~3,将溶解的酰化试剂在磁力搅拌器上缓慢加入壳聚糖酸溶液中,室温下充分混合均匀,在10~90℃条件下反应,优选为40~90℃,反应时间2~10h。反应结束后经过透析2~4天,冷冻干燥2~4天,得到修饰后壳聚糖产物。
在一种或多种实施方式中,所述含有壳聚糖的酸溶液和所述含有酰化试剂的溶液进行反应的反应温度为10~90℃,优选为40~90℃,反应时间为2~10h。
在一种或多种实施方式中,溶解壳聚糖的酸溶液可以是有机酸溶液,优选乙酸溶液,更优选地,乙酸溶液的质量分数为0.01~30%。溶解酰化试剂的极性溶剂包括丙酮、丁酮、水、DMSO、DMF等,优选丙酮,且丙酮的用量为保证将酰化试剂完全溶解即可。通过将壳聚糖链上游离氨基与酰化试剂的羧基摩尔比设置为1:1~3,使得酰化试剂的酸酐能够更好的与壳聚糖链上游离氨基进行反应,从而对壳聚糖修饰得到α-β不饱和酰基化壳聚糖。同时,将溶解的酰化试剂在磁力搅拌器上搅拌,并缓慢加入壳聚糖酸溶液中,能够使得二者能够更加充分地进行接触,进而达到更好的反应效果。在反应结束后对反应液进行透析和冷冻干燥操作,可以将α-β不饱和酰基化壳聚糖中残留的小分子杂质更好的去除,以得到纯度更高的α-β不饱和酰基化壳聚糖,进而促进后续Michael加成反应的更好进行。
(2)制备巯基化壳聚糖(CS-SH)。
巯基化壳聚糖(CS-SH)由以下反应式生成:
在一种或多种实施方式中,巯基化壳聚糖(CS-SH)的制备方法是将含有壳聚糖的溶液与含有巯基化合物的溶液在羧基活化剂的作用下进行反应。
在一种或多种实施方式中,当采用含有羧基的巯基化合物作为原料时,巯基化壳聚糖(CS-SH)可以通过以下方法制备:
a.制备含有壳聚糖的溶液:将壳聚糖溶解于质量分数为0.01~30%的酸溶液中。
b.制备含巯基化合物的溶液:将巯基化合物,根据其溶解性,溶解于双蒸水或碱溶液或酸溶液中。
c.将羧基活化剂加入到上述含巯基化合物的溶液中,混合均匀后,调节其PH值至4.5~6.5,继续混合搅拌。
d.将活化后的含有巯基化合物的溶液与含有壳聚糖的溶液进行混合,巯基化合物与壳聚糖上氨基的摩尔比为1~10:1,充分搅拌均匀,优选转移至圆底烧瓶中,放置在温度为50~60℃以下进行恒温反应。
e.将反应后得到的反应液进行透析2~5天,再将得到的透析产物进行冷冻干燥2~5天后,得到巯基化壳聚糖。通过透析的手段可以除去残留的小分子杂质,提高所得的壳聚糖巯基化衍生物的纯度。
需要说明的是,上述过程中也可以先进行步骤b和c,再进行步骤a,其不会影响最终产物的生成。
在一种或多种实施方式中,巯基化壳聚糖(CS-SH)制备过程中的酸溶液可以是有机酸溶液,优选乙酸溶液。即优选将壳聚糖溶解于质量分数为0.01~30%的乙酸溶液中。
在一种或多种实施方式中,碱溶液可以为强碱溶液,例如氢氧化钠溶液、氢氧化钾溶液、氢氧化钙溶液等,也可以为弱碱溶液,例如氨水溶液、碳酸钠溶液、碳酸氢钠溶液等,优选氢氧化钠溶液。双蒸水可以使得溶解后的溶液杂质含量少,纯度更高,后续反应效果越好。溶解巯基化合物的酸溶液可以是有机酸溶液,优选乙酸溶液。
在一种或多种实施方式中,羧基活化剂包括EDC和NHS,EDC即1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐,EDC是可溶于水的碳二亚胺,在酰胺合成中用作羧基的活化试剂,也用于活化磷酸酯基团、蛋白质与核酸的交联和免疫偶联物的制取,常和N-羟基琥珀酰亚胺(NHS)或N-羟基硫代琥珀酰亚胺连用,以提高偶联效率。NHS即N-羟基琥珀酰亚胺,活化羧基以利于酰胺键的形成。用于合成氨基酸保护剂、半合成卡那霉素及医药中间体。本公开实施例中的羧基活化剂可按照EDC和NHS的质量比为1~10:1的比例进行配制,这种比例有利于二者达到更好的偶联效率,使得对巯基化合物的羧基活化效果更好。
在一种或多种实施方式中,可以将EDC和NHS在磁力搅拌器作用下加入到上述含巯基化合物的溶液中,以达到更好的混合效果。
在一种或多种实施方式中,混合羧基活化剂后,用碱或酸溶液调节加有羧基活化剂的巯基化合物溶液的PH值,使得其PH值为4.5~6.5。优选地,用1M氢氧化钠或1M盐酸溶液进行调节PH值。EDC和NHS在PH值为4.5~6.5的条件下能够达到对羧基最好的活化效果。
在一种或多种实施方式中,调节PH值后混合搅拌的时间为10~150min,使得羧基活化剂能够对巯基化合物的羧基进行充分的活化,以更好地对壳聚糖进行修饰。
其中,反应温度优选55~60℃,进一步优选55℃,反应时间可以为1~8h,优选2~6h,更优选地4~5h。
(3)将α-β不饱和酰基化壳聚糖溶液与巯基化壳聚糖溶液进行Michael加成反应。
Michael加成反应的反应式为:
在一种或多种实施方式中,进行Michael加成反应可以是将α-β不饱和酰基化壳聚糖溶液与巯基化壳聚糖溶液混合后挤出至碱溶液中反应成型。将α-β不饱和酰基化壳聚糖溶液与巯基化壳聚糖溶液使得二者按照一定比例进行充分混合,从而更加有利于反应的进行以及对生成的壳聚糖水凝胶的性能的调节。
在一种或多种实施方式中,首先,将步骤(1)得到的α-β不饱和酰基化壳聚糖溶解在质量分数为0.1~30%的酸溶液中,配成浓度10~50mg/ml的溶液;其中,酸溶液可以有机酸溶液,优选乙酸溶液。
其次,将步骤(2)得到的巯基化壳聚糖溶解在酸溶液中配成10~50mg/ml的溶液,并将溶液中的巯基化壳聚糖经还原剂处理。
还原剂为金属Zn、二硫苏糖醇或对苯二酚。进行还原处理的时间优选5~50min。
然后,将配置的酰化试剂酰化壳聚糖溶液和巯基化壳聚糖溶液充分混合,并挤出至碱溶液中反应成型,实现新型3D打印壳聚糖水凝胶的制备。
其中,碱溶液可以为强碱溶液,例如氢氧化钠溶液、氢氧化钾溶液、氢氧化钙溶液等,也可以为弱碱溶液,例如氨水溶液、碳酸钠溶液、碳酸氢钠溶液等,优选氢氧化钠溶液。
值得注意的是,壳聚糖本身在碱溶液中会析出得到的不溶物,但是,壳聚糖的析出是由于碱溶液改变其电荷分布而聚集析出的不溶物,在用酸中和掉碱后不溶物会重新溶解。相比之下,本公开实施例制备得到的壳聚糖水凝胶因为是化学交联,性质稳定,在强酸中不会溶解。这一点间接说明了本公开实施例提供的壳聚糖水凝胶在形成过程中成功发生了加成反应,而不是简单的物理变化。
上述制备过程分别在壳聚糖分子链上接枝α-β不饱和酰基化结构,在壳聚糖分子链接枝巯基基团,通过对两种生物材料的活性结构进行修饰,利用Michael加成反应,以化学交联机制来实现壳聚糖水凝胶的快速固化,以实现材料的3D可打印性,并通过改变经修饰后材料的浓度及壳聚糖衍生物的接枝率来调节壳聚糖水凝胶的力学强度及弹性模量。本公开实施例制备的3D生物打印壳聚糖水凝胶固化速度快,生物相容性好、力学强度可调、在培养基中的稳定性好,生物降解速度可调,应用范围大。其与目前的紫外光固化壳聚糖水凝胶、PH响应壳聚糖水凝胶、温敏型壳聚糖水凝胶、离子响应壳聚糖水凝胶等相比,既提高了固化速度,也改善了壳聚糖水凝胶的机械强度和弹性,使得3D打印的壳聚糖水凝胶 具备良好的支撑性和保真性,防止胶体在短时间内坍塌和极大程度的形变。本公开还提供一种新的巯基与α-β不饱和结构接枝的产物,该产物在生物医学及组织工程领域具有重要用途。
本公开的一些实施方式还提供了上述壳聚糖水凝胶在制作生物医用材料或组织工程材料或3D生物打印材料上的应用。
本公开所提供的双交联壳聚糖水凝胶,是在前期壳聚糖水凝胶研究(具体可参见CN201810291744.1)的基础上,进一步采用乙醇处理的方法,所得到的具有化学交联和物理交联微观结构的双交联壳聚糖水凝胶材料,相较于现有技术中的双交联几丁质水凝胶而言,不仅制备反应更为快捷,而且无需使用具有较高毒性的交联剂,而且所得到的水凝胶材料力学性能更为优异。
本公开一方面提供了一种双交联壳聚糖水凝胶的制备方法,本公开所提供的制备方法主要包括:
将α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶在乙醇溶液中浸泡处理,得到双交联壳聚糖水凝胶。
在一种或多种实施方式中,所述α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶为本公开前述的壳聚糖水凝胶。
如上的制备方法中,采用化学交联和物理交联方法制备双交联壳聚糖水凝胶。不同于现有技术中所采用的以环氧氯丙烷为交联剂的制备方法,本公开中第一步交联是采用巯基点击加成的方式进行,加成反应前利用马来酸酐和巯基丁二酸分别对壳聚糖进行化学改性,利用改性后的壳聚糖衍生自身互为交联剂,在催化剂的催化下发生加成反应,以实现化学交联。巯基的点击加成反应立体选择性高,反应速度快,可以实现快速成型,便于各种三维结构的制备;第二步交联采用乙醇处理,可以提高水凝胶分子侧链的分子间氢键作用力以及分子主链的疏水作用力,从而大大提高壳聚糖水凝胶的力学强度,通过改变乙醇的浓度(可以以浓度:0%<vol%≤100%的乙醇作为处理试剂)以及壳聚糖衍生物的接枝率,可以有效调节胶体力学强度。进一步的,由本公开方法所得到的壳聚糖水凝胶力学强度最大断裂强度可达10.8MPa,最大弹性模量可达1.32MPa。
在一种或多种实施方式中,化学交联所得水凝胶在乙醇溶液中的浸泡处理的时间(t)为0<t≤48h(例如可以为,但不限于14,16,20,24,30,32,36或者42h等);
在一种或多种实施方式中,所述浸泡处理的时间为24h。
在一种或多种实施方式中,α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应包括:
在溶液条件下,将α-β不饱和酰基化壳聚糖与巯基化壳聚糖混合反应,然后与碱溶液反应,得到水凝胶;
作为优选,此步骤中,是将α-β不饱和酰基化壳聚糖溶于酸溶液(优选为有机酸溶液,更优选为乙酸溶液,特别是0.1-30%(m/m)的乙酸溶液)中;
作为优选,此步骤中,是将巯基化壳聚糖溶于酸溶液(优选为有机酸溶液,更优选为乙酸溶液,特别是0.1-30%(m/m)的乙酸溶液)中;
作为优选,此步骤中,还包括对巯基化壳聚糖进行还原处理的步骤;
优选的,所述还原处理包括:向巯基化壳聚糖溶液中加入还原剂,进行还原处理;
更优选的,所述还原剂包括:锌(金属锌),二硫苏糖醇,以及对苯二酚中的至少一种;
更优选的,所述还原处理的时间为5-50min(例如可以为,但不限于10、15、20、25、30、35、40,或者45min等);
作为优选,此步骤中,是将α-β不饱和酰基化壳聚糖与巯基化壳聚糖混合反应后,所得产物挤出至碱溶液中反应成型,得到化学交联的水凝胶;
在一种或多种实施方式中,所述用以成型的碱溶液可以为:例如氢氧化钠、氢氧化钾,或者氢氧化钙溶液等强碱性溶液;或者为:氨水、碳酸钠、碳酸氢钠溶液等弱碱性溶液;
在一种或多种实施方式中,所述碱溶液为氢氧化钠溶液(优选浓度为0.01-10M,例如,但不限于0.05,0.1,1,3,5,7,或者9M等)。
在一种或多种实施方式中,所述制备方法中还包括:
将巯基化壳聚糖还原后,再与α-β不饱和酰基化壳聚糖反应;
优选的,巯基化壳聚糖还原包括:将巯基化壳聚糖以还原剂处理的步骤;
更优选的,所述还原剂包括:锌,二硫苏糖醇,以及对苯二酚中的至少一种。
在一种或多种实施方式中,作为水凝胶制备原料的α-β不饱和酰基化壳聚糖包括如下式(i)化合物:
其中,式(i)中,R
1为壳聚糖除去氨基的残基部分;
R
2、R
3、R
4分别独立的为氢,取代或非取代烷基,取代或非取代的烷氧基,取代或非取代的芳基,以及取代或非取代的杂芳基;
在一种或多种实施方式中,R
2、R
3、R
4分别独立的为氢,碳原子数为1-20的取代或非取代烷基(优选为碳原子数为1-12的取代或非取代烷基,更优选为碳原子数为1-6的取代或非取代烷基,例如,但不限于:取代或非取代烷的甲基、乙基、丙基、异丙基、丁基、特丁基、戊基、异戊基、己基等),碳原子数为1-20的取代或非取代的烷氧基(优选为碳原子数为1-12的取代或非取代烷氧基,更优选为碳原子数为1-6的取代或非取代烷氧基,例如,但不限于:取代或非取代的甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、特丁氧基、戊氧基、异戊氧基、己氧基等),碳原子数为5-20的取代或非取代的芳基(优选为碳原子数5-12的取代或非取代芳基,例如,但不限于:取代或非取代的苯基、萘基、联苯基等),以及碳原子数为5-20的取代或非取代的杂芳基(优选为碳原子数5-12的取代或非取代杂芳基,例如,但不限于:取代或非取代的吡咯、吲哚、吡唑、吲唑、咪唑、苯丙吡唑、***、苯并***等);
在一种或多种实施方式中,当R
2、R
3、R
4中任意的R基为取代的烷基,取代的烷氧基,取代的芳基或者取代的杂芳基时,该取代的烷基,取代的烷氧基,取代的芳基或者取代的杂芳基中至少一个氢原子可以为烷基(优选碳原子数为1-20的烷基,更优选为碳原子数1-12的烷基,进一步优选为碳原子数1-6的烷基,例如,但不限于:甲基,乙基,丙基,异丙基,丁基,特丁基,戊基,异戊基,己基等),羧基,氨基,烷氧基(优选碳原子数为1-20的烷氧基,更优选为碳原子数为1-12的烷氧基,进一步优选为碳原子数为1-6的烷氧基,例如,但不限于:甲氧基,乙氧基,丙氧基,异丙氧基,丁氧基,特丁氧基,戊氧基,异戊氧基,己氧基等),芳基(优选碳原子数5-20的芳基,更优选为碳原子数5-12的芳基,例如,但不限于:苯基、萘基,联苯基等),杂芳基(优选碳原子数5-20的杂芳基,优选为碳原子数5-12的取代或非取代杂芳基,例如,但不限于:取代或非取代的吡咯,吲哚,吡唑,吲唑,咪唑,苯丙吡唑,***,苯并***等),酯基或者卤素(氟,氯,溴或碘)所取代;
其中,当取代基的数量大于1时,不同的取代基可以任选的为相同或者不同;
R
5为羰基,羧基,酯基,酰胺基,取代或非取代烷基(优选为C1-C12取代或非取代烷基,更优选为C1-C6取代或非取代烷基),取代或非取代的烷氧基(优选为C1-C12取代或非取代氧烷基,更优选为C1-C6取代或非取代氧烷基),取代或非取代的芳基(优选为C5-C20取代或非取代芳基,更优选为C5-C12取代或非取代芳基),以及取代或非取代的杂芳基(优选为C1-C12取代或非取代杂芳基,更优选为C1-C6取代或非取代杂芳基);
在一种或多种实施方式中,当R
5为羰基时,所述羰基结构为:
其中R可以为取代或非取代烷基(优选为C1-C12取代或非取代烷基,更优选为C1-C6取代或非取代烷基),取代或非取代的烷氧基(优选为C1-C12取代或非取代氧烷基,更优选为C1-C6取代或非取代氧烷基),取代或非取代的芳基(优选为C5-C20取代或非取代芳基,更优选为C5-C12取代或非取代芳基),以及取代或非取代的杂芳基(优选为C1-C12取代或非取代杂芳基,更优选为C1-C6取代或非取代杂芳基);
在一种或多种实施方式中,当R
5为酯基时,所述酯基的结构为:
或者
其中R′可以为取代或非取代烷基(优选为C1-C12取代或非取代烷基,更优选为C1-C6 取代或非取代烷基),取代或非取代的烷氧基(优选为C1-C12取代或非取代氧烷基,更优选为C1-C6取代或非取代氧烷基),取代或非取代的芳基(优选为C5-C20取代或非取代芳基,更优选为C5-C12取代或非取代芳基),以及取代或非取代的杂芳基(优选为C1-C12取代或非取代杂芳基,更优选为C1-C6取代或非取代杂芳基);
在一种或多种实施方式中,当R
5为酰胺基时,所述酰胺基的结构为:
其中,R″和R″′分别独立的为氢,取代或非取代烷基(优选为C1-C12取代或非取代烷基,更优选为C1-C6取代或非取代烷基),取代或非取代的烷氧基(优选为C1-C12取代或非取代氧烷基,更优选为C1-C6取代或非取代氧烷基),取代或非取代的芳基(优选为C5-C20取代或非取代芳基,更优选为C5-C12取代或非取代芳基),以及取代或非取代的杂芳基(优选为C1-C12取代或非取代杂芳基,更优选为C1-C6取代或非取代杂芳基)。
在一种或多种实施方式中,所述α-β不饱和酰基化壳聚糖的合成方法包括:
将壳聚糖与酰基化试剂反应,得到α-β不饱和酰基化壳聚糖;
其中,所述酰基化试剂包括:α-β不饱和酸,α-β不饱和酸酐,α-β不饱和酰卤,以及α-β不饱和酯中的至少一种;
作为优选,所述壳聚糖的分子量0.1-1000万,例如,但不限于1,5,10,50,100,300,500,700或者900万等;
更优选的,所述壳聚糖的分子量为5万,脱乙酰度为80%,其结构可参考如下:
作为优选,所述α-β不饱和酸包括:β-甲基丙烯酸,以及β-异丙基丙烯酸中的至少一种;
所述α-β不饱和酸酐包括:马来酸酐;
所述α-β不饱和酰卤包括:丙烯酰氯,以及甲基丙烯酰氯中的至少一种;
所述α-β不饱和酯包括:甲基丙烯酸甲酯,以及甲基丙烯酸乙酯中的至少一种;
作为优选,所述合成方法还包括:将所得α-β不饱和酰基化壳聚糖进行纯化的步骤;
更优选的,所述纯化包括:透析,通过采用透析纯化,也能够将残余的修饰剂(酰化试剂)除去,从而避免修饰剂对于后续反应的影响。
在一种或多种实施方式中,所述α-β不饱和酰基化壳聚糖的合成方法包括:
将壳聚糖与酰基化试剂反应,得到α-β不饱和酰基化壳聚糖;
其中,所述酰基化试剂包括:α-β不饱和酸,α-β不饱和酸酐,α-β不饱和酰卤,以及α-β不饱和酯中的至少一种;
优选的,所述合成方法还包括:将所得α-β不饱和酰基化壳聚糖进行纯化的步骤。
在一种或多种实施方式中,所述α-β不饱和酰基化壳聚糖的合成方法包括:
向溶于酸溶液中的壳聚糖中,加入酰化试剂(可以以溶液的形式加料),室温下搅拌混合后,在10-90℃(例如,但不限于20、30、40、50、60、70或者80℃等,优选为40-90℃)下反应2-10h(例如,但不限于3、4、5、6、7、8,或者9h等);
然后,将产物进行透析(优选透析2-4d),干燥(优选采用冷冻干燥),得到α-β不饱和酰基化壳聚糖;
作为优选,壳聚糖溶液的浓度为10~100mg/ml;
作为优选,用于溶解壳聚糖的酸溶液包括有机酸溶液,优选为乙酸溶液(特别是浓度0.01-30%的乙酸溶液);
作为优选,用于溶解酰化试剂的溶液包括:丙酮,丁酮,水,DMSO和DMF等极性溶剂中的至少一种(特别是丙酮);
作为优选,壳聚糖与酰化试剂的摩尔比为1:1-1:3。
在一种或多种实施方式中,作为水凝胶制备原料的巯基化壳聚糖包括如下式(ii)所示化合物:
其中,式(ii)中,R
1为壳聚糖除去氨基的残基部分;
R
6为取代或非取代基的亚烷基,取代或非取代的亚芳基,以及取代或非取代的亚杂芳基;
作为优选,R
6为碳原子数为1-20的取代或非取代烷基(优选为碳原子数为1-12的取代或非取代烷基,更优选为碳原子数为1-6的取代或非取代烷基,例如,但不限于:取代或非取代烷的甲基、乙基、丙基、异丙基、丁基、特丁基、戊基、异戊基、己基等),碳原子数为1-20的取代或非取代的烷氧基(优选为碳原子数为1-12的取代或非取代烷氧基,更优选为碳原子数为1-6的取代或非取代烷氧基,例如,但不限于:取代或非取代的甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、特丁氧基、戊氧基、异戊氧基、己氧基等),碳原子数为5-20的取代或非取代的芳基(优选为碳原子数5-12的取代或非取代芳基,例如,但不限于:取代或非取代的苯基、萘基、联苯基等),以及碳原子数为5-20的取代或非取代的杂芳基(优选为碳原子数5-12的取代或非取代杂芳基,例如,但不限于:取代或非取代的吡咯、吲哚、吡唑、吲唑、咪唑、苯丙吡唑、***、苯并***等);
作为优选,当R
6为取代的烷基,取代的烷氧基,取代的芳基或者取代的杂芳基时,该取代的烷基,取代的烷氧基,取代的芳基或者取代的杂芳基中至少一个氢原子可以为烷基(优选碳原子数为1-20的烷基,更优选为碳原子数1-12的烷基,进一步优选为碳原子数1-6的烷基,例如,但不限于:甲基,乙基,丙基,异丙基,丁基,特丁基,戊基,异戊基,己基等),羧基,氨基,烷氧基(优选碳原子数为1-20的烷氧基,更优选为碳原子数为1-12的烷氧基,进一步优选为碳原子数为1-6的烷氧基,例如,但不限于:甲氧基,乙氧基,丙氧基,异丙氧基,丁氧基,特丁氧基,戊氧基,异戊氧基,己氧基等),芳基(优选碳原子数5-20的芳基,更优选为碳原子数5-12的芳基,例如,但不限于:苯基、萘基,联苯基等),杂芳基(优选碳原子数5-20的杂芳基,优选为碳原子数5-12的取代或非取代杂芳基,例如,但不限于:取代或非取代的吡咯,吲哚,吡唑,吲唑,咪唑,苯丙吡唑,***,苯并***等),酯基或者卤素(氟,氯,溴或碘)所取代;
其中,当取代基的数量大于1时,不同的取代基可以任选的为相同或者不同。
在一种或多种实施方式中,所述巯基化壳聚糖的合成方法包括:
将壳聚糖与巯基化试剂反应,得到巯基化壳聚糖;
优选的,所述巯基化试剂包括:具有巯基和羧基的化合物;
更优选的,所述巯基化试剂包括:二巯基丁二酸、巯基丁二酸、巯基丙酸、硫代乙酸,以及2-巯基-3-吡啶甲酸中的至少一种;
优选的,所述合成方法还包括:将所得巯基化壳聚糖进行纯化的步骤。
在一种或多种实施方式中,所述巯基化壳聚糖的合成方法包括:
将壳聚糖与巯基化试剂反应,得到巯基化壳聚糖;
作为优选,所述巯基化试剂包括:具有巯基和羧基的化合物;
更优选的,所述巯基化试剂包括:二巯基丁二酸、巯基丁二酸、巯基丙酸、硫代乙醇酸,以及2-巯基-3-吡啶甲酸中的至少一种;
作为优选,所述壳聚糖的分子量为0.1-1000万,例如,但不限于1,5,10,50,100,300,500,700或者900万等;
更优选的,所述壳聚糖的分子量为5万,脱乙酰度为80%;
优选的,所述合成方法还包括:将所得巯基化壳聚糖进行纯化的步骤;
更优选的,所述纯化包括:透析,同样的,通过采用透析的方式进行巯基化壳聚糖的纯化,也能够避免修饰剂(巯基化试剂)在产物巯基化壳聚糖中的残留;
作为优选,巯基化壳聚糖是在羧基活化剂存在的条件下进行反应;
更优选的,所述羧基活化剂包括:EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)以及NHC(N-羟基琥珀酰亚胺);
作为优选,所述巯基化试剂包括:同时具有羧基和巯基的化合物;
更优选的,所述巯基化试剂包括:二巯基丁二酸,巯基丁二酸,巯基丙酸,硫代乙醇酸,以及2-巯基-3-吡啶甲酸等中的至少一种;
作为优选,壳聚糖与巯基化试剂的摩尔比为1:1-10:1。
在一种或多种实施方式中,所述巯基化壳聚糖的合成方法包括:
(a)将壳聚糖溶于酸溶液(优选为有机酸溶液,更优选为乙酸溶液,特别是浓度为0.01-30%的乙酸溶液)中;
作为优选,所得壳聚糖溶液的浓度为10~100mg/ml;
(b)将巯基化试剂(根据溶解性不同)溶于水(优选为双蒸水),碱溶液(例如氢氧化钠、氢氧化钾,或者氢氧化钙溶液等强碱性溶液,或者氨水、碳酸钠、碳酸氢钠溶液等弱碱性溶液,优选为氢氧化钠溶液)或者酸溶液(优选为有机酸溶液,更优选为乙酸溶液,特别是浓度为0.01~30%的乙酸溶液)中;
(c)将羧基活化剂加入巯基化试剂溶液中,混合均匀,并优选的在调节pH至4.5-6.5后,继续混合;
(d)将步骤(a)和步骤(c)溶液进行混合,然后优选的在50-60℃(例如,但不限于52、55、58℃等)条件下进行反应1-8h(例如,但不限于2、3、4、5、6,或者7h,优选为2-6h,更优选为4-5h)。
(e)步骤(d)反应液透析(优选透析2-4d),干燥(优选采用冷冻干燥),得到巯基化壳聚糖。
本公开还提供了一种双交联壳聚糖水凝胶,所述双交联壳聚糖水凝胶由本公开如上制备方法得到。
本公开所制备的双交联壳聚糖水凝胶的微观化学结构,既存在化学键交联,同时也存在物理交联,因而使得本公开双交联壳聚糖水凝胶具有良好的力学性能,相较于现有技术中的几丁质水凝胶或者未经物理交联处理的水凝胶而言,在力学性能上都有显著的提高。
又一方面,本公开也提供了一种本公开双交联壳聚糖水凝胶在生物材料制备中的应用;
进一步的,本公开也能够提供一种包含本公开双交联壳聚糖水凝胶的生物材料;
如上所述的生物材料优选的包括生物纤维。
一种壳聚糖巯基化衍生物,其通式为:
其中,R为亚烷基或取代亚烷基。
在一种或多种实施方式中,R为取代亚烷基时,该取代亚烷基可以为至少一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羟基和卤代烷基中至少一种基团取代的亚烷基。即可以是亚烷基中有一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羟基和卤代烷基中的一种基团取代;也可以是亚烷基中有两个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羟基和卤代烷基中的两种基团取 代;也可以是亚烷基中有两个以上的氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羟基和卤代烷基中的两种以上的基团取代;也可以是亚烷基中多个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羟基和卤代烷基中的多个同一种基团取代或被其中多个不同基团的组合对应进行取代。
在一种或多种实施方式中,R的碳原子数可为1~20个,优选1~15个,更优选1~10个。即R可以为C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20的亚烷基或取代亚烷基。
在一种或多种实施方式中,所述取代亚烷基为至少一个氢原子被烷基、羧基、氨基、烷氧基、芳香基、酯基、羧基和卤代烷基中至少一种基团取代的亚烷基。
在一种或多种实施方式中,R的碳原子数为1~20个。
由于壳聚糖分子链上存在游离的氨基和羟基,从而可以通过与羧基进行反应,达到对壳聚糖分子链进行修饰改性的目的。要在壳聚糖分子中引入巯基,最直接的方式就是将同时具有羧基和巯基的化合物与壳聚糖进行反应。但是,上述反应中,由于羧基的亲电性不足,其只能与壳聚糖分子中亲核性较强的氨基进行反应,而不会与羟基发生反应。而在本公开实施例中,采用同时具有羧基和磺酸基的磺酸基化合物与壳聚糖进行反应,由于磺酸的强吸电子性,磺酸基化合物的羧基亲电性较强,可以同时与壳聚糖中的氨基和伯羟基发生反应,在壳聚糖分子链中引入磺酸基,再通过与还原剂反应,即可将磺酸基还原成巯基,得到氨基和伯羟基均被巯基化后的壳聚糖巯基化衍生物。同时具有羧基和磺酸基的化合物可以通过经水解反应、氨解反应等直接生成,或者可通过含二硫键经还原后再剧烈氧化直接得到。例如,同时具有羧基和磺酸基的化合物可以为:二磺酸基丁二酸、磺酸基乙酸、3-磺酸基丙酸、磺酸基丁二酸等。
在一种或多种实施方式中,壳聚糖巯基化衍生物由以下反应式生成:
本公开一些实施方式涉及壳聚糖巯基化衍生物的制备方法,包括壳聚糖与磺酸基化合物在羧基活化剂的存在下进行反应,再将磺酸基还原成巯基。
在一种或多种实施方式中,壳聚糖巯基化衍生物的制备方法包括:将含有所述壳聚糖的溶液与含有所述磺酸基化合物的溶液在羧基活化剂的作用下进行反应。
在一种或多种实施方式中,本公开实施方式中的壳聚糖巯基化衍生物可以通过以下方法制备:
(1)制备含有壳聚糖的溶液:将壳聚糖溶解于0.01~30%的酸溶液中。
(2)制备含修饰剂化合物的溶液:将磺酸基化合物根据其溶解性,溶解于双蒸水或碱溶液或酸溶液中,修饰剂本身为溶液的为便于反应操作可用相应溶剂稀释。
(3)将羧基活化剂加入到上述含磺酸基化合物的溶液中,混合均匀后,调节其PH值至4.5~6.5,继续混合搅拌。
(4)将活化后的含有磺酸基化合物的溶液与含有壳聚糖的溶液进行混合,充分搅拌均匀,优选转移至圆底烧瓶中,放置在温度为50~60℃以下进行恒温反应。
(5)向反应后的反应液中加入适量还原剂,室温下搅拌24h。
(6)将反应后得到的反应液进行透析2~5天,再将得到的透析产物进行冷冻干燥2~5天后,得到壳聚糖巯基化衍生物。通过透析的手段可以除去残留的小分子杂质,从而得到纯度更高的壳聚糖巯基化衍生物。
在一种或多种实施方式中,含有所述壳聚糖的溶液是酸溶液。根据一些实施方式,酸溶液可以是有机酸溶液,优选乙酸溶液。即优选将述壳聚糖溶解于0.01~30%的乙酸溶液中。
在一种或多种实施方式中,含有所述磺酸基化合物的溶液是双蒸水或碱溶液或酸溶液。根据一些实施方式,碱溶液可以为强碱溶液,例如氢氧化钠溶液、氢氧化钾溶液、氢氧化钙溶液等,也可以为弱碱溶液,例如氨水溶液、碳酸钠溶液、碳酸氢钠溶液等,优选氢氧化钠溶液。双蒸水将经过一次蒸馏后的水,再次蒸馏所得到的水,其可以使得溶解后的溶液纯度更高,后续反应效果越好。溶解磺酸基化合物的酸溶液可以是有机酸溶液,优选乙酸溶液。
在一种或多种实施方式中,羧基活化剂包括1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)。EDC是个可溶于水的碳二亚胺,在酰胺合成中用作羧基的活化试剂,也用于活化磷酸酯基团、蛋白质与核酸的交联和免疫偶联物的制取,常和NHS或N-羟基硫代琥珀酰亚胺连用,以提高偶联效率。NHS即N-羟基琥珀酰亚胺,活化羧基以用于酰胺键的形成。用于合成氨基酸保护剂、半合成卡那霉素及医药中间体。本公开实施方式中的羧基活化剂可按照EDC和NHS的质量比为10:1~1:1的比例进行配制,这种比例有利于二者达到更好的偶联效率,使得对磺酸基化合物的羧基活化效果更好。
在一种或多种实施方式中,可以取EDC和NHS在磁力搅拌器作用下加入到上述含磺酸基化合物的溶液中,以达到更好的混合效果。
在一种或多种实施方式中,混合羧基活化剂后,用碱或酸溶液调节加有羧基活化剂的磺酸基化合物溶液的PH值,使得其PH值为4.5~6.5。优选地,用1M氢氧化钠或1M盐酸溶液进行调节PH值。EDC和NHS在PH值为4.5~6.5的条件下能够达到对羧基最好的活化效果。
在一种或多种实施方式中,调节PH值后混合搅拌的时间为10~150min,使得羧基活化剂能够对磺酸基化合物的羧基进行充分的活化,以更好地对壳聚糖进行修饰。
在一种或多种实施方式中,反应温度优选55~60℃,进一步优选55℃。反应时间可以为1~8h,优选2~6h,更优选地4~5h。
需要说明的是,上述过程中也可以先进行步骤(2)和(3),再进行步骤(1),不会影响最终产物的生成。
在一种或多种实施方式中,将含有所述壳聚糖的溶液与含有所述磺酸基化合物的溶液混合并在羧基活化剂的存在下进行反应;再通过还原剂将磺酸基还原为巯基;其中,含有所述壳聚糖的溶液优选酸溶液;含有所述磺酸基化合物的溶液优选双蒸水或碱溶液或酸溶液。
在一种或多种实施方式中,所述羧基活化剂包括EDC和NHS,所述EDC和所述NHS的质量比为10:1~1:1。
在一种或多种实施方式中,利用所述羧基活化剂对含有所述磺酸基化合物的溶液中待反应的羧基进行活化,然后混合含有所述磺酸基化合物的溶液和含有所述壳聚糖的溶液进行反应,优选反应时间为1~8h。
在一种或多种实施方式中,所述壳聚糖巯基化衍生物由以下反应式生成:
在一种或多种实施方式中,所述磺酸基化合物为同时具有羧基和磺酸基的化合物。
本公开实施方式中制备壳聚糖巯基化衍生物的方法操作工艺简单,反应条件温和。其为制备一种新的壳聚糖巯基化衍生物以及水凝胶提供了一种简单可行的新方法。所得的壳聚糖巯基化衍生物可用于再生医学、组织工程支架以及医药卫生领域,应用范围广泛。
需要说明的是,壳聚糖巯基化衍生物也可以与小分子或纳米粒子等发生反应来制备其他化学材料。
上述制备方法得到的壳聚糖巯基化衍生物可在制备水凝胶中进行应用,可以将壳聚糖巯基化衍生物与马来酰化壳聚糖混合后制备水凝胶。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数为0.01%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声30min。准确称取酰化试剂(马来酸酐)1.8g,加入5ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌20min左右使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度40℃,恒温加热反应2h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到MCS产物。
(2)准确称取巯基丁二酸1.5g,加入到一定量双蒸水中进行溶解。称取1.2g EDC和300mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至6.5,置磁力搅拌器上搅拌30min。准确称取1.5g壳聚糖,溶于质量分数为0.01%的乙酸溶液中,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度55℃,恒温加热反应5h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到壳聚糖巯基产物。
(3)准确称取MCS 60mg,用质量分数1%的乙酸溶液溶解,依次进行超声震荡、氮气除气泡,配成浓度为10mg/ml的溶液。再准确称取CS-SH 60mg,用质量分数1%的乙酸溶液进行溶解,再进行超声震荡、氮气除气泡,配成浓度为10mg/ml的溶液,用Zn处理10min,震荡混合均匀。将配好的MCS溶液和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例2
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数为0.01%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声30min。准确称取酰化试剂(β-甲基丙烯酸)1.8g,加入5ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌20min左右使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度40℃, 恒温加热反应2h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到MCS产物。
(2)准确称取巯基丁二酸1.5g,加入到一定量双蒸水中进行溶解。称取1.2g EDC和300mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至6.5,置磁力搅拌器上搅拌30min。准确称取1.5g壳聚糖,溶于质量分数为0.01%的乙酸溶液中,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度55℃,恒温加热反应5h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到壳聚糖巯基产物。
(3)准确称取MCS 120mg,用质量分数10%的乙酸溶液溶解,依次进行超声震荡、氮气除气泡,配成浓度为50mg/ml的溶液。再准确称取CS-SH 120mg,用质量分数10%的乙酸溶液进行溶解,再进行超声震荡、氮气除气泡,配成浓度为50mg/ml的溶液,用Zn处理10min,震荡混合均匀。将配好的MCS溶液和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例3
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数为30%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声35min。准确称取酰化试剂(马来酸酐)4.5g,加入15ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌30min使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度90℃,恒温加热反应10h。反应停止后透析两天,每隔4h换一次透析液。冷冻干燥两天得到MCS产物。
(2)准确称取2-巯基烟酸1.5g,加入到一定量双蒸水中进行溶解。称取1.2g EDC和300mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至5.5左右,置磁力搅拌器上搅拌30min。准确称取1.5g壳聚糖,溶于质量分数为10%的乙酸溶液中,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度50℃,恒温加热反应8h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到壳聚糖巯基产物。
(3)准确称取MCS 60mg,用质量分数为10%的乙酸溶液溶解,依次进行超声震荡、氮气除气泡,配成浓度为10mg/ml的溶液。再准确称取CS-SH 60mg,用质量分数10%的乙酸溶液进行溶解,再进行超声震荡、氮气除气泡,配成浓度为10mg/ml的溶液,用Zn处理15min,震荡混合均匀。将配好的MCS溶液和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例4
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数为30%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声35min。准确称取酰化试剂(β-异丙基丙烯酸)4.5g,加入15ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌30min使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度90℃,恒温加热反应10h。反应停止后透析两天,每隔4h换一次透析液。冷冻干燥两天得到MCS产物。
(2)准确称取2-巯基烟酸1.5g,加入到一定量双蒸水中进行溶解。称取1.2g EDC和300mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至5.5左右,置磁力搅拌器上搅拌30min。准确称取1.5g壳聚糖,溶于质量分数为10%的乙酸溶液中,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度50℃,恒温加热反应8h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到壳聚糖巯基产物。
(3)准确称取MCS 120mg,用质量分数为10%的乙酸溶液溶解,依次进行超声震荡、氮气除气泡,配成浓度为50mg/ml的溶液。再准确称取CS-SH 120mg,用质量分数10%的乙酸溶液进行溶解,再进行超声震荡、氮气除气泡,配成浓度为50mg/ml的溶液,用Zn处理15min,震荡混合均匀。将配好的MCS溶液和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例5
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数30%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声150min。准确称取酰化试剂(马来酸酐)5.4g,加入20ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌150min使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度90℃,恒温加热反应10h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到MCS产物。
(2)准确称取1.5g 3-巯基丙酸溶液,加入一定量双蒸水进行稀释。称取1.2g EDC和400mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至5左右,置磁力搅拌器上搅拌60min。准确称取1.5g壳聚糖,溶于0.1%质量分数的乙酸溶液,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度60℃,恒温加热反应3h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到CS-SH产物。
(3)准确称取MCS 240mg,用质量分数为1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为100mg/ml的溶液。准确称取CS-SH 240mg,用质量分数1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为100mg/ml的溶液,用还原剂二硫苏糖醇处理100min,震荡混合均匀。将配好的MCS和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例6
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数30%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声150min。准确称取酰化试剂(β-甲基丙烯酸)5.4g,加入20ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌150min使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度90℃,恒温加热反应10h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到MCS产物。
(2)准确称取1.5g 3-巯基丙酸溶液,加入一定量双蒸水进行稀释。称取700mg EDC和700mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至5左右,置磁力搅拌器上搅拌60min。准确称取1.5g壳聚糖,溶于0.1%质量分数的乙酸溶液,将壳聚糖溶液缓慢均匀倒入上述混合 液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度60℃,恒温加热反应3h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天得到CS-SH产物。
(3)准确称取MCS 60mg,用质量分数为1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为10mg/ml的溶液。准确称取CS-SH 60mg,用质量分数1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为10mg/ml的溶液,用还原剂二硫苏糖醇处理100min,震荡混合均匀。将配好的MCS和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实施例7
本实施例提供的壳聚糖水凝胶的制备方法包括:
(1)准确称取壳聚糖1.5g,溶于质量分数15%的乙酸中。在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声150min。准确称取酰化试剂(马来酸酐)1.2g,加入6ml丙酮使其完全溶解。将溶解后的酰化试剂匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌40min使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度60℃,恒温加热反应6h。反应停止后透析三天,每隔4h换一次透析液。冷冻干燥三天得到MCS产物。
(2)准确称取1.5g巯基乙酸溶液,加入一定量双蒸水进行稀释。称取1.5g EDC和150mg NHS同时加入上述溶液中,充分混合后,用氢氧化钠溶液调节混合溶液PH至6.5,置磁力搅拌器上搅拌8min。准确称取1.5g壳聚糖,溶于2%质量分数的乙酸溶液,将壳聚糖溶液缓慢均匀倒入上述混合液,搅拌使混合完全。将上述混合液转移至250ml圆底烧瓶中,置于集热式磁力搅拌器中,设置温度55℃,恒温加热反应5h。反应停止后透析三天,每隔4h换一次透析液。冷冻干燥三天得到CS-SH产物。
(3)准确称取MCS 240mg,用质量分数为1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为100mg/ml的溶液。准确称取CS-SH 240mg,用质量分数1%的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为100mg/ml的溶液,用还原剂对苯二酚处理100min,震荡混合均匀。将配好的MCS和CS-SH溶液充分混合后,挤出至NaOH溶液中反应成胶,得到所需壳聚糖水凝胶。
化学反应式:
实验例1
对实施例1中的壳聚糖、巯基化壳聚糖以及α-β不饱和酰基化壳聚糖进行傅里叶红外分析图谱,得到图1,图1中壳聚糖与巯基化壳聚糖红外图谱对比分析可以看出,无论是MCS还是CS-SH在波数3400cm
-1附近,均可以看到引入羧基后,产生的-OH伸缩振动;在2900cm
-1附近,表示支链上的C-H伸缩振动;1500cm
-1-1650cm
-1附近的酰胺Ⅰ带和酰胺Ⅱ带,可以看到无论是MCS还是CS-SH的酰胺带均有加强,特别是其中的MCS,由于C=C双键的引入,产生的C=C伸缩振动峰与酰胺带重叠,在该波段的峰强有明显的增加;在1100cm
-1附近,MCS和CS-SH都因为有羧基的引入,在该位置出现明显的C-O伸缩振动峰;在2100cm
-1附近的-NH
3
+的吸收带,壳聚糖被修饰后游离氨基减少,质子化氨基含量减少,-NH
3
+的吸收带峰强度明显减小,以上现象表明巯基化合物和酰化试剂被成功引入到壳聚糖链上。同时,通过扫描电镜对实施例1中的壳聚糖水凝胶的表面结构和表面孔径做扫描电镜(SEM)分析。结果依次如图2,图3所示。SEM分析表明固化后壳聚糖水凝胶表面出现很多交联后的孔洞,说明交联反应进行得很完全,进一步说明制备得到的巯基化壳聚糖可以成功制备快速固化壳聚糖水凝胶。
进一步地,通过用instron力学测试机对实施例1中得到的壳聚糖水凝胶进行测试,使用10N传感器,置于传感器压缩基底上,根据样品的大小设置好测试参数,测试曲线出现突变后,停止压缩,***自动得出弹性模量值。
测试参数为:长10mm,宽8mm,高5mm,压缩速率0.8mm/min。测试结果如图4所示,在压缩率为75%,压缩应力约为700kPa时发生断裂,因此,可以看出该水凝胶具有较好的机械强度和弹性性能。
综上所述,通过将酰化试剂酰化的壳聚糖与巯基化的壳聚糖作为原材料,通过Michael加成反应可实现原材料由液态到固态的快速转变,大大提高了材料的可打印性,通过调节二者的浓度可实现固化后壳聚糖水凝胶弹性模量的调节,可扩大材料的应用范围,该方法制备的壳聚糖水凝胶在生物医学及组织工程领域具有重要用途,其固化速度快,生物相容性好、机械强度可调、在培养基中的稳定性好,生物降解速度可调,应用范围大。
实施例8
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入0.01%(m/m)的乙酸溶液中,搅拌均匀后超声30min;
将1.8g马来酸酐加入10ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在40℃条件下反应2h;
反应结束后,将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
步骤1)反应式可参考如下:
2)将1.5g巯基丁二酸加入双蒸水中搅拌溶解,然后加入1.2g EDC以及300mg NHS,充分混合后,以1M NaOH调节溶液pH至6.5,继续搅拌混合;
将1.5g壳聚糖加入0.01%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有巯基丁二酸的混合溶液中,55℃条件下反应5h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
步骤2)反应式可参考如下:
3)将60mg MCS加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的MCS溶液;
将60mg CS-SH加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的CS-SH溶液;
向所得CS-SH溶液中加入金属锌,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例8的双交联壳聚糖水凝胶。
实施例8反应式如图5所示,反应流程如图6所示。
实施例9
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入0.01%(m/m)的乙酸溶液中,搅拌均匀后超声30min;
将1.8gβ-甲基丙烯酸加入5ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在40℃条件下反应2h;
反应结束后,将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g巯基丁二酸加入双蒸水中搅拌溶解,然后加入1.2g EDC以及300mg NHS,充分混合后,以1M NaOH调节溶液pH至6.5,继续搅拌混合;
将1.5g壳聚糖加入0.01%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有巯基丁二酸的混合溶液中,55℃条件下反应5h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将120mg MCS加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到50mg/L的MCS溶液;
将120mg CS-SH加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到50mg/L 的CS-SH溶液;
向所得CS-SH溶液中加入金属锌,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例9的双交联壳聚糖水凝胶。
实施例10
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入0.30%(m/m)的乙酸溶液中,搅拌均匀后超声35min;
将4.5g马来酸酐加入15ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在40℃条件下反应2h;
反应结束后,将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g 2-巯基烟酸加入双蒸水中搅拌溶解,然后加入1.2g EDC以及300mg NHS,充分混合后,以1M NaOH调节溶液pH至5.5,继续搅拌混合;
将1.5g壳聚糖加入10%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有2-巯基烟酸的混合溶液中,50℃条件下反应8h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将60mg MCS加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的MCS溶液;
将60mg CS-SH加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的CS-SH溶液;
向所得CS-SH溶液中加入金属锌,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例10的双交联壳聚糖水凝胶。
实施例11
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入30%(m/m)的乙酸溶液中,搅拌均匀后超声35min;
将4.5gβ-异丙基丙烯酸加入15ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在90℃条件下反应10h;
反应结束后,将反应液透析2d,期间每4h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g 2-巯基烟酸加入双蒸水中搅拌溶解,然后加入1.2g EDC以及300mg NHS,充分混合后,以1M NaOH调节溶液pH至5.5,继续搅拌混合;
将1.5g壳聚糖加入10%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有2-巯基烟酸的混合溶液中,50℃条件下反应8h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将120mg MCS加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到50mg/L的MCS溶液;
将120mg CS-SH加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到50mg/L的CS-SH溶液;
向所得CS-SH溶液中加入金属锌,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例11的双交联壳聚糖水凝胶。
实施例12
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入30%(m/m)的乙酸溶液中,搅拌均匀后超声150min;
将5.4g马来酸酐加入20ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在90℃条件下反应10h;
反应结束后,将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g 3-巯基丙酸加入双蒸水中搅拌溶解,然后加入1.2g EDC以及400mg NHS,充分混合后,以1M NaOH调节溶液pH至5,继续搅拌混合;
将1.5g壳聚糖加入0.1%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有3-巯基丙酸的混合溶液中,60℃条件下反应3h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将240mg MCS加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到100mg/L的MCS溶液;
将60mg CS-SH加入10%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的CS-SH溶液;
向所得CS-SH溶液中加入二硫苏糖醇,震荡反应100min,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例12的双交联壳聚糖水凝胶。
实施例13
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入30%(m/m)的乙酸溶液中,搅拌均匀后超声150min;
将5.4gβ-甲基丙烯酸加入20ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在90℃条件下反应10h;
反应结束后,将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g 3-巯基丙酸加入双蒸水中搅拌溶解,然后加入700mg EDC以及700mg NHS,充分混合后,以1M NaOH调节溶液pH至5,继续搅拌混合;
将1.5g壳聚糖加入0.1%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有3-巯基丙酸的混合溶液中,60℃条件下反应3h;
将反应液透析3d,期间每5h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将60mg MCS加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的MCS溶液;
将60mg CS-SH加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到10mg/L的CS-SH溶液;
向所得CS-SH溶液中加入二硫苏糖醇,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例13的双交联壳聚糖水凝胶。
实施例14
1)将1.5g壳聚糖(分子量为50,000左右,脱酰度为80%左右)加入15%(m/m)的乙酸溶液中, 搅拌均匀后超声150min;
将1.2gβ-甲基丙烯酸加入6ml丙酮中搅拌溶解,然后将所得溶液加入壳聚糖溶液中,搅拌混合均匀,在60℃条件下反应6h;
反应结束后,将反应液透析3d,期间每4h进行一次透析液替换,所得产物冷冻干燥,得到产品α-β不饱和酰基化壳聚糖(简记为MCS)。
2)将1.5g巯基乙酸加入双蒸水中搅拌溶解,然后加入1.5g EDC以及700mg NHS,充分混合后,以1M NaOH调节溶液pH至6.5,继续搅拌混合;
将1.5g壳聚糖加入2%(m/m)的乙酸溶液搅拌溶解,然后将所得壳聚糖溶液加入含有巯基乙酸的混合溶液中,60℃条件下反应3h;
将反应液透析3d,期间每4h进行一次透析液替换,所得产物冷冻干燥,得到产品巯基化壳聚糖(简记为CS-SH)。
3)将240mg MCS加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到100mg/L的MCS溶液;
将240mg CS-SH加入1%(m/m)的乙酸溶液中搅拌溶解,然后超声振荡和氮气除泡,得到100mg/L的CS-SH溶液;
向所得CS-SH溶液中加入对苯二酚,震荡反应,然后过滤,得到还原处理后的CS-SH溶液;
将MCS溶液与还原处理后的CS-SH溶液混合反应,然后挤出至NaOH溶液中反应,得到化学交联的水凝胶。
将所得化学交联的水凝胶加入无水乙醇中,浸泡24h,得到实施例14的双交联壳聚糖水凝胶。
实验例2
将未经修饰的壳聚糖置于无水乙醇中浸泡24h,得到物理交联水凝胶,记为CSH-E10;
参照实施例8中的方法,按照马来酸酐与壳聚糖摩尔比为1.2:1,巯基丁二酸与壳聚糖摩尔比为1.2:1的摩尔比例,得到化学交联水凝胶,记为M4S4-E0;
同时,参照实施例8中的方法,按照马来酸酐与壳聚糖摩尔比为1.2:1,巯基丁二酸与壳聚糖摩尔比为1.2:1的摩尔比例,得到化学交联水凝胶,然后,将所得化学交联水凝胶分别置于浓度为20%,40%,60%,80%的乙醇溶液以及无水乙醇中,浸泡24h,得到对应的双交联水凝胶,所得水凝胶分别记为M4S4-E2,M4S4-E4,M4S4-E6,M4S4-E8,M4S4-E10;
参照实施例8方法,按照马来酸酐与壳聚糖摩尔比为0.2:1,巯基丁二酸与壳聚糖摩尔比为0.2:1的摩尔比例,得到化学交联水凝胶,然后,将所得化学交联水凝胶置于无水乙醇中,浸泡24h,得到对应的双交联水凝胶,记为M1S1-E10;
参照实施例8方法,按照马来酸酐与壳聚糖摩尔比为0.5:1,巯基丁二酸与壳聚糖摩尔比为0.5:1的摩尔比例,得到化学交联水凝胶,然后,将所得化学交联水凝胶置于无水乙醇中,浸泡24h,得到对应的双交联水凝胶,记为M2S2-E10;
参照实施例8方法,按照马来酸酐与壳聚糖摩尔比为1:1,巯基丁二酸与壳聚糖摩尔比为1:1的摩尔比例,得到化学交联水凝胶,然后,将所得化学交联水凝胶置于无水乙醇中,浸泡24h,得到对应的双交联水凝胶,记为M3S3-E10;
不同水凝胶测试数据如下表所示:
由图7、图8所示检测结果,以及如上表格数据可知,相较于单一物理交联或者化学交联的水凝胶而言,本公开双交联水凝胶在力学性能上有着显著的提高,无论是压缩模量和断裂强度,都明显提高;
同时,由M4S4-E2,M4S4-E4,M4S4-E6,M4S4-E8,M4S4-E10组双交联水凝胶性能对比可知,当原料配比相同时,采用无水乙醇进行物理交联的水凝胶物理性能最优;
进一步的,由M4S4-E10,M1S1-E10,M2S2-E10,M3S3-E10组双交联水凝胶性能对比可知,原料酰基化试剂和巯基化试剂与壳聚糖配比对于最终产品双交联壳聚糖力学性能也有着明显的影响,按照1.2:1的摩尔比例所制备的酰基化壳聚糖以及巯基化壳聚糖反应后,能够得到力学性能更优的双交联壳聚糖。
实验例3
按照实施例8方法,分别得到MCS溶液与还原处理后的CS-SH溶液,将二者混合均匀后,吸入注射器中,利用注射泵进行出样,混合液在含有NaOH的接收皿中快速成型,得到壳聚糖生物纤维;
然后,将所得壳聚糖生物纤维在无水乙醇中浸泡24h,得到双交联壳聚糖生物纤维,其扫描电镜图如图9所示。
实施例15
本实施例中的壳聚糖巯基化衍生物的反应方程式为:
具体的反应步骤如下:
(1)将0.3g的壳聚糖溶解于质量分数0.01%的乙酸溶液中,得到壳聚糖乙酸溶液。
(2)取5ml磺酸基丁二酸溶液,加双蒸水稀释至50ml。
(3)取0.8g EDC和0.2g NHS在磁力搅拌器作用下一次加入上述磺酸基丁二酸溶液中,充分混合均匀,再用1M稀盐酸调节上述混合溶液的PH至6.5,将混合溶液搅拌30min,使羧基充分活化。
(4)将羧基活化后的磺酸基丁二酸溶液与壳聚糖乙酸溶液混合后搅拌10min时间,充分搅拌均匀,将混合溶液转移至圆底烧瓶中,在温度为55℃下恒温反应数5h。
(5)将用盐酸处理过表面氧化锌的金属锌颗粒加入到上述反应液中,室温搅拌24h。
(6)经过反应后的反应液进行透析3d、冷冻干燥3d后,得到壳聚糖巯基化衍生物。
实施例16
本实施例中的壳聚糖巯基化衍生物的反应方程式为:
具体的反应步骤如下:
(1)将4.5g的壳聚糖溶解于质量分数30%的乙酸溶液中,得到壳聚糖乙酸溶液。
(2)取2.8g 2-磺酸基烟酸,加双蒸水稀释至50ml。
(3)取1.6g EDC和0.4g NHS在磁力搅拌器作用下一次加入上述2-磺酸基烟酸溶液中,充分混合均匀,再用1M稀盐酸调节上述混合溶液的PH至6.5,将混合溶液搅拌30min,使羧基充分活化。
(4)将羧基活化后的2-磺酸基烟酸溶液与壳聚糖乙酸溶液混合后搅拌10min时间,充分搅拌均匀,将混合溶液转移至圆底烧瓶中,在温度为60℃下恒温反应数3h。
(5)将用盐酸处理过表面氧化锌的金属锌颗粒加入到上述反应液中,室温搅拌24h。
(6)经过反应后的反应液进行透析3d、冷冻干燥3d后,得到壳聚糖巯基化衍生物。
实施例17
本实施例中的壳聚糖巯基化衍生物的反应方程式为:
具体的反应步骤如下:
(1)将2.1g的壳聚糖溶解于质量分数2%的乙酸溶液中,得到壳聚糖乙酸溶液。
(2)取4.5g 3-磺酸基丙酸,加双蒸水稀释至50ml。
(3)取1.0g EDC和1.0g NHS在磁力搅拌器作用下一次加入上述3-磺酸基丙酸溶液中,充分混合均匀,再用1M稀盐酸调节上述混合溶液的PH至5.5,将混合溶液搅拌60min,使羧基充分活化。
(4)将羧基活化后的3-磺酸基丙酸溶液与壳聚糖乙酸溶液混合后搅拌10min时间,充分搅拌均匀,将混合溶液转移至圆底烧瓶中,在温度为50℃下恒温反应数8h。
(5)将用盐酸处理过表面氧化锌的金属锌颗粒加入到上述反应液中,室温搅拌24h。
(6)经过反应后的反应液进行透析3d、冷冻干燥3d后,得到壳聚糖巯基化衍生物。
实施例18
本实施例中的壳聚糖巯基化衍生物的反应方程式为:
具体的反应步骤如下:
(1)将0.15g的壳聚糖溶解于质量分数0.01%的乙酸溶液中,得到壳聚糖乙酸溶液。
(2)取0.09g磺酸基乙酸,加双蒸水稀释至50ml。
(3)取0.090g EDC和0.009g NHS在磁力搅拌器作用下一次加入上述磺酸基乙酸溶液中,充分混合均匀,用1M稀盐酸调节上述混合溶液的PH,使其PH为6.5,再搅拌30min时间,使羧基充分活化。
(4)将活化后的磺酸基乙酸溶液加入壳聚糖乙酸溶液中拌150min时间,充分搅拌均匀,并将混合液转移至圆底烧瓶中,在温度55℃以下恒温反应数5h。
(5)将用盐酸处理过表面氧化锌的金属锌颗粒加入到上述反应液中,室温搅拌24h。
(6)经过反应的反应液经透析3d、冷冻干燥3d后,得到壳聚糖巯基化衍生物。
实施例19
准确称取壳聚糖1.5g溶于质量分数0.01%质量分数的乙酸溶液中,在磁力搅拌器作用下基本溶解均匀后,置于超声振荡器中超声30min左右。准确称取马来酸酐1.8g,加入5ml丙酮使其完全溶解。将溶解后的马来酸酐匀速缓慢的加入壳聚糖乙酸溶液中,在磁力搅拌器下搅拌20min左右使其充分混匀。将混合均匀的液体转入150ml圆底烧瓶中,置集热式磁力搅拌器中,设置温度40℃,恒温加热反应2h。反应停止后透析三天,每隔5h换一次透析液。冷冻干燥三天左右得到MCS产物。
化学反应式:
准确称取MCS 60mg,用一定质量分数的乙酸溶液溶解,超声震荡,氮气除气泡,配成浓度为10mg/ml的溶液。准确称取实施例15中的壳聚糖巯基化衍生物(TCS)60mg,用氢氧化钠溶液溶解,超声震荡,氮气除气泡,配成浓度为10mg/ml的溶液,用还原剂处理10min左右,震荡混合均匀。将配好的MCS和TCS溶液在3D打印机上由进样***进样,计算机程序控制出样速度和打印模式,均匀混合制备3D生物打印水凝胶。
对实施例15中的壳聚糖以及壳聚糖巯基化衍生物傅里叶红外分析图谱,分别得到图10,图10中壳聚糖与巯基化壳聚糖红外图谱对比分析可以看出,巯基化壳聚糖在波数3400cm
-1附近,可以看到羧基上的-OH伸缩振动;在2900cm
-1附近,表示支链上引入的C-H伸缩振动;在1500cm
-1~1650cm
-1附近的酰胺Ⅰ带和酰胺Ⅱ带,修饰后的壳聚糖酰胺带加强;在1100cm
-1附近,修饰后的壳聚糖因为羧基的引入,在该位置出现明显的C-O伸缩振动峰;在2100cm
-1附近的-NH
3
+的吸收带,壳聚糖被修饰后游离氨基减少,质子化氨基含量减少,-NH
3
+的吸收带峰强度明显减小,以上现象表明巯基成功引入到壳聚糖链上。同时,通过扫描电镜对固化后的水凝胶的表面结构和表面孔径做扫描电镜(SEM)分析。结果依次如图11,图12所示。SEM分析表明固化后水凝胶表面出现很多交联后的孔洞,说明交联反应进行得很完全,进一步说明制备得到的壳聚糖巯基化衍生物可以成功制备快速固化水凝胶。
进一步地,通过用instron力学测试机对实施例19中得到的水凝胶进行测试,使用10N传感器,置于传感器压缩基底上,根据样品的大小设置好测试参数,测试曲线出现突变后,停止压缩,***自动得出弹性模量值。
测试参数为:长10mm,宽8mm,高5mm,压缩速率0.8mm/min。测试结果如图13所示,在压缩率为75%,压缩应力约为700kPa时发生断裂,因此,可以看出该水凝胶具有较好的机械强度和弹性性能。
综上所述,本公开实施方式中,通过将同时含有磺酸基和羧基的磺酸基化合物作为修饰剂,在羧基活化剂的作用下,在壳聚糖分子链上的氨基和伯羟基成功引入磺酸基,并进一步通过还原得到巯基。该制备方法的路线设计合理,操作简单可行,对设备要求低,能够高效高产率地得到壳聚糖巯基化衍生物。该壳聚糖巯基化衍生物由于巯基侧链的作用,而具有很好的亲核性能、抗氧化性能以及丰富的衍生性,其可以进一步通过亲核反应、交联反应等进行衍生,其应用范围十分广泛。例如,将该壳聚糖巯基化衍生物与含马来酰亚胺、乙烯砜、α-β不饱和醛、酮、酸、酯等结构的其他高分子衍生物发生化学反应,来制备快速固化的水凝胶,在再生医学和组织工程领域有重要的应用。又比如,由于该壳聚糖巯基化衍生物较好的亲核性能以及生物相容性,可以将该壳聚糖巯基化衍生物用于制备药物载体,其良好的亲核性能,利于其与靶细胞的结合,可以达到增加药效的目的;或者,利用该壳聚糖巯基化衍生物良好的抗氧化性能,将其与天然聚合物制成高分子膜,在储存保鲜领域,发挥其作用。
以上所描述的实施例是本公开一部分实施例,而不是全部的实施例。本公开的实施例的详细描述并非旨在限制要求保护的本公开的范围,而是仅仅表示本公开的选定实施例。该壳聚糖巯基化衍生物具有丰富的变化和衍生性,其适用广泛,此处仅仅列举了其可能的部分用途,并不能穷举,基于本公开中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例或者其它可能的应用方式,都属于本公开保护的范围。
(1)本公开方法制备的壳聚糖水凝胶在生物医学及组织工程领域具有重要用途,其可打印性好, 固化速度快,生物相容性好、机械强度可调、在培养基中的稳定性好,生物降解速度可调,应用范围大。
(2)本公开中既包含化学交联、又包含物理交联结构的双交联壳聚糖水凝胶,不仅制备方法简便、快捷,而且制备反应速度较快,同时无需使用毒性较强的化学交联剂,制备过程绿色、环保、安全。由本公开方法所得到的双交联壳聚糖水凝胶的固化速度快,力学强度高、生物相容性好、在培养基中的稳定性好,应用范围大。与目前常见的紫外光固化壳聚糖水凝胶、pH响应壳聚糖水凝胶、温敏型壳聚糖水凝胶、以及离子响应壳聚糖水凝胶等相比,本公开双交联壳聚糖水凝胶既提高了固化速度,也改善了壳聚糖水凝胶的机械强度和弹性。
(3)本公开实施方式的壳聚糖巯基化衍生物的制备方法的路线设计合理,操作简单可行,对设备要求低,能够高效高产率地得到壳聚糖巯基化衍生物。该壳聚糖巯基化衍生物由于巯基侧链的作用,而具有很好的亲核性能、抗氧化性能以及丰富的衍生性,其可以进一步通过亲核反应、交联反应等进行衍生,应用范围十分广泛。改性后的形成的壳聚糖巯基化衍生物在碱性条件下巯基质子离去可产生硫负离子,可与含马来酰亚胺、乙烯砜、α-β不饱和醛、酮、酸、酯等结构的其他高分子衍生物发生化学反应来制备得到水凝胶,提高了水凝胶的固化速度,也改善了水凝胶的机械强度和弹性。
Claims (20)
- 根据权利要求1所述的壳聚糖水凝胶的制备方法,其中,进行Michael加成反应之前对所述巯基化壳聚糖通过还原剂进行处理,优选地,所述还原剂为金属Zn、二硫苏糖醇或对苯二酚。
- 根据权利要求1或2所述的壳聚糖水凝胶的制备方法,其中,将α-β不饱和酰基化壳聚糖溶液与巯基化壳聚糖溶液进行Michael加成反应,优选地,所述α-β不饱和酰基化壳聚糖溶液的浓度为10~100mg/ml,PH值小于7;优选地,所述巯基化壳聚糖溶液的浓度的10~100mg/ml,PH值小于7。
- 根据权利要求1-3中任一项所述的壳聚糖水凝胶的制备方法,其中,进行所述Michael加成反应是将所述α-β不饱和酰基化壳聚糖溶液与所述巯基化壳聚糖溶液混合后,与碱溶液反应成胶。
- 根据权利要求1-4中任一项所述的壳聚糖水凝胶的制备方法,其中,所述α-β不饱和酰基化壳聚糖的制备包括以下步骤:将含有壳聚糖的酸溶液和含有酰化试剂的溶液混合后进行反应,再进行透析、冷冻干燥;其中,所述酰化试剂包括α-β不饱和酸、α-β不饱和酸酐、α-β不饱和酰氯和α-β不饱和酯中的至少一种;优选地,所述壳聚糖链上游离氨基与所述酰化试剂的摩尔比为1:1~10。
- 根据权利要求1-5中任一项所述的壳聚糖水凝胶的制备方法,其中,所述巯基化壳聚糖的制备包括以下步骤:将含有壳聚糖的溶液与含有巯基化合物的溶液在羧基活化剂的作用下进行反应;优选地,含有壳聚糖的溶液是将壳聚糖溶解于质量分数为0.01~30%的酸溶液中,含有所述巯基化合物的溶液是将所述巯基化合物,根据其溶解性,溶解于双蒸水或碱溶液或酸溶液中,所述羧基活化剂包括EDC和NHS,所述EDC和所述NHS的质量比为1~10:1;进一步优选地,所述巯基化合物与壳聚糖上氨基的摩尔比为1~10:1;优选地,反应温度为50~60℃,进一步优选为15~20℃,反应时间为4~12h,且将经过反应后的混合液进行透析、冷冻干燥;优选地,所述巯基化合物为同时含有羧基和巯基的化合物,更优选地,所述巯基化合物包括乙酰半胱氨酸、半胱氨酸、巯基丁二酸、二巯基丁二酸、2-巯基-3-吡啶甲酸中的任一种。
- 根据权利要求1-6中任一项所述的壳聚糖水凝胶的制备方法,其中,所述巯基化壳聚糖的制备是先利用所述羧基活化剂对含有所述巯基化合物的溶液中的羧基进行活化,然后混合含有所述巯基化合物的溶液和含有壳聚糖的溶液进行反应。
- 根据权利要求8所述的壳聚糖水凝胶在制作生物医用材料或组织工程材料或3D生物打印材料上的应用。
- 一种双交联壳聚糖水凝胶的制备方法,其中,所述制备方法包括:将α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶在乙醇溶液中浸泡处理,得到双交联壳聚糖水凝胶。
- 根据权利要求10所述的制备方法,其中,所述浸泡处理的时间为0-48h,但不包括0h;优选的,所述浸泡处理的时间为24h。
- 根据权利要求10-13中任一项所述的制备方法,其中,包括:在溶液条件下,将α-β不饱和酰基化壳聚糖与巯基化壳聚糖混合反应,然后与碱溶液反应,得到水凝胶。
- 根据权利要求10或11所述的制备方法,其中,所述α-β不饱和酰基化壳聚糖与巯基化壳聚糖反应所得水凝胶为根据权利要求8所述的壳聚糖水凝胶。
- 根据权利要求10-15中任一项所述的制备方法得到的双交联壳聚糖水凝胶。
- 权利要求16所述的双交联壳聚糖水凝胶在生物材料制备中的应用;优选的,所述生物材料包括:生物纤维;以及/或者,包含权利要求16所述的双交联壳聚糖水凝胶的生物材料;优选的,所述生物材料包括:生物纤维。
- 一种如权利要求18所述的壳聚糖巯基化衍生物的制备方法,其中,壳聚糖与磺酸基化合物在羧基活化剂的存在下进行反应;再通过还原剂将磺酸基还原为巯基;所述磺酸基化合物为同时具有羧基和磺酸基的化合物。
- 如权利要求18所述的壳聚糖巯基化衍生物在制备水凝胶中的应用。
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