阅读说明：本技术 一种甲壳素多层水凝胶材料的制备方法 (Preparation method of chitin multilayer hydrogel material ) 是由 贺盟 薛友财 张新疆 冯萌娜 张萌 王成双 王金山 吴刘益 张伟 贾正涛 于 2020-05-06 设计创作，主要内容包括：本发明公开了一种甲壳素多层水凝胶材料的制备方法,将琼脂棒先置于戊二醛-醋酸溶液中浸泡,再放入甲壳素溶液中浸泡；重复浸入甲壳素溶液和戊二醛-醋酸溶液,得到需要水凝胶的层数。该方法通过戊二醛对甲壳素分子链上氨基的化学交联以及甲壳素分子链间强氢键相互作用,可以备出具有清晰层结构的甲壳素凝胶层。通过间歇式凝固法和调控实验条件,可制得层厚可控的多层甲壳素水凝胶。多层甲壳素水凝胶在医疗和药物控制释放等领域具有应用前景。本发明的方法所制得多层水凝胶具有良好的生物相溶性,并且可以控制层厚,规整的多层结构可降解。本发明的方法工艺过程简单无污染,易于工业化。(The invention discloses a preparation method of a chitin multilayer hydrogel material, which comprises the steps of soaking an agar rod in glutaraldehyde-acetic acid solution, and then soaking the agar rod in chitin solution; repeatedly immersing the hydrogel into chitin solution and glutaraldehyde-acetic acid solution to obtain the number of layers of hydrogel. The method can prepare the chitin gel layer with a clear layer structure through chemical crosslinking of amino groups on a chitin molecular chain by glutaraldehyde and strong hydrogen bond interaction among chitin molecular chains. The multilayer chitin hydrogel with controllable layer thickness can be prepared by an intermittent solidification method and regulating and controlling experimental conditions. The multilayer chitin hydrogel has application prospects in the fields of medical treatment, drug controlled release and the like. The multilayer hydrogel prepared by the method has good biocompatibility, can control the thickness of the layer, and has a regular multilayer structure and is degradable. The method has simple process without pollution and is easy for industrialization.)

1. A preparation method of a chitin multilayer hydrogel material is characterized by comprising the following steps:

s1, preparing a chitin solution with the mass concentration of 2-3% and a glutaraldehyde-acetic acid solution with the mass concentration of 0.5-4%;

s2, placing the agar template in a glutaraldehyde-acetic acid solution for soaking, taking out, then placing in a chitin solution for soaking, and taking out after solidification;

and S3, repeating the step S2 for a plurality of times to obtain the required number of layers.

2. The method of claim 1, wherein the chitin solution comprises NaOH, urea, chitin and water, wherein: the mass ratio of NaOH, urea, chitin and water is 11:4:2:83 or 11:4:3: 82.

3. The method for preparing the chitin multilayer hydrogel material according to claim 2, wherein the preparation method of the chitin solution comprises the following steps: dispersing chitin in an aqueous solution of NaOH/urea, putting the chitin into an ultrasonic cleaning machine, performing ultrasonic treatment until the chitin is completely dispersed, and then freezing the solution; taking out every 2 hours, stirring the mixture on a powerful electric stirrer until no ice cluster exists, and then continuously freezing the mixture; repeatedly stirring for 3-4 times, centrifuging the clear and transparent chitin solution in a centrifuge, and taking out the supernatant obtained after centrifugation, namely the chitin solution for later use.

4. The method of claim 3, wherein the preparation of the chitin solution comprises subjecting the chitin solution to ultrasonic treatment in an ultrasonic cleaning machine for 5 minutes.

5. The method of claim 3, wherein the temperature of the freezing environment is-30 ℃ during the preparation of the chitin solution.

6. The method for preparing the chitin multilayer hydrogel material according to claim 3, wherein the preparation method of the chitin solution comprises centrifuging at-5 deg.C and 7000 rpm for 10 minutes.

7. The method for preparing the chitin multilayer hydrogel material according to claim 1, wherein in the step S2, the agar template is soaked in glutaraldehyde-acetic acid solution for 1-9 minutes.

8. The method for preparing the chitin multilayer hydrogel material according to claim 1, wherein in the step S2, the agar template is soaked in the chitin solution for 1-9 minutes.

9. The method for preparing the chitin multilayer hydrogel material according to claim 1, wherein in step S2, the agar template is a rod-shaped agar, and the diameter of the rod-shaped agar is 2-5 mm.

10. The method of claim 1, wherein in step S2, the agar template is formed by solidifying an agar aqueous solution at 95 ℃ in a refrigerator at 5 ℃.

Technical Field

The invention belongs to the field of renewable resources of natural high polymer materials, and particularly relates to a preparation method of a chitin multilayer hydrogel material.

Background

The multilayer hydrogel is a novel gel material, has an inner cavity and a complex internal structure, and can be used for storing medicines and cells. Moreover, it generally has controlled physical and chemical properties. Therefore, multilayered hydrogels have important applications in the biomedical field. By simulating the structures of blood vessels and onions, the multi-layer hydrogel similar to a rod shape and an onion shape can be successfully prepared, and can be used in the fields of tissue engineering, nerve cell culture micro-patterns, cell biosensors, drug release and the like. In the field of drug delivery, multi-layered gels have long been precedent and can be used as embedding materials to reach deeper sites with drugs. Compared with the traditional medicine carrying system, the medicine carrying system with the multilayer embedding materials has the following advantages: (1) controlling the release of the drug, controlling the duration of the action of the drug and avoiding the side effects of the drug release as required; (2) the medicine can reach the affected part accurately, and has high curative effect and no medicine waste and side effect on non-affected part.

Chitin is a renewable natural polymer with rich content, and has been successfully prepared into a series of functional materials, such as fibers, microspheres, films and hydrogels, and chitin-based materials have excellent biocompatibility and biodegradability. However, because the preparation difficulty of the multilayer chitin hydrogel is large, no report is found at present.

Disclosure of Invention

The invention provides a preparation method of a chitin multilayer hydrogel material, which aims to solve the problems in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of a chitin multilayer hydrogel material comprises the following steps:

s1, preparing a chitin solution with the mass concentration of 2-3% and a glutaraldehyde-acetic acid solution with the mass concentration of 0.5-4%;

s2, placing the agar template in a glutaraldehyde-acetic acid solution for soaking, taking out, then placing in a chitin solution for soaking, and taking out after solidification;

and S3, repeating the step S2 for a plurality of times to obtain the required number of layers.

As a preferred mode, the chitin solution comprises NaOH, urea, chitin and water, wherein: the mass ratio of NaOH, urea, chitin and water is 11:4:2:83 or 11:4:3: 82. The mass concentration of chitin in the chitin solution is 2-3%, and after multiple attempts, the mass concentration of the chitin is too low, and the thickness of gel is too small; the mass concentration of the chitin is too high, and the viscosity is relatively high, so that a regular multilayer structure is not favorably formed.

As a preferred mode, the preparation method of the chitin solution comprises the following steps: dispersing chitin in an aqueous solution of NaOH/urea, putting the chitin into an ultrasonic cleaning machine, performing ultrasonic treatment until the chitin is completely dispersed, and then freezing the solution; taking out every 2 hours, stirring the mixture on a powerful electric stirrer until no ice cluster exists, and then continuously freezing the mixture; repeatedly stirring for 3-4 times, centrifuging the clear and transparent chitin solution in a centrifuge, and taking out the supernatant obtained after centrifugation, namely the chitin solution for later use.

As a preferable mode, in the preparation method of the chitin solution, the time of ultrasound in an ultrasonic cleaning machine is 5 minutes.

As a preferred mode, in the preparation method of the chitin solution, the temperature of a freezing environment is-30 ℃.

As a preferred mode, in the preparation method of the chitin solution, the centrifugal environment temperature is-5 ℃, the rotating speed is 7000 r/min, and the centrifugal time is 10 minutes.

In a preferable mode, in step S2, the agar template is soaked in the glutaraldehyde-acetic acid solution for 1-9 minutes.

In a preferable mode, in the step S2, the agar template is soaked in the chitin solution for 1-9 minutes.

In a preferred embodiment, in step S2, the agar template is a bar-shaped agar, and the diameter of the bar-shaped agar is 2-5 mm.

In a preferred embodiment, in step S2, the agar template is formed by solidifying an agar aqueous solution at 95 ℃ in a refrigerator at 5 ℃.

The method is characterized in that the solidification and assembly of the chitin hydrogel solution layer by layer are regulated and controlled by an agar template loaded with glutaraldehyde-acetic acid, and the thickness of the gel layer is regulated and controlled by changing experimental conditions.

Compared with the prior art, the invention has the following beneficial effects:

the chitin solution obtained in the invention is regulated and controlled by an agar template loaded with glutaraldehyde-acetic acid solution to solidify and assemble each layer of hydrogel, and multilayer tubular gel is prepared. The thickness of each layer of chitin hydrogel is regulated and controlled by adjusting the concentration of chitin and glutaraldehyde-acetic acid solution and the soaking time of the agar template in the two solutions.

The method of the invention adopts chitin to dissolve to obtain the solution, and utilizes an agar rod loaded with glutaric acid-acetic acid as a template to regulate and control the layer-by-layer gelation and assembly of the chitin solution. In the gelation process, through the induction of the concentration gradients of glutaric acid-acetic acid solution inside and outside the agar plate and chitin solution (the concentration of glutaric acid-acetic acid in the agar plate is high, and the concentration of chitin outside the agar plate is high), glutaraldehyde continuously migrates into the chitin solution from the agar rod, glutaraldehyde can form C = N double bond with amino on a chitin molecular chain to crosslink chitin through a chemical method, and meanwhile, acetic acid can gradually destroy a tubular inclusion compound around the chitin molecules in the solution to expose the hydroxyl of the chitin molecules. The thickness of each layer of hydrogel is controlled by the difference of the concentration of the chitin solution and the concentration of the glutaric acid-acetic acid solution, and the thickness can also be changed by the soaking time. It has now been possible to prepare multi-layered hydrogels with each layer having a thickness of about 20-33 μm. The drug delivery device has good application prospect in medical treatment and drug release utilization due to the fact that the drug delivery device has an internal cavity for storing drugs and cells and a complex internal structure.

The multilayer hydrogel prepared by the method has good biocompatibility, can control the thickness of the layer, and has a regular multilayer structure and is degradable. The method has simple process without pollution and is easy for industrialization.

Drawings

FIG. 1 shows the thickness of the chitin gel layer observed after the agar stick is soaked in 2wt% glutaraldehyde-acetic acid solution for two minutes and then soaked in 2wt% chitin solution for different times; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 1 minute; FIG. b is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 2 minutes; FIG. c is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 3 minutes; FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 4 minutes; FIG. e is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 5 minutes; FIG. f is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 6 minutes; FIG. g is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 7 minutes; FIG. h is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 8 minutes; FIG. (i) is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 2wt% chitin for 9 minutes; graph (j) is a line graph obtained by corresponding data in graphs (a) - (i).

FIG. 2 is a graph showing the thickness of a gel layer of chitin observed after an agar stick is first soaked in a 2wt% glutaraldehyde-acetic acid solution for two minutes and then soaked in a 3wt% chitin solution for different times; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 1 minute; FIG. b is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 2 minutes; FIG. c is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 3 minutes; FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 4 minutes; FIG. e is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 5 minutes; FIG. f is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 6 minutes; FIG. g is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 7 minutes; FIG. h is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 8 minutes; FIG. (i) is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for two minutes and 3wt% chitin for 9 minutes; graph (j) is a line graph obtained by corresponding data in graphs (a) - (i).

FIG. 3 shows the thickness of a single chitin gel layer observed after an agar stick is immersed in glutaric acid-acetic acid solutions of different concentrations for 2 minutes and immersed in a 2wt% chitin solution for 2 minutes; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 0.5wt% glutaraldehyde-acetic acid for 2 minutes and 2wt% chitin for 2 minutes; FIG. b is a photograph of a single-layer hydrogel obtained by soaking 1wt% glutaraldehyde-acetic acid for 2 minutes and 2wt% chitin for 2 minutes; FIG. (c) is a photograph of a single-layer hydrogel obtained by soaking 1.5wt% glutaraldehyde-acetic acid for 2 minutes and 2wt% chitin for 2 minutes; FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes and 2wt% chitin for 2 minutes; and (e) is a line graph obtained by corresponding data in the four graphs in the graphs (a-d).

FIG. 4 shows the thickness of a single chitin gel layer observed after an agar stick is immersed in glutaric acid-acetic acid solutions of different concentrations for 3 minutes and immersed in a 2wt% chitin solution for 2 minutes; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 0.5wt% glutaraldehyde-acetic acid for 3 minutes and 2wt% chitin for 2 minutes; FIG. b is a photograph of a single-layer hydrogel obtained by soaking 1wt% glutaraldehyde-acetic acid for 3 minutes and 2wt% chitin for 2 minutes; FIG. (c) is a photograph of a single-layer hydrogel obtained by soaking 1.5wt% glutaraldehyde-acetic acid for 3 minutes and 2wt% chitin for 2 minutes; FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 3 minutes and 2wt% chitin for 2 minutes; and (e) is a line graph obtained by corresponding data in the four graphs in the graphs (a-d).

FIG. 5 shows the thickness of a single chitin gel layer observed after an agar stick is immersed in glutaric acid-acetic acid solutions of different concentrations for 4 minutes and immersed in a 2wt% chitin solution for 2 minutes; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 0.5wt% glutaraldehyde-acetic acid for 4 minutes and 2wt% chitin for 2 minutes; FIG. b is a photograph of a single-layer hydrogel obtained by soaking 1wt% glutaraldehyde-acetic acid for 4 minutes and 2wt% chitin for 2 minutes; FIG. (c) is a photograph of a single-layer hydrogel obtained by soaking 1.5wt% glutaraldehyde-acetic acid for 4 minutes and 2wt% chitin for 2 minutes; FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 4 minutes and 2wt% chitin for 2 minutes; and (e) is a line graph obtained by corresponding data in the four graphs in the graphs (a-d).

FIG. 6 is a graph showing the thickness of a single-layer chitin gel layer formed by immersing an agar rod in a 2wt% glutaraldehyde-acetic acid solution for 2 minutes, then immersing the agar rod in a 2wt% chitin solution for 2 minutes, taking out the agar rod, and then immersing the agar rod in a 2wt% glutaraldehyde-acetic acid solution for different times; wherein: FIG. a is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes, 2wt% chitin for 2 minutes, and 2wt% glutaraldehyde-acetic acid for 1 minute, FIG. b is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes, 2wt% chitin for 2 minutes, and 2wt% glutaraldehyde-acetic acid for 2 minutes, FIG. c is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes, 2wt% chitin for 3 minutes, and 2wt% glutaraldehyde-acetic acid for 1 minute, FIG. d is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes, 2wt% chitin for 2 minutes, and 2wt% glutaraldehyde-acetic acid for 4 minutes, and FIG. e is a photograph of a single-layer hydrogel obtained by soaking 2wt% glutaraldehyde-acetic acid for 2 minutes, 2wt% chitin is soaked for 2 minutes, 2wt% glutaraldehyde-acetic acid is soaked for 5 minutes to obtain a single-layer hydrogel photo, the graph (f) is the single-layer hydrogel photo obtained by 2wt% glutaraldehyde-acetic acid being soaked for 2 minutes, 2wt% chitin is soaked for 2 minutes, 2wt% glutaraldehyde-acetic acid is soaked for 6 minutes, the graph (g) is the single-layer hydrogel photo obtained by 2wt% glutaraldehyde-acetic acid being soaked for 2 minutes, 2wt% chitin is soaked for 2 minutes, 2wt% glutaraldehyde-acetic acid is soaked for 7 minutes, the graph (h) is the single-layer hydrogel photo obtained by 2wt% glutaraldehyde-acetic acid being soaked for 2 minutes, 2wt% chitin is soaked for 2 minutes, 2wt% glutaraldehyde-acetic acid is soaked for 8 minutes, the graph (i) is 2wt% glutaraldehyde-acetic acid being soaked for 2 minutes, 2wt% chitin is soaked for 2 minutes, and (f) taking a photo of the monolayer hydrogel obtained by soaking the monolayer hydrogel for 9 minutes in 2wt% glutaraldehyde-acetic acid, wherein the picture (j) is a line graph obtained by corresponding data of the nine pictures (a-i).

Fig. 7 is a comparison of 3 layers of chitin hydrogel immersed in different reagents, respectively, wherein: (a) the reagents (a) and (f) are 10wt% NaOH solutions, the reagents (b) and (g) are 10wt% acetic acid solutions, the reagents (c) and (h) are ethanol, the reagents (d) and (i) are N-dimethylformamide, the reagents (e) and (j) are acetone, the pictures obtained before soaking for 24 hours are (a) to (e), and the pictures obtained after soaking for 24 hours are (f) to (j).

Detailed Description

The present invention will be further described with reference to the following examples.

The starting materials used in this embodiment are all known compounds and are commercially available. The thickness of the multilayer gel was measured by light microscopy.