阅读说明：本技术 一种壳聚糖-石墨烯复合凝胶及其制备方法 (Chitosan-graphene composite gel and preparation method thereof ) 是由 胡堃 胡苗苗 杨桂娟 王海波 李路海 危岩 于 2020-12-16 设计创作，主要内容包括：本发明开发了一种壳聚糖-石墨烯复合凝胶及其制备方法。本发明先将壳聚糖基复合水凝胶和石墨烯各自的优势结合起来,开发具有层次结构和排列纤维结构的复合水凝胶。首先对凝胶交联剂原料PEG进行双端醛基化,壳聚糖作为多官能团大分子,nHAC掺杂其中增强了壳聚糖基复合水凝胶的粘度,石墨烯材料保留复合水凝胶可注射性能的同时增强了力学性能。(The invention discloses a chitosan-graphene composite gel and a preparation method thereof. According to the invention, the advantages of chitosan-based composite hydrogel and graphene are combined to develop the composite hydrogel with a hierarchical structure and a fiber arrangement structure. Firstly, double-end aldehyde group is carried out on PEG which is a raw material of a gel cross-linking agent, chitosan is used as a polyfunctional macromolecule, nHAC is doped in the chitosan-based composite hydrogel to enhance the viscosity of the chitosan-based composite hydrogel, and the graphene material maintains the injectable performance of the composite hydrogel and enhances the mechanical property of the composite hydrogel.)

1. The preparation method of the chitosan-graphene composite gel is characterized by comprising the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, and placing the solution in a constant-temperature water bath kettle to be stirred and dissolved to obtain a chitosan solution;

s2, preparation of mineralized collagen powder: slowly dripping 0.055mol of PO into the collagen solution₄ ^3-The solution of (1); stirring the mixture at 25 ℃, and simultaneously dropwise adding a calcium ion aqueous solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process; standing for 4 hours, removing supernatant, washing, filtering, freeze-drying and grinding to obtain mineralized collagen powder;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

2. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: the mass ratio of the mineralized collagen powder to the chitosan solution in the step S3 is 1% -4%.

3. The method for preparing chitosan-graphene composite gel according to claim 1, wherein the temperature of the constant temperature water bath in the step S1 is 20-30 ℃.

4. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: the stirring time of the step S1 is 1-2 hours.

5. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: in step S2, an aqueous solution of sodium hydroxide is added dropwise so that the pH value of the solution becomes 6 to 8.

6. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66.

7. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: PO in the step S3₄ ^3-The solution is phosphoric acid solution.

8. The preparation method of chitosan-graphene composite gel according to claim 1, characterized in that: the calcium ion aqueous solution in step S3 is a calcium chloride solution.

9. A chitosan-graphene composite gel prepared by the preparation method of any one of claims 1 to 8.

Technical Field

The invention belongs to the technical field of materials, and particularly relates to chitosan-graphene composite gel and a preparation method thereof.

Background

The hydrogel has the capability of accurately controlling the physical and chemical properties and easily integrating nano materials, and has become a key platform for soft tissue engineering application of skeletal muscle and the like. They are popular biomaterials that provide an ideal microenvironment for the initial survival and proliferation of cells. Hydrogels can add biological or physical factors without affecting their flexibility and mechanical properties, which can offset the limitations of other scaffolding methods in bone tissue engineering. However, new strategies are needed to simultaneously implement combinations of biophysical factors and complex hierarchies. Chitosan-based hydrogels have been extensively studied as an injectable, degradable, bioactive bone regeneration system. Chitosan has cationic properties due to the presence of pendant amine groups in its molecular structure, making it soluble in water under mildly acidic conditions. The chitosan hydrogel has elasticity and elasticity, is similar to natural tissues, and can easily adopt the provided space geometry. One disadvantage of chitosan hydrogels is poor mechanical properties, but this disadvantage can be overcome by compounding with other materials.

Recently, more and more work is found to explore the potential of the application of graphene, a two-dimensional carbon allotrope, in biomedicine and regeneration engineering. The prototypes proposed to date encompass imaging, drug delivery, antimicrobial properties and tissue engineering. Among the various three-dimensional macrostructures, graphene hydrogels are composed of an interconnected porous network with a large specific surface area, and are of particular interest. These hydrogels provide multidimensional ion/electron transport pathways with inherent properties of graphene, making them potential candidates for supercapacitor electrodes. The biocompatibility and unique electrical, mechanical and thermal properties of graphene-based materials have made them show great potential in biomedical applications. Graphene is a two-dimensional (2D) material consisting of sp2 hybridized carbon atoms with a single atomic layer, with a large number of oxygen-containing groups on the basal plane, such as hydroxyl, epoxy, and carboxyl groups, and is one of the most valuable materials found in the 21 st century. The specific structure of the high specific surface area graphene, as well as the interactions and polymer chains between the graphene, make it suitable for development as a nanocomposite, which can promote cell proliferation, adhesion and differentiation with little or no cytotoxicity.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a chitosan-graphene composite gel and a preparation method thereof. According to the invention, the advantages of chitosan-based composite hydrogel and graphene are combined to develop the composite hydrogel with a hierarchical structure and a fiber arrangement structure. Firstly, double-end aldehyde group is carried out on PEG which is a raw material of a gel cross-linking agent, chitosan is used as a polyfunctional macromolecule, nHAC is doped in the chitosan-based composite hydrogel to enhance the viscosity of the chitosan-based composite hydrogel, and the graphene material maintains the injectable performance of the composite hydrogel and enhances the mechanical property of the composite hydrogel.

The purpose of the invention is realized by the following scheme:

the invention provides a preparation method of chitosan-graphene composite gel, which comprises the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, and placing the solution in a constant-temperature water bath kettle to be stirred and dissolved to obtain a chitosan solution;

s2, preparation of mineralized collagen powder: slowly dripping 0.055mol of PO into the collagen solution₄ ^3-The solution of (1); stirring the mixture at 25 ℃, and simultaneously dropwise adding a calcium ion aqueous solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process; standing for 4 hr, removing supernatant, washing, filtering,freeze drying and grinding to obtain mineralized collagen powder;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

Further, the mass ratio of the mineralized collagen powder to the chitosan solution in the step S3 is 1% -4%.

Further, the temperature of the constant temperature water bath in the step S1 is 20-30 ℃.

Further, the stirring time of step S1 is 1-2 hours.

Further, in step S2, an aqueous solution of sodium hydroxide is added dropwise so that the pH of the solution becomes 6 to 8.

Further, the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66.

Further, PO in the step S3₄ ^3-The solution is phosphoric acid solution.

Further, in the step S3, the calcium ion aqueous solution is a calcium chloride solution.

The invention also discloses the chitosan-graphene composite gel prepared by the preparation method.

The invention has the beneficial effects that:

the beneficial effects and principles of the present application are written.

1. The invention firstly carries out one-step esterification reaction based on chitosan with excellent biocompatibility and polyethylene glycol with excellent biological safety, the two ends of the polyethylene glycol are conjugated to generate polyethylene glycol (DF-PEG) with two benzaldehyde end caps, then the chitosan composite hydrogel is upgraded, and a cross-linked composite network is formed by chitosan and polyethylene glycol modifier (DF-PEG). By controlling the system proportion of each component, mineralized collagen (nHAC) is doped in the system, and sol-gel transformation is formed within 30 +/-5 seconds, so that the injectable self-healing chitosan-based hydrogel is prepared. Through FT-IR and nuclear magnetism characterization, the two ends of the gel cross-linking agent polyethylene glycol are successfully conjugated with aldehyde groups. The chitosan-mineralized collagen hydrogel constructed based on Schiff base bonds is quick in gelling and has a certain self-healing characteristic. The hydrogel has shear thinning characteristics and better viscoelasticity through rheological characterization. SEM shows that the hydrogel has compact pore structure, continuous and stable three-dimensional network structure, and is beneficial to cell adhesion and growth. The cell compatibility test result of the composite hydrogel shows that the hydrogel has good biocompatibility and has potential application value in tissue engineering materials.

2. The pH of the solution during the preparation of mineralized collagen is between 6 and 8. Precipitation occurred at pH 5 to 6, and the solution became a white suspension at pH 7.

Drawings

FIG. 1 is an SEM image of a CS/nHAC composite hydrogel of example 1-example 4 a) CS; b) c), d) 1%, 2%, 4% of CS/nHAC.

FIG. 2a) viscosity-shear rate curves for chitosan composite hydrogels; b) and c) respectively representing the elastic modulus (G ') and the loss modulus (G') change of the chitosan composite hydrogel in different proportions in a frequency range (0.1-100 rad/s).

FIG. 3 is a graph showing the swelling ratio of the CS/nHAC composite hydrogel as a function of time.

FIG. 4 shows OD value a) and intracellular protein content b) of the L929 cell-CS/nHAC composite hydrogel.

Detailed Description

In order to better understand the present invention, the following examples are further provided for illustration, which are only used for explaining the present invention and do not limit the present invention in any way.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products. The collagen solution is purchased from the institute of Innovation of Tianjin Diwu purple.

Example 1

A preparation method of chitosan-graphene composite gel comprises the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, placing the solution in a constant-temperature water bath kettle, and stirring for 1.5 hours to dissolve the chitosan to obtain a chitosan solution; the temperature of the constant-temperature water bath kettle is 25 DEG C

S2, preparation of mineralized collagen powder: slowly dripping 0.055mol of phosphoric acid solution into the collagen solution; stirring the mixture at 25 ℃, and simultaneously dropwise adding a calcium chloride solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process to ensure that the pH value of the solution is 7; standing for 4 hours, removing supernatant, washing, filtering, freeze-drying and grinding to obtain mineralized collagen powder; the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution; the mass ratio of the mineralized collagen powder to the chitosan solution is 0%;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

Example 2

A preparation method of chitosan-graphene composite gel comprises the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, placing the solution in a constant-temperature water bath kettle, and stirring for 1.5 hours to dissolve the chitosan to obtain a chitosan solution; the temperature of the constant-temperature water bath kettle is 25 DEG C

S2, preparation of mineralized collagen powder: slowly dripping 0.055mol of phosphoric acid solution into the collagen solution; stirring the mixture at 25 ℃, and simultaneously dropwise adding a calcium chloride solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process to ensure that the pH value of the solution is 7; standing for 4 hours, removing supernatant, washing, filtering, freeze-drying and grinding to obtain mineralized collagen powder; the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution; the mass ratio of the mineralized collagen powder to the chitosan solution is 1%;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

Example 3

A preparation method of chitosan-graphene composite gel comprises the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, placing the solution in a constant-temperature water bath kettle, and stirring for 1.5 hours to dissolve the chitosan to obtain a chitosan solution; the temperature of the constant-temperature water bath kettle is 25 DEG C

S2, preparation of mineralized collagen powder: slowly dripping 0.055mol of phosphoric acid solution into the collagen solution; stirring the mixture at the temperature of 25 ℃,simultaneously dropwise adding a calcium chloride solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process to ensure that the pH value of the solution is 7; standing for 4 hours, removing supernatant, washing, filtering, freeze-drying and grinding to obtain mineralized collagen powder; the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution; the mass ratio of the mineralized collagen powder to the chitosan solution is 2%;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

Example 4

A preparation method of chitosan-graphene composite gel comprises the following steps:

s1, weighing 0.3g of chitosan at room temperature, dissolving the chitosan in 9.2g of dilute acetic acid solution with the volume fraction of 2%, placing the solution in a constant-temperature water bath kettle, and stirring for 1.5 hours to dissolve the chitosan to obtain a chitosan solution; the temperature of the constant-temperature water bath kettle is 25 DEG C

S2, preparation of mineralized collagen powder: slowly dripping 0.055mol of phosphoric acid solution into the collagen solution; stirring the mixture at 25 ℃, and simultaneously dropwise adding a calcium chloride solution; after the dropwise addition, dropwise adding a sodium hydroxide aqueous solution in the stirring process to ensure that the pH value of the solution is 7; standing for 4 hours, removing supernatant, washing, filtering, freeze-drying and grinding to obtain mineralized collagen powder; the PO₄ ^3-The ratio of the molar number of calcium to phosphorus of the solution and the calcium ion aqueous solution is 1.66;

s3, preparing a chitosan-mineralized collagen composite solution: adding the mineralized collagen powder prepared in the step (2) into the chitosan solution prepared in the step S1 in batches, and fully stirring and dissolving; then, adding 6mL of graphene dispersion liquid with the concentration of 0.02g/mL into the solution, and fully stirring to uniformly disperse the graphene dispersion liquid; obtaining chitosan-mineralized collagen composite solution; the mass ratio of the mineralized collagen powder to the chitosan solution is 4%;

s4, weighing 0.3g of double-ended benzaldehyde polyethylene glycol powder, and dissolving in 1.2g of deionized water to obtain a DF-PEG cross-linking agent solution;

s5, mixing the chitosan-mineralized collagen composite solution prepared in the step S4 and the DF-PEG cross-linking agent solution prepared in the step S2 together, and uniformly stirring to obtain the chitosan-graphene composite gel.

Characterization and Performance testing

And (3) testing:

1. the microporous morphology of the hydrogel was observed by SEM (test method: by SEM (Hitachi SU-8010, 5 KV.) Prior to SEM observation, the composite hydrogel frozen overnight at-55 ℃ was cut into an appropriate size, the cross section was fixed on a sample stage with a conductive tape facing up for observation, then gold was sprayed on the cross section by a vacuum coater for 240s, and the internal morphology of the hydrogel was observed by SEM.

2. FIG. 2 test method of FIG. 3: rheological measurements were made on CS/GR composite hydrogels using a rotational rheometer (AR-G2) operating under controlled stress. The instrument was equipped with a 25mm diameter stainless steel parallel plate geometry and water collector to keep the hydrogel moisture constant and minimize water evaporation. All samples were approximately 5 μm thick at room temperature (25 ℃). The applied strain was 1%. The shear frequency varied from 0.1 to 100rad/s and the heating rate was 2 ℃/min. The viscosity curve was determined by log shear rate scanning, with shear rates ranging from 0.1s-1 to 100 s-1. Uniformly spreading the CS/GR composite hydrogel on a sample table, removing bubbles in the gel to obtain a flat and uniform-thickness surface, contacting an upper plate of a parallel plate with the sample gel, and then carrying out data acquisition.

3. The test method comprises the following steps: parameters were measured using a microplate reader at 562nm light.

As a result:

1. as can be seen from fig. 1, the pore sizes of the pure CS scaffold and the CS/nHAC blended system are significantly different, and the nHAC-containing hydrogel shows a uniform and large pore structure, confirming that nHAC plays an important role in controlling the pore size of the hydrogel. Overall, these results indicate that the CS/nHAC hydrogel has a suitable pore size, approaching that of the ideal pores of scaffolds for tissue engineering.

2. As can be seen from FIG. 2a), the chitosan-based composite hydrogel has the property of shear thinning, and the composite gel belongs to non-Newtonian fluid. In contrast, as the concentration of mineralized collagen increases, the viscosity of the composite gel increases and then decreases. The gel viscosity was highest for 2% mineralized collagen in four experiments. The storage modulus G 'and the loss modulus G' of the chitosan-based composite hydrogel are respectively shown in FIGS. 2b) and c). It can be seen that G' is much higher than G ", indicating that the hydrogel exhibits a pronounced solid-like behavior. The G 'of the composite hydrogel is stable and does not change depending on the change of the angular velocity, and the storage modulus and the mechanical strength of the G' of the composite hydrogel added with the mineralized collagen are increased.

3. As can be seen from fig. 3, the equilibrium swelling ratio of the crosslinked hydrogel increased from 264% for the pure chitosan hydrogel to 312% for the hydrogel containing 2% nHAC. These findings indicate that nHAC content can increase the water absorption capacity of the crosslinked network.

4. As can be seen from figure 4, for all these measurements, the values for all samples increased with time, indicating that L929 cells continued to grow with longer cell culture times, with a growth rate that increased and then decreased with increasing mineralized collagen concentration. This indicates that appropriate mineralized collagen better promotes cell proliferation. In the case of higher concentration, the gel has dense internal network and limited cell living space. Therefore, the CS/nHAC composite hydrogel has good cell compatibility, and lays an experimental foundation for realizing the application of the chitosan-based composite hydrogel in the biomedical field

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.