Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Those whose specific conditions are not specified in the embodiment or examples are carried out according to the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The thiolated derivatives of chitosan according to embodiments of the present invention, and methods for preparing and using the same will be described in detail below.
A thiolated derivative of chitosan, having the general formula:
wherein R is an alkylene or substituted alkylene group.
Wherein, when R is 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 aromatic group, an ester group, a hydroxyl group and a halogenated alkyl group. That is, one hydrogen atom in the alkylene group is substituted by one of alkyl, carboxyl, amino, alkoxy, aryl, ester, hydroxyl and halogenated alkyl; or two hydrogen atoms in the alkylene are replaced by two groups of alkyl, carboxyl, amino, alkoxy, aryl, ester, hydroxyl and halogenated alkyl; two or more hydrogen atoms in the alkylene group may be substituted by two or more groups selected from the group consisting of an alkyl group, a carboxyl group, an amino group, an alkoxy group, an aromatic group, an ester group, a hydroxyl group and a halogenated alkyl group; or a plurality of hydrogen atoms in the alkylene are replaced by a plurality of same groups of alkyl, carboxyl, amino, alkoxy, aryl, ester, hydroxyl and halogenated alkyl or a plurality of different groups thereof correspondingly.
Wherein, the number of carbon atoms of R is 1 to 20, preferably 1 to 15, and more preferably 1 to 10. That is, R may be an alkylene group or a substituted alkylene group of C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20.
Because free amino and hydroxyl exist on the chitosan molecular chain, the chitosan molecular chain can be modified through reaction with carboxyl. To introduce a thiol group into a chitosan molecule, the most direct way is to react a compound having both a carboxyl group and a thiol group with chitosan. However, in the above reaction, the carboxyl group is not electrophilic enough, and therefore, it can react only with the amino group having a strong nucleophilicity in the chitosan molecule, and does not react with the hydroxyl group. In the embodiment of the invention, a sulfonic compound with both carboxyl and sulfonic group is adopted to react with chitosan, and the carboxyl of the sulfonic compound has strong electrophilicity due to the strong electron withdrawing property of sulfonic acid, and can simultaneously react with amino and primary hydroxyl in chitosan, so that the sulfonic group is introduced into a molecular chain of the chitosan, and then the sulfonic group is reduced into sulfydryl through reaction with a reducing agent, and the sulfydryl derivative of the chitosan with both the amino and the primary hydroxyl being sulfydryl is obtained. The compound having both carboxyl group and sulfonic acid group can be directly produced by hydrolysis reaction, aminolysis reaction, etc., or can be directly obtained by reducing a disulfide bond-containing compound and then severely oxidizing the reduced disulfide bond-containing compound. For example, the compound having both a carboxyl group and a sulfonic acid group may be: disulfonic acid succinic acid, sulfonic acid acetic acid, 3-sulfonic acid propionic acid, sulfonic acid succinic acid, and the like.
Specifically, the thiolated derivatives of chitosan are formed by the following reaction formula:
some embodiments of the present invention relate to a method for preparing a thiolated derivative of chitosan, which includes reacting chitosan with a sulfonic acid group compound in the presence of a carboxyl group activating agent, and then reducing the sulfonic acid group to a thiol group.
According to some embodiments, the method of preparing the thiolated derivative of chitosan comprises: and reacting the solution containing the chitosan with the solution containing the sulfonic acid group compound under the action of a carboxyl activating agent.
Specifically, the thiolated derivative of chitosan in the present embodiment may be prepared by the following method:
(1) preparation of chitosan-containing solution: dissolving chitosan in 0.01-30% acid solution.
(2) Preparation of a solution containing modifier compound: the sulfonic acid group compound is dissolved in double distilled water or alkali solution or acid solution according to the solubility, and the modifier is self solution and can be diluted by corresponding solvent for reaction operation.
(3) Adding a carboxyl activating agent into the solution containing the sulfonic acid group compound, uniformly mixing, adjusting the pH value to 4.5-6.5, and continuously mixing and stirring.
(4) And mixing the activated solution containing the sulfonic acid group compound and the solution containing the chitosan, fully and uniformly stirring, preferably transferring the mixture into a round-bottom flask, and standing the mixture at the temperature of below 50-60 ℃ for constant-temperature reaction.
(5) Adding a proper amount of reducing agent into the reaction solution after the reaction, and stirring for 24 hours at room temperature.
(6) Dialyzing the reaction solution obtained after the reaction for 2-5 days, and freeze-drying the obtained dialysis product for 2-5 days to obtain the chitosan sulfhydrylation derivative. Residual small molecular impurities can be removed by means of dialysis, so that the sulfhydryl chitosan derivative with higher purity can be obtained.
According to some embodiments, the solution containing chitosan is an acid solution. According to some embodiments, the acid solution may be an organic acid solution, preferably an acetic acid solution. Namely, the chitosan is preferably dissolved in 0.01 to 30% acetic acid solution.
According to some embodiments, the solution containing the sulfonic acid group compound is double distilled water or an alkali solution or an acid solution. According to some embodiments, the alkali solution may be a strong alkali solution, such as a sodium hydroxide solution, a potassium hydroxide solution, a calcium hydroxide solution, etc., or a weak alkali solution, such as an aqueous ammonia solution, a sodium carbonate solution, a sodium bicarbonate solution, etc., preferably a sodium hydroxide solution. The double distilled water is water obtained after primary distillation and water obtained after secondary distillation, so that the purity of the dissolved solution is higher, and the subsequent reaction effect is better. The acid solution dissolving the sulfonic acid group compound may be an organic acid solution, preferably an acetic acid solution.
According to some embodiments, the carboxyl activating agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC is a water-soluble carbodiimide, used as a carboxyl activating reagent in amide synthesis, and also used for activating phosphate groups, crosslinking proteins and nucleic acids, and preparing immunoconjugates, and is often used in combination with NHS or N-hydroxythiosuccinimide to improve coupling efficiency. NHS, N-hydroxysuccinimide, activates the carboxyl group for amide bond formation. Is used for synthesizing amino acid protective agent, semi-synthetic kanamycin and medical intermediate. The carboxyl activating agent in the embodiment of the invention can be prepared by mixing EDC and NHS according to the mass ratio of 10: 1-1: 1, which is beneficial to the two to achieve better coupling efficiency, so that the carboxyl activating effect on the sulfonic acid group compound is better.
According to some embodiments, EDC and NHS can be added to the solution containing sulfonic acid group compound under the action of a magnetic stirrer to achieve better mixing effect.
According to some embodiments, after mixing the carboxyl activating agent, the pH of the sulfonic acid group compound solution to which the carboxyl activating agent is added is adjusted with an alkali or acid solution so that the pH is 4.5 to 6.5. Preferably, the pH is adjusted with 1M sodium hydroxide or 1M hydrochloric acid solution. EDC and NHS can achieve the best activation effect on carboxyl under the condition that the pH value is 4.5-6.5.
According to some embodiments, the time for mixing and stirring is 10-150 min after the pH value is adjusted, so that the carboxyl activating agent can fully activate the carboxyl of the sulfonic compound, and the chitosan can be better modified.
According to some embodiments, the reaction temperature is preferably 55 to 60 ℃, and more preferably 55 ℃. The reaction time can be 1-8 h, preferably 2-6 h, and more preferably 4-5 h.
In the above process, the steps (2) and (3) may be performed first, and then the step (1) may be performed, without affecting the formation of the final product.
The method for preparing the thiolated derivative of chitosan in the embodiment of the invention has the advantages of simple operation process and mild reaction conditions. It provides a simple and feasible new method for preparing a new sulfhydryl derivative of chitosan and hydrogel. The obtained sulfhydryl derivatives of chitosan can be used in the fields of regenerative medicine, tissue engineering scaffolds and medical health, and have wide application range.
The thiolated derivatives of chitosan may also be reacted with small molecules or nanoparticles to produce other chemical materials.
The chitosan sulfhydrylation derivative obtained by the preparation method can be applied to the preparation of hydrogel, and the chitosan sulfhydrylation derivative and the maleylation chitosan can be mixed to prepare the hydrogel.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The reaction equation of the thiolated derivative of chitosan in this example is:
the specific reaction steps are as follows:
(1) 0.3g of chitosan is dissolved in 0.01 percent of acetic acid solution by mass fraction to obtain chitosan acetic acid solution.
(2) 5ml of sulfonic acid succinic acid solution is taken and diluted to 50ml by adding double distilled water.
(3) Adding 0.8g EDC and 0.2g NHS into the sulfonic succinic acid solution under the action of a magnetic stirrer, mixing uniformly, adjusting the pH of the mixed solution to 6.5 with 1M dilute hydrochloric acid, and stirring the mixed solution for 30min to activate carboxyl sufficiently.
(4) Mixing the sulfonic succinic acid solution after carboxyl activation with the chitosan acetic acid solution, stirring for 10min, stirring uniformly, transferring the mixed solution into a round-bottom flask, and reacting for 5h at the constant temperature of 55 ℃.
(5) The metallic zinc particles with the surface zinc oxide treated by hydrochloric acid are added into the reaction solution and stirred for 24 hours at room temperature.
(6) Dialyzing the reacted reaction solution for 3d, and freeze-drying for 3d to obtain the chitosan sulfhydrylation derivative.
Example 2
The reaction equation of the thiolated derivative of chitosan in this example is:
the specific reaction steps are as follows:
(1) 4.5g of chitosan was dissolved in an acetic acid solution with a mass fraction of 30% to obtain a chitosan acetic acid solution.
(2) 2.8g of 2-sulfonic acid nicotinic acid is taken and diluted to 50ml by adding double distilled water.
(3) Adding 1.6g EDC and 0.4g NHS into the 2-sulfonic niacin solution under the action of a magnetic stirrer, fully and uniformly mixing, adjusting the pH of the mixed solution to 6.5 by using 1M dilute hydrochloric acid, and stirring the mixed solution for 30min to fully activate carboxyl.
(4) Mixing the 2-sulfonic acid group nicotinic acid solution after carboxyl activation and the chitosan acetic acid solution, stirring for 10min, stirring uniformly, transferring the mixed solution into a round bottom flask, and reacting for 3h at the constant temperature of 60 ℃.
(5) The metallic zinc particles with the surface zinc oxide treated by hydrochloric acid are added into the reaction solution and stirred for 24 hours at room temperature.
(6) Dialyzing the reacted reaction solution for 3d, and freeze-drying for 3d to obtain the chitosan sulfhydrylation derivative.
Example 3
The reaction equation of the thiolated derivative of chitosan in this example is:
the specific reaction steps are as follows:
(1) 2.1g of chitosan was dissolved in an acetic acid solution with a mass fraction of 2% to obtain a chitosan acetic acid solution.
(2) 4.5g of 3-sulfonic propionic acid was diluted to 50ml with double distilled water.
(3) Adding 1.0g EDC and 1.0g NHS into the 3-sulfonic propionic acid solution under the action of a magnetic stirrer, mixing uniformly, adjusting the pH of the mixed solution to 5.5 with 1M dilute hydrochloric acid, and stirring the mixed solution for 60min to activate carboxyl sufficiently.
(4) Mixing the 3-sulfonic propionic acid solution after carboxyl activation with the chitosan acetic acid solution, stirring for 10min, stirring uniformly, transferring the mixed solution into a round-bottom flask, and reacting for 8h at a constant temperature of 50 ℃.
(5) The metallic zinc particles with the surface zinc oxide treated by hydrochloric acid are added into the reaction solution and stirred for 24 hours at room temperature.
(6) Dialyzing the reacted reaction solution for 3d, and freeze-drying for 3d to obtain the chitosan sulfhydrylation derivative.
Example 4
The reaction equation of the thiolated derivative of chitosan in this example is:
the specific reaction steps are as follows:
(1) 0.15g of chitosan was dissolved in 0.01% by mass of acetic acid solution to obtain a chitosan acetic acid solution.
(2) 0.09g of sulfonic acid acetic acid was diluted to 50ml with double distilled water.
(3) 0.090g EDC and 0.009g NHS were added into the above sulfonic acid group acetic acid solution at a time under the action of a magnetic stirrer, mixed well, the pH of the above mixed solution was adjusted to 6.5 with 1M dilute hydrochloric acid, and stirred for another 30min to activate the carboxyl group sufficiently.
(4) Adding the activated sulfonic acid group acetic acid solution into chitosan acetic acid solution, stirring for 150min, stirring uniformly, transferring the mixed solution into a round-bottom flask, and reacting for 5h at constant temperature below 55 ℃.
(5) The metallic zinc particles with the surface zinc oxide treated by hydrochloric acid are added into the reaction solution and stirred for 24 hours at room temperature.
(6) Dialyzing the reacted reaction solution for 3d, and freeze-drying for 3d to obtain the chitosan sulfhydrylation derivative.
Example 5
Accurately weighing 1.5g of chitosan, dissolving the chitosan in 0.01 mass percent acetic acid solution, basically uniformly dissolving the chitosan under the action of a magnetic stirrer, and then placing the chitosan in an ultrasonic oscillator for ultrasonic treatment for about 30 min. 1.8g of maleic anhydride was weighed out accurately, and 5ml of acetone was added to dissolve completely. Slowly adding the dissolved maleic anhydride into the chitosan acetic acid solution at a constant speed, and stirring for about 20min under a magnetic stirrer to fully and uniformly mix. Transferring the uniformly mixed liquid into a 150ml round-bottom flask, placing the flask in a heat collection type magnetic stirrer, setting the temperature at 40 ℃, and heating at constant temperature for reaction for 2 hours. Dialyzing for three days after the reaction is stopped, and changing the dialyzate every 5 hours. The MCS product was obtained after about three days of lyophilization.
The chemical reaction formula is as follows:
accurately weighing MCS 60mg, dissolving with acetic acid solution with a certain mass fraction, ultrasonically oscillating, and removing bubbles with nitrogen to prepare solution with the concentration of 10 mg/ml. 60mg of the thiolated derivative (TCS) of chitosan in example 1 was weighed out accurately, dissolved in sodium hydroxide solution, subjected to ultrasonic oscillation, bubbled with nitrogen gas, prepared into a solution with a concentration of 10mg/ml, treated with a reducing agent for about 10min, and mixed uniformly by oscillation. And (3) injecting the prepared MCS and TCS solution on a 3D printer by using a sample injection system, controlling the sample injection speed and the printing mode by using a computer program, and uniformly mixing to prepare the 3D bioprinting hydrogel.
The Fourier infrared analysis spectra of the chitosan and the thiolated chitosan derivative in the example 1 are respectively shown in the figure 1, and the comparison analysis of the chitosan and the thiolated chitosan infrared spectra in the figure 1 shows that the thiolated chitosan has the wave number of 3400cm-1Nearby, the-OH stretching vibration on the carboxyl group can be seen; at 2900cm-1Nearby, C-H stretching vibration introduced on the branched chain is shown; at 1500cm-1~1650cm-1The modified chitosan amide band is strengthened by the nearby amide I band and amide II band; at 1100cm-1In the vicinity, the modified chitosan has obvious C-O stretching vibration peak at the position due to the introduction of carboxyl; at 2100cm-1nearby-NH3 +The free amino group is reduced and the protonated amino group content is reduced after the chitosan is modified, and-NH is reduced3 +The peak intensity of the absorption band of (a) is significantly reduced, and the above phenomenon indicates that the thiol group is successfully introduced into the chitosan chain. And meanwhile, carrying out Scanning Electron Microscope (SEM) analysis on the surface structure and the surface aperture of the cured hydrogel through a SEM. The results are shown in FIG. 2 and FIG. 3 in sequence. SEM analysis shows that a plurality of crosslinked holes appear on the surface of the cured hydrogel, which indicates that the crosslinking reaction is completely performed, and further indicates that the prepared sulfhydryl chitosan derivative can successfully prepare the fast-curing hydrogel.
Further, the hydrogel obtained in example 5 was tested with an instron mechanical testing machine, a 10N sensor was used, placed on a sensor compression substrate, test parameters were set according to the size of the sample, compression was stopped after a sudden change in the test curve occurred, and the system automatically obtained the elastic modulus value.
The test parameters are: length 10mm, width 8mm, height 5mm, compression rate 0.8 mm/min. As shown in FIG. 4, the test results showed that the hydrogel was broken at a compression rate of 75% and a compressive stress of about 700kPa, and thus, it was seen that the hydrogel had superior mechanical strength and elastic properties.
In summary, in the embodiment of the present invention, a sulfonic acid group compound containing both a sulfonic acid group and a carboxyl group is used as a modifier, and under the action of a carboxyl group activator, the sulfonic acid group is successfully introduced into amino groups and primary hydroxyl groups on a molecular chain of chitosan, and is further reduced to obtain thiol groups. The preparation method has the advantages of reasonable route design, simple and feasible operation, low equipment requirement and capability of obtaining the thiolated chitosan derivative with high efficiency and high yield. The chitosan sulfhydrylation derivative has good nucleophilic performance, oxidation resistance and rich derivatization due to the action of sulfhydryl side chains, can be further derivatized through nucleophilic reaction, crosslinking reaction and the like, and has a wide application range. For example, the thiol-modified chitosan derivative reacts with other high molecular derivatives containing maleimide, vinyl sulfone, alpha-beta unsaturated aldehyde, ketone, acid, ester, etc. to prepare fast-curing hydrogel, which has important application in regenerative medicine and tissue engineering fields. For another example, due to the good nucleophilic performance and biocompatibility of the thiolated derivative of chitosan, the thiolated derivative of chitosan can be used for preparing a drug carrier, the good nucleophilic performance of the thiolated derivative of chitosan is beneficial to the combination of the thiolated derivative of chitosan and target cells, and the purpose of increasing the drug effect can be achieved; or the chitosan sulfhydrylation derivative and natural polymers are prepared into a macromolecular membrane by utilizing the good oxidation resistance of the chitosan sulfhydrylation derivative, and the function of the macromolecular membrane is played in the field of storage and preservation.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. The thiol-modified chitosan derivative has rich variations and derivations, and is widely applicable, and only some possible applications thereof are listed here, but not exhaustive, and all other embodiments or other possible application modes obtained by those skilled in the art without creative efforts will fall within the protection scope of the present invention based on the embodiments of the present invention.