阅读说明：本技术 一种可注射自愈合抗菌水凝胶的制备方法 (Preparation method of injectable self-healing antibacterial hydrogel ) 是由 蔡杰 谢芳 陈毅军 陆艺文 于 2019-11-04 设计创作，主要内容包括：本发明涉及一种可注射自愈合抗菌水凝胶的制备方法,采用氢氧化钾/尿素水溶液为溶剂,在常温下快速搅拌并超声后使天然蚕丝剥离为纳米纤维,然后经过分离制得均匀的蚕丝纳米纤维溶液。将蚕丝纳米纤维选择性氧化后与甲壳素季铵盐溶液或壳聚糖季铵盐溶液混合,得到可注射自愈合抗菌蚕丝纳米纤维/甲壳素或壳聚糖季铵盐水凝胶。所得水凝胶同时具有优异的生物活性、生物可降解性。(The invention relates to a preparation method of injectable self-healing antibacterial hydrogel, which adopts potassium hydroxide/urea aqueous solution as a solvent, quickly stirs at normal temperature and carries out ultrasonic treatment to peel natural silk into nano-fibers, and then the uniform silk nano-fiber solution is prepared by separation. Selectively oxidizing the silk nano-fiber, and mixing the oxidized silk nano-fiber with a chitin quaternary ammonium salt solution or a chitosan quaternary ammonium salt solution to obtain the injectable self-healing antibacterial silk nano-fiber/chitin or chitosan quaternary ammonium salt hydrogel. The obtained hydrogel has excellent bioactivity and biodegradability.)

1. A preparation method of injectable self-healing antibacterial hydrogel is characterized by comprising the following steps:

(1) adopting potassium hydroxide/urea aqueous solution as a solvent, wherein the concentration of potassium hydroxide is 10-25 wt%, the concentration of urea is 5-30 wt%, rapidly stirring for 1 min-6 h at room temperature, peeling natural silk into nanofibers after ultrasonic treatment, separating to obtain uniform silk nanofiber solution, and then selectively oxidizing the silk nanofibers;

(2) mixing the obtained silk nanofiber solution with a chitin quaternary ammonium salt solution or a chitosan quaternary ammonium salt solution at the temperature of 4-37 ℃ to gelatinize the mixture to obtain the injectable self-healing antibacterial silk nanofiber/chitin or chitosan quaternary ammonium salt hydrogel.

2. The preparation method according to claim 1, wherein the degree of substitution of the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt is 0.2-0.6, the mass fraction of the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt is 1-4 wt%, and the molar ratio of amino groups in the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt to aldehyde groups in the silk nanofibers is 0.7-1.8.

3. The preparation method according to claim 1, wherein the silk nanofiber solution obtained in the step (1) has a solid content of silk of 1 wt% to 12 wt%, and the silk nanofiber solution obtained before mixing in the step (2) is diluted or concentrated to have a solid content of 1 wt% to 3 wt%.

4. The preparation method according to claim 1, wherein the power of the ultrasonic treatment in the step (1) is 100-1200 w, and the ultrasonic time is not more than 6 hours.

5. The method of claim 1, wherein the quaternary ammonium salt of chitin is prepared by the following steps: preparing an aqueous solution containing potassium hydroxide and urea, adding chitin, and mechanically stirring at a low temperature until a transparent and clear chitin solution is obtained; dropwise adding a quaternizing agent into the chitin solution to ensure that the molar ratio of the quaternizing agent to the chitin unit is 4: 1-16: 1, reacting at the temperature of-10-40 ℃, stirring for 3-48 hours, and adding HCl to adjust to neutrality; dialyzing the reaction solution by distilled water, and freeze-drying to obtain the chitin quaternary ammonium salt.

6. The method for preparing the quaternary ammonium salt of the chitosan according to claim 1, wherein the quaternary ammonium salt of the chitosan is prepared by the following method: preparing an aqueous solution containing potassium hydroxide and urea, adding chitosan, and mechanically stirring at a low temperature until a transparent and clear chitosan solution is obtained; dropwise adding a quaternizing agent into the chitosan solution to ensure that the molar ratio of the quaternizing agent to the chitin unit is 4: 1-16: 1, the reaction temperature is-10-40 ℃, stirring for 3-48 hours, and then adding HCl to adjust to neutrality; dialyzing the reaction solution by distilled water, and freeze-drying to obtain the chitosan quaternary ammonium salt.

7. The production method according to claim 5 or 6, characterized in that: the quaternizing agent is any one or more of 2, 3-epoxypropyltrimethylammonium chloride, 2, 3-epoxypropyltripropylammonium chloride and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

8. The production method according to claim 5 or 6, characterized in that: and adding other derivatization reagents while adding the quaternization reagent, wherein the molar ratio of the other derivatization reagents to the chitin monomer unit is 4: 1-16: 1.

9. The method of claim 8, wherein: the other derivatization reagent comprises any one or more of chloroacetic acid, chloropropionic acid, chlorobutyric acid, sodium chloroacetate, ethylene oxide dimethyl sulfate, p-methyl benzene sulfonyl chloride, succinic anhydride, maleic anhydride, acrylamide and acrylonitrile.

Technical Field

The invention relates to a preparation method of injectable self-healing antibacterial hydrogel, belonging to the field of nano materials and biological application tissue engineering.

Background

In recent years, due to the fact that the dynamic chemical reversible property has wide application in aspects such as gas adsorption and separation, nanocomposite preparation, new material construction and the like, the dynamic chemical reversible property is receiving more and more attention of researchers. In addition, dynamic chemistry is also used to design and construct hydrogels with injectability or self-repair capability, and injectable hydrogels have wide applications in drug loading, cell culture, tissue engineering, and other aspects due to their outstanding advantages. In the reported construction of hydrogel suitable for cell encapsulation and biomedical tissue engineering, the injectable hydrogel with self-repairing capability has great advantages.

The quaternized chitin and quaternized chitosan are natural polysaccharide derivatives, and have excellent biodegradability, antibacterial property and bioactivity. The silk is natural animal silk protein and has excellent mechanical performance and physical and chemical properties, such as flexibility, tensile strength, air permeability, moisture permeability, slow release property and the like. The surface of the nano-scale silk nanofiber has rich aldehyde groups, thiol groups, hydroxyl groups, carboxyl groups and other groups, and the application of the silk nanofiber is expanded. The preparation method of the silk nanofiber comprises two modes of top-down and bottom-up. The bottom-up method first dissolves silk proteins using solvents such as inorganic salt solutions (lithium bromide aqueous solution (adv. mater.2017,29,1702769.), calcium chloride/ethanol aqueous solution (sci. rep.2017,7,2107.), concentrated acids (adv. sci.2017,4,1700191.) or ionic liquids (j. phys. chem.b 2017,121, 6108) 6116.) and then by self-assembly into silk protein nanofibers. In the method, hydrogen bonds among fibroin molecules are destroyed, the structure of the regenerated silk nanofiber is different from that of natural silk due to the destruction of the original silk fiber structure in the dissolving process, and the performance of the material prepared from the silk nanofiber is often inferior to that of the natural silk. On the other hand, the top-down approach involves selective dissolution of silk into silk nanofibers using weaker solvents such as hydrochloric acid/formic acid (mater. sci. eng., C2015, 48, 444-. In addition, the degummed silk is added into sodium hydroxide/urea solution precooled to-12 ℃, and is dialyzed to be neutral after being frozen and unfrozen for a plurality of times, and then is treated by ultrasonic wave, so that the silk nanofiber solution (ACS Nano 2018,12,11860-11870) can be obtained. Although the chemical properties of sodium hydroxide and potassium hydroxide are similar, silk fibers can be effectively stripped to obtain nano fibers only by adopting a circulating freezing-thawing method in a sodium hydroxide/urea aqueous solution, time and energy are consumed, the stripping efficiency is not obvious, and a plurality of micron-sized silk fibers still cannot be stripped, so that the silk fibers are difficult to be practically applied. The silk fiber can be completely stripped into the nano fiber at room temperature by selecting the potassium hydroxide/urea aqueous solution, so that the method is a 'green' process with high efficiency, low cost and energy saving. And further selectively oxidizing the silk nano-fiber to form a dynamic covalent bond with the quaternized chitin or quaternized chitosan with antibacterial property, thereby obtaining the injectable self-healing antibacterial silk nano-fiber/chitin or chitosan quaternary ammonium salt hydrogel. The novel hydrogel has wide application prospect in the biomedical material fields of wound repair, tissue engineering scaffolds, drugs, protein sustained and controlled release and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a preparation method of biodegradable silk nanofiber/chitin or chitosan quaternary ammonium salt hydrogel which has the advantages of injectability, self-healing, antibacterial property and good biological activity.

The scheme adopted by the invention for solving the technical problems is as follows:

a preparation method of injectable self-healing antibacterial hydrogel is characterized by comprising the following steps:

(1) adopting potassium hydroxide/urea aqueous solution as a solvent, wherein the concentration of potassium hydroxide is 10-25 wt%, the concentration of urea is 5-30 wt%, rapidly stirring for 1 min-6 h at room temperature, peeling natural silk into nanofibers after ultrasonic treatment, separating to obtain uniform silk nanofiber solution, and then selectively oxidizing the silk nanofibers;

(2) and mixing the obtained silk nanofiber solution with a chitin quaternary ammonium salt solution or a chitosan quaternary ammonium salt solution at the temperature of 4-37 ℃ to gelatinize the mixture to obtain the silk nanofiber reinforced injectable self-healing antibacterial hydrogel.

Preferably, the substitution degree of the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt is 0.2-0.6, the mass fraction of the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt solution is 1 wt% -4 wt%, and the ratio of amino groups in the chitin quaternary ammonium salt or the chitosan quaternary ammonium salt to aldehyde groups in the silk nanofiber is 0.7-1.8.

Preferably, the silk nano-fiber solution obtained in the step (1) has a solid content of 1 wt% to 12 wt%, and the silk nano-fiber solution obtained before mixing in the step (2) is diluted or concentrated to have a solid content of 1 wt% to 3 wt%.

Preferably, the power of the ultrasonic treatment in the step (1) is 100 w-1200 w, and the ultrasonic time is not more than 6 hours.

Preferably, before the obtained silk nano-fiber solution is mixed with chitin quaternary ammonium salt or chitosan quaternary ammonium salt, the silk nano-fibers are selectively oxidized, so that the silk nano-fibers are subjected to oxidation reaction to obtain the silk nano-fiber solution with more aldehyde groups, and the aldehyde group content in the obtained oxidized silk nano-fibers can reach 25-45%.

Preferably, the chitin quaternary ammonium salt is prepared by the following method: preparing an aqueous solution containing potassium hydroxide and urea, adding chitin, and mechanically stirring at a low temperature until a transparent and clear chitin solution is obtained; dropwise adding a quaternizing agent into the chitin solution to ensure that the molar ratio of the quaternizing agent to the chitin unit is 4: 1-16: 1, reacting at the temperature of-10-40 ℃, stirring for 3-48 hours, and adding HCl to adjust to neutrality; dialyzing the reaction solution by distilled water, and freeze-drying to obtain the chitin quaternary ammonium salt.

Preferably, the chitosan quaternary ammonium salt is prepared by the following method: preparing an aqueous solution containing potassium hydroxide and urea, adding chitosan, and mechanically stirring at a low temperature until a transparent and clear chitosan solution is obtained; dropwise adding a quaternizing agent into the chitosan solution to ensure that the molar ratio of the quaternizing agent to the chitin unit is 4: 1-16: 1, the reaction temperature is-10-40 ℃, stirring for 3-48 hours, and then adding HCl to adjust to neutrality; dialyzing the reaction solution by distilled water, and freeze-drying to obtain the chitosan quaternary ammonium salt.

Preferably, the quaternising agents are: 2, 3-epoxypropyltrimethylammonium chloride, 2, 3-epoxypropyltripropylammonium chloride, and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

Preferably, the other derivatizing agent comprises any one or more of chloroacetic acid, chloropropionic acid, chlorobutyric acid, sodium chloroacetate, ethylene oxide dimethyl sulfate, p-methyl benzene sulfonyl chloride, succinic anhydride, maleic anhydride, acrylamide, acrylonitrile.

The silk is ultrasonically dispersed by stirring at room temperature in a potassium hydroxide/urea solution system, the silk nanofiber can be simply, efficiently and quickly peeled off, the chemical structure of the silk is not damaged in the dissolving process, the surface of the obtained silk nanofiber has rich chemical groups such as-SH, -COO, -OH and-CHO, and more aldehyde groups can be obtained on the surface of the silk nanofiber through oxidation reaction. Because the surface of the quaternized chitin or chitosan molecule contains rich amino (-NH)₂) The hydrogel can react with aldehyde groups on the surface of the silk nanofiber through Schiff base to form dynamic chemical bonds, so that the injectable self-healing antibacterial hydrogel can be obtained, and meanwhile, due to the good mechanical property of the silk nanofiber, the hydrogel can be endowed with excellent mechanical strength and biological activity. The gelation time and mechanical strength of the mixed solution under the condition of body temperature can be controlled by adjusting the concentration of the material and the proportion of the functional groups. Can be added to the solution to rapidly form at body temperatureThe hydrogel can be used as a scaffold material of cells, a carrier of medicines and the like; the chitin, the chitosan derivative and the silk nanofiber have good biocompatibility and bioactivity, and the formed hydrogel can be used as a wound dressing for wound healing; therefore, the silk nanofiber/chitin or chitosan quaternary ammonium salt hydrogel is very suitable for being applied to the fields of biological application and tissue engineering.

Drawings

FIG. 1 is a diagram of the quaternized chitosan solution obtained in example 6;

FIG. 2 shows the Tyndall effect of the silk nanofiber solution obtained in example 18 under the illumination test;

fig. 3 is the injectable self-healing antimicrobial hydrogel reinforced by silk nanofibers obtained from example 22, wherein a is quaternized chitosan solution, b is silk nanofiber solution, and c is a gel rapidly formed after mixing a and b.

Detailed Description

The following examples are provided to further illustrate the present invention for better understanding, but the present invention is not limited to the following examples.

Example 1

Preparing 98g of an aqueous solution containing 20 wt% of potassium hydroxide and 4 wt% of urea, precooling to-30 ℃, adding 2g of chitin at room temperature, and mechanically stirring for 30 minutes to obtain a transparent and clear 2 wt% chitin solution; adding 5.96g of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride into the chitin solution to ensure that the molar ratio of the quaternizing agent to the chitin unit is 4:1, reacting at 25 ℃, stirring for 24 hours, and adding HCl to neutralize the reaction solution; dialyzing the reaction solution by distilled water for 7 days, freeze-drying to obtain a white fibrous chitin quaternary ammonium salt sample, and measuring the degree of substitution DQ of the sample to be 0.18, NH₂The content is 15%.

Examples 2-6 were prepared similarly to example 1, with the differences shown in Table 1 below.

TABLE 1 EXAMPLES 1-6 preparation parameters and degree of substitution and NH of the resulting quaternary ammonium salt samples₂Content (wt.)

Example 7

Preparing 98g of aqueous solution containing 18 wt% of potassium hydroxide and 6 wt% of urea, precooling to-30 ℃, adding 2g of chitosan at room temperature, and mechanically stirring for 30 minutes to obtain a transparent and clear 2 wt% chitosan solution; adding 7.53g of 3-chloro-2-hydroxypropyl trimethyl ammonium chloride into the chitosan solution, so that the molar ratio of the quaternizing agent to the chitosan unit is 4:1, reacting at-30 ℃, stirring for 24 hours, and adding HCl to neutralize the reaction solution; dialyzing the reaction solution with distilled water for 7 days, and freeze-drying to obtain a white fibrous chitosan quaternary ammonium salt sample (DQ is 0.39, NH)₂＝78％)。

Examples 8-12 were prepared similarly to example 7 with the following differences as shown in Table 2.

TABLE 2 preparation parameters of examples 7-12 and degree of substitution and NH of the resulting quaternary ammonium salt samples₂Content (wt.)

Example 13:

preparing 98g of an aqueous solution containing 20 wt% of potassium hydroxide and 4 wt% of urea, precooling to-30 ℃, adding 2g of chitin at room temperature, and mechanically stirring for 30 minutes to obtain a transparent and clear 2 wt% chitin solution; adding 3-chloro-2-hydroxypropyl trimethyl ammonium chloride into a chitin solution, then adding chloroacetic acid to ensure that the molar ratio of a quaternizing agent to a chitin unit is 4:1 and the molar ratio of chloroacetic acid to the chitin unit is 4:1, reacting at 40 ℃, stirring for 24 hours, and then adding HCl to neutralize the reaction solution; dialyzing the reaction solution by distilled water for 7 days, freezing and drying to obtain a white fibrous quaternized carboxymethyl chitin sample, and measuring the substitution degree DQ of the quaternary ammonium salt of the obtained sample to be 0.18, the substitution degree DS of carboxymethyl to be 0.15 and NH₂The content is 35%.

Example 14:

preparing 98g of aqueous solution containing 20 wt% of potassium hydroxide and 4 wt% of urea, precooling to-30 ℃, and adding 2g of formaldehyde at room temperatureMechanically stirring chitin for 30 min to obtain transparent and clear chitin solution of 2 wt%; adding 3-chloro-2-hydroxypropyl trimethyl ammonium chloride into a chitin solution, then adding ethylene oxide to ensure that the molar ratio of a quaternizing agent to a chitin unit is 4:1 and the molar ratio of the ethylene oxide to the chitin unit is 4:1, reacting at 40 ℃, stirring for 24 hours, and then adding HCl to neutralize the reaction solution; dialyzing the reaction solution by distilled water for 7 days, freezing and drying to obtain a white fibrous quaternized hydroxypropyl chitin sample, and measuring the substitution degree DQ of the quaternary ammonium salt of the obtained sample to be 0.18, the substitution degree DS of hydroxyethyl to be 0.16 and NH₂The content was 34%.

Example 15:

preparing 98g of an aqueous solution containing 20 wt% of potassium hydroxide and 4 wt% of urea, precooling to-30 ℃, adding 2g of chitin at room temperature, and mechanically stirring for 30 minutes to obtain a transparent and clear 2 wt% chitin solution; adding 2, 3-epoxypropyltripropylammonium chloride into a chitin solution, then adding chloroacetic acid to ensure that the molar ratio of a quaternizing agent to a chitin unit is 4:1 and the molar ratio of ethylene oxide to the chitin unit is 4:1, reacting at 40 ℃, stirring for 24 hours, and then adding HCl to neutralize a reaction solution; dialyzing the reaction solution by distilled water for 7 days, and freeze-drying to obtain a white fibrous quaternized hydroxypropyl chitin sample, wherein the substitution degree of the quaternary ammonium salt DQ of the obtained sample is 0.17, the substitution degree of the carboxymethyl DS is 0.16, and NH is measured₂The content was 34%.

Example 16

Preparing 98g of aqueous solution containing 18 wt% of potassium hydroxide and 6 wt% of urea, precooling to-30 ℃, adding 2g of chitosan at room temperature, and mechanically stirring for 30 minutes to obtain a transparent and clear 2 wt% chitosan solution; adding 3-chloro-2-hydroxypropyl trimethyl ammonium chloride into the chitosan solution, then adding chloroacetic acid to ensure that the molar ratio of a quaternizing agent to chitosan units is 4:1 and the molar ratio of chloroacetic acid to chitin units is 4:1, reacting at 25 ℃, stirring for 24 hours, and then adding HCl to neutralize the reaction solution; dialyzing the reaction solution by distilled water for 7 days, and freeze-drying to obtain a white fibrous carboxymethyl chitosan quaternary ammonium salt sample, wherein the substitution degree of the quaternary ammonium salt DQ of the obtained sample is 0.39, the substitution degree of the carboxymethyl DS is 0.36, and NH is measured₂The content was 78%.

Example 17:

adding 4g of silk raw material into a 10 wt% potassium hydroxide/20 wt% urea solution with the total mass of 100g, rapidly stirring for 3h at room temperature to obtain a uniformly dispersed silk fiber solution, transferring the silk fiber solution into a dialysis bag, and dialyzing with deionized water to be neutral. Then transferred to a beaker and sonicated in a cell disruptor at 600w for 30 minutes to give a light blue colored silk nanofiber solution. The resulting solution was centrifuged at 8000r/min, and the supernatant liquid was poured into a jar and stored in a refrigerator at 4 ℃ under sealed conditions. And (3) taking part of the solution for freeze-drying, and calculating the content of the silk nano-fibers to be eight thousandths of the total weight by using a weighing method. The total mass of the silk in the silk nanofiber solution is calculated according to the total volume, and then the conversion rate of the nanofiber is calculated to be 66%, and the aldehyde group content is calculated to be 9.5%.

Examples 18-21 were prepared similarly to example 17, with the differences shown in Table 3 below.

TABLE 3 preparation parameters and product parameters of examples 18-21

Example 22

The silk nanofiber solution obtained in example 21 was prepared into a 1 wt% solution, and NaIO pre-dissolved in 10mL of water was added dropwise₄(0.1g), stirring at 25 ℃ in the dark for 2h, adding 0.4mL of ethylene glycol to terminate the reaction, stirring for 1h, and dialyzing to obtain the silk nanofiber with higher aldehyde group content, wherein the aldehyde group content is 24.5%.

Example 23

The silk nanofiber solution obtained in example 21 was prepared into a 1 wt% solution, and NaIO pre-dissolved in 10mL of water was added dropwise₄(0.2g), stirring at 40 ℃ in the dark for 2h, adding 0.4mL of glycol to terminate the reaction, stirring for 1h, and dialyzing to obtain the silk nanofiber with higher aldehyde group content, wherein the aldehyde group content is 33.8%.

Example 24

Obtained by working example 21The silk nano-fiber solution is prepared into 1 wt% solution, NaIO pre-dissolved in 10mL water is added dropwise₄(0.3g), stirring at 25 ℃ in the dark for 2h, adding 0.4mL of ethylene glycol to terminate the reaction, stirring for 1h, and dialyzing to obtain the silk nanofiber with higher aldehyde group content, wherein the aldehyde group content is 42%.

Example 25

Preparing the quaternized chitin prepared in the embodiment 6 into a 2 wt% solution, taking the silk nanofiber obtained in the embodiment 14, preparing a silk nanofiber solution with a certain concentration according to the content ratio of amino groups to aldehyde groups of 0.7-1.8, mixing the two solutions with 37 ℃, and quickly curing the solution within 5min to form gel.

Examples 26-35 were prepared similarly to example 25, with the differences shown in Table 3 below.

TABLE 3 preparation parameters and product gel times for examples 22-35

While the foregoing is directed to the preferred embodiment of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.