WO2020156291A1

WO2020156291A1 - Physical and chemical double cross-linked network high-strength gelatin hydrogel and preparation method therefor

Info

Publication number: WO2020156291A1
Application number: PCT/CN2020/073067
Authority: WO
Inventors: 汪少芸; 何庆燕; 黄彦; 陈惠敏
Original assignee: 福州大学
Priority date: 2019-01-30
Filing date: 2020-01-19
Publication date: 2020-08-06
Also published as: CN109749098B; CN109749098A

Abstract

The present invention relates to a physical and chemical double cross-linked network high-strength gelatin hydrogel and a preparation method therefor, comprising: using Traut's reagent and methacrylic anhydride reagent to respectively perform modification of gelatin and chitosan; preparing a hydrogel; and using a method of alkaline sulphate solution immersion to introduce physical and chemical cross-linking to obtain a double cross-linked network high-strength gelatin-chitosan hydrogel. The present invention uses a physical and chemical double cross-linking method to obtain the high-strength gelatin-chitosan hydrogel; the method is simple and convenient, fast and efficient, and avoids the use of synthetic polymers and toxic cross-linking agents; and the obtained high-strength gelatin-chitosan hydrogel has excellent mechanical properties and biocompatibility. The present invention provides a new idea and method for preparing a high-strength biocompatible protein-based hydrogel, and helps broaden the scope of application of protein-based hydrogel materials in fields such as biomaterials and tissue engineering.

Description

Physical and chemical double-crosslinked network high-strength gelatin hydrogel and preparation method thereof

Technical field

The invention relates to a high-strength gelatin hydrogel with a physical/chemical double crosslinking network and a preparation method thereof, and belongs to the field of biological materials.

Background technique

Hydrogel is a three-dimensional network structure material rich in water (>50%) formed by hydrophilic polymer. Because of its unique physical and chemical properties, such as soft texture, controllable mechanical properties, good tissue compatibility, and human soft tissue Similarly, hydrogel is known as the most ideal biomedical material. However, due to the large amount of water and uneven network structure, the mechanical properties of hydrogel are far inferior to human soft tissues, which greatly limits its application in the field of biomedical materials. The methods that have been reported to prepare high-strength hydrogels, such as dual-network hydrogels, dual-crosslinked hydrogels, etc., use one of the material networks/crosslinked networks to support deformation and disperse stress, while the other Material network/cross-linking network to dissipate energy, so as to achieve excellent mechanical properties of hydrogel. However, these methods all contain bio-incompatible synthetic polymers, toxic cross-linking agents, and complex operating procedures. Therefore, natural sources of polymers are used to develop a hydrogel with excellent mechanical properties and biocompatibility. It is a challenge that needs to be solved urgently.

Gelatin is a hydrolysate of collagen, which has attracted wide attention due to its excellent biocompatibility and biodegradability. However, poor mechanical properties are the biggest obstacle to the use of gelatin-based hydrogels in the biomedical field. With its mechanical properties, the material will be a class of biomedical materials with great potential. In recent years, many reported gelatin-based hydrogels have adopted synthetic polymers, toxic cross-linking agents, or irritating operating procedures while improving mechanical properties, resulting in a greatly reduced biocompatibility of the hydrogels. Therefore, it is of great significance to prepare gelatin-based high-strength, biocompatible hydrogels composed of natural-source polymers.

technical problem

Technical solutions

The purpose of the present invention is to provide a high-strength gelatin hydrogel with a physical/chemical double-crosslinked network and a preparation method for the deficiencies in the above-mentioned research fields, and the obtained physical/chemical double-crosslinked gelatin hydrogel has excellent properties. The mechanical properties and good biocompatibility.

To achieve the above objectives, the following technical solutions are adopted:

The present invention uses the immersion method to prepare a high-strength gelatin hydrogel with a physical/chemical double-crosslinked network, which includes the following steps:

(1) Preparation of mercapto gelatin: take gelatin, mix with deionized water, heat to dissolve, add a certain amount of Traut’s reagent, after reaction under certain conditions, dialyze and freeze-dry to obtain mercapto gelatin;

(2) Preparation of olefin chitosan: take chitosan, mix with acetic acid solution, add an equal volume of ethanol after dissolving, stir well, ultrasonic wave to remove bubbles, add a certain amount of methacrylic anhydride, and after reaction under certain conditions, dialyze , Freeze-drying to obtain olefin chitosan;

(3) Preparation of gelatin-chitosan hydrogel: dissolve olefin chitosan in acetic acid solution, add a certain amount of sulfhydryl gelatin after complete dissolution, stir and dissolve at 50°C, remove air bubbles with ultrasound, and prepare a preformed gel; Pour the pre-gluing liquid into the mold and form a gel at low temperature to obtain gelatin-chitosan hydrogel;

(4) Preparation of high-strength gelatin-chitosan hydrogel: immerse the gelatin-chitosan hydrogel in a sulfate solution with a pH of 10.0 by the immersion method to produce a physical/chemical double-crosslinked network hydrogel; Rinse continuously with phosphate buffer to remove excess sulfate in the gel to obtain a high-strength gelatin-chitosan hydrogel with a physical/chemical double cross-linked network.

Further, the preparation of sulfhydryl gelatin in step (1) is as follows: take a certain amount of gelatin, add deionized water and stir and dissolve at 50°C to prepare a gelatin solution with a mass concentration of 1%, add Traut's reagent, and protect under nitrogen. After 24 hours of reaction at room temperature in the environment, first dialyze with 5 mM HCl solution at room temperature twice for 12 hours each time, and then dialyze twice with 1 mM HCl solution for 12 hours each time. The molecular weight cut-off of the dialysis bag is 3500 Da. After dialysis, The sulfhydryl gelatin is obtained by freeze-drying method.

Further, the preparation of olefin chitosan in step (2) is as follows: take a certain amount of chitosan, add 2.0% acetic acid solution, stir to dissolve, add an equal volume of ethanol solution, stir evenly and ultrasonically remove bubbles, add a certain amount The amount of methacrylic anhydride was stirred for 12 hours at room temperature. After the reaction, the reaction was dialyzed with 15 mM NaCl solution for 12 hours, and then dialyzed with deionized water for 4 times, each 12 hours. The molecular weight cut-off of the dialysis bag was 3500 Da. After the dialysis, use The freeze-drying method obtains olefin chitosan.

Further, the specific operation of the preparation of gelatin-chitosan hydrogel in step (3) is as follows: take a certain amount of olefin chitosan and dissolve it in 1.0% acetic acid solution, add a certain amount of mercapto gelatin after complete dissolution, and stir to dissolve at 50°C , The pre-gelling liquid is obtained; then the pre-gluing liquid is ultrasonically removed from the bubbles, then poured into the mold, and placed at 4°C for 2 hours to form a gel to obtain a gelatin-chitosan hydrogel.

Further, the specific operations for preparing the high-strength gelatin-chitosan hydrogel in step (4) are as follows: weigh a certain amount of sulfate, add deionized water and stir to dissolve, adjust the pH to 10.0 with NaOH solution, and change step (3) Soak the gelatin-chitosan hydrogel in the salt solution at 25°C for 12h, take it out, and rinse with 0.1M pH 7.4 phosphate buffer to remove the sulfate in the hydrogel, which is a physical/chemical double cross Linked network gelatin high-strength hydrogel.

Beneficial effect

The present invention uses pigskin gelatin as raw material, auxiliary addition of chitosan, uses sulfhydryl-olefin Michael addition reaction to introduce chemical crosslinking, and uses sulfate and alkaline conditions to introduce physical crosslinking of chitosan and gelatin to obtain physical/chemical Double cross-linked network high-strength gelatin hydrogel. The invention adopts a simple soaking method to produce a physical and chemical cross-linking network for natural polymer hydrogels, avoids the use of synthetic polymers and toxic cross-linking agents, and obtains high-strength gelatin hydrogels with excellent mechanical properties And biocompatibility, it provides a new idea and method for preparing high-strength protein-based hydrogels, and helps the development and utilization of protein-based hydrogel materials, so that they can be used in biomaterials, tissue engineering and other fields.

Description of the drawings

Figure 1 is the modification chemical reaction formula of gelatin and chitosan;

Figure 2 shows the compression and tensile mechanical properties of the high-strength gelatin hydrogel with a physical/chemical double cross-linked network;

Figure 3 is a graph showing the results of biocompatibility of a high-strength gelatin hydrogel with a physical/chemical double cross-linked network.

Embodiments of the invention

实施例Example 11

Step 1: Weigh 1.0g of pigskin gelatin, add 99.0mL of deionized water, stir at 50°C until it is completely dissolved to prepare a gelatin solution with a mass concentration of 1.0%, add 200mg Traut's reagent, and room temperature under nitrogen protection Stir for 24h. After the reaction, the solution was put into a dialysis bag with a molecular weight cut-off of 3500Da, first dialyzed with 5 mM HCl solution at room temperature for 12 hours each time, and then dialyzed with 1 mM HCl solution twice, each time 12 hours, after the end of dialysis After taking the sample out and moving it to a refrigerator at -20°C for 24 hours, then moving it to a refrigerator at -80°C for 2 hours and freeze-drying to obtain mercapto gelatin. The reaction chemical formula is shown in Figure 1.

Step 2: Weigh 3.0g of chitosan, dissolve it in 100mL of 2.0% acetic acid solution, add an equal volume of ethanol solution, stir well and ultrasonically remove bubbles, add 0.305g of methacrylic anhydride, stir at room temperature for 12h, and the reaction is complete Pack the solution into a dialysis bag with a molecular weight cut-off of 3500 Da, first dialyzed with 15 mM NaCl solution at room temperature for 12 hours, and then dialyzed with deionized water 4 times for 12 hours each time. After the dialysis is completed, remove the sample and move it to -20°C After being placed in the refrigerator for 24 hours, it was then moved to -80°C for 2 hours and freeze-dried to obtain olefin chitosan. The chemical formula of the reaction is shown in Figure 1.

Step 3: Weigh 0.2g of olefin chitosan and dissolve it in 10mL 1.0% acetic acid solution, add 1.0g of mercapto gelatin after complete dissolution, stir and dissolve at 50℃, so that the mass concentrations of olefin chitosan and mercapto gelatin are 2.0 respectively %, 10.0%. After the mixed solution is ultrasonically degassed, it is poured into a mold and placed at 4° C. for 2 hours to form a gel to obtain a gelatin-chitosan hydrogel.

Step 4: Weigh 20.0g of ammonium sulfate and add 80mL of deionized water to prepare an ammonium sulfate solution with a mass concentration of 20%, adjust the pH to 10.0 with NaOH solution, and soak the gelatin-chitosan hydrogel at 25℃ In the salt solution for 12 hours, take it out, and continuously rinse with 0.1M pH 7.4 phosphate buffer to remove the sulfate in the hydrogel to obtain a high-strength gelatin hydrogel with a physical/chemical double cross-linked network.

Step 5: Use the texture analyzer to test the compression and tensile mechanical properties of the hydrogel in the Return to Start mode. For the compression experiment, the hydrogel was made into a cylindrical shape with a diameter of 8mm and a height of 10mm, and the test speed was 5mm/min; for the tensile experiment, the hydrogel was made into a "dumbbell shape" (positioning pin length, width, The thickness is 30, 3, and 2mm, the length, width, and thickness of the bell are 10, 15, and 2mm respectively), the test speed is 50mm/min; the strain is calculated by changing the length of the hydrogel to its initial length, Calculate the stress by dividing the force on the hydrogel by its initial cross-sectional area, and draw the hydrogel stress-strain graph (Figure 2).

It can be seen from Figure 2 that by introducing physical/chemical double crosslinking, the resulting high-strength gelatin hydrogel has excellent mechanical properties, the fracture deformation is as high as 80% when compressed, and the fracture stress is as high as 1.8 MPa; the fracture deformation becomes 142% when stretched. , The fracture stress is 0.4MPa, which is much higher than the physical crosslinked hydrogel and the chemical crosslinked hydrogel.

Step 6: Human skin fibroblasts are cultured in DMEM medium containing 10% fetal bovine serum and 1.0% penicillin-streptomycin, and can be used when they are passaged to 2-3 generations; physical/chemical double cross-linked gelatin is hydrogel The gel was placed in a 6-well plate and immersed in sterile phosphate buffer. After sterilization under UV light for 1 hour, the phosphate buffer was sucked out, and DMEM medium was added to infiltrate at 37°C for 12 hours. After the DMEM medium was removed, 1 mL of human skin fibroblast suspension (5×10 ⁴ cells/well) was added and cultured at 37°C under 5.0% CO ₂ conditions. The viability and morphology of the cells on the hydrogel were stained and observed with the Calcein-AM/PI live cell/dead cell double staining kit. The medium in the 6-well plate was aspirated, washed with phosphate buffer, and the dye was added After incubating at 37°C for 30 min, and washing again with phosphate buffer, the hydrogel was observed under a fluorescent inverted microscope and photographed and imaged.

It can be seen from Figure 3 that fibroblasts multiply and grow well on the hydrogel, and no dead cells appear, indicating that the high-strength gelatin hydrogel with a physical/chemical double cross-linked network has good biocompatibility.

The above are only preferred implementation examples of the present invention, and any equivalent changes and modifications made in accordance with the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims

A method for preparing a high-strength gelatin hydrogel with a physical/chemical double-crosslinked network, characterized in that: the method specifically includes the following steps:

(1) Preparation of mercapto gelatin: take gelatin, mix with deionized water, heat to dissolve, add a certain amount of Traut’s reagent, after reaction under certain conditions, dialyze and freeze-dry to obtain mercapto gelatin;

(2) Preparation of olefin chitosan: take chitosan, mix with acetic acid solution, add an equal volume of ethanol after dissolving, stir well, ultrasonic wave to remove bubbles, add a certain amount of methacrylic anhydride, and after reaction under certain conditions, dialyze , Freeze-drying to obtain olefin chitosan;

(3) Preparation of gelatin-chitosan hydrogel: dissolve olefin chitosan in acetic acid solution, add a certain amount of sulfhydryl gelatin after complete dissolution, stir and dissolve at 50°C, remove air bubbles with ultrasound, and prepare a preformed gel; Pour the pre-gluing liquid into the mold and form a gel at low temperature to obtain gelatin-chitosan hydrogel;

(4) Preparation of high-strength gelatin-chitosan hydrogel: soak the gelatin-chitosan hydrogel in a sulfate solution with a pH of 10.0 by the immersion method to produce a physical/chemical double-crosslinked network hydrogel; Rinse continuously with phosphate buffer to remove excess sulfate in the gel to obtain a high-strength gelatin-chitosan hydrogel with a physical/chemical double cross-linked network.
The preparation method according to claim 1, characterized in that: the specific operation of preparing the mercapto gelatin in step (1) is as follows: take a certain amount of gelatin, add deionized water and stir and dissolve at 50°C to obtain a mass concentration It is a 1% gelatin solution, added with Traut's reagent and reacted at room temperature under nitrogen protection for 24 hours, then dialyzed with 5 mM HCl solution at room temperature twice for 12 hours each time, and then dialyzed with 1 mM HCl solution twice, each time After 12h, the molecular weight cut-off of the dialysis bag was 3500 Da. After the dialysis, the sulfhydryl gelatin was obtained by freeze-drying.
The preparation method according to claim 1, wherein the specific operation of the preparation of the olefin chitosan in step (2) is as follows: take a certain amount of chitosan, add 2.0% acetic acid solution, and after dissolving, add an equal volume After stirring the ethanol solution, add methacrylic anhydride and stir at room temperature for 12 hours. After 12 hours of reaction, dialyze with 15 mM NaCl solution for 12 hours, then dialyze with deionized water 4 times, each 12 hours, the molecular weight cut off by the dialysis bag After dialysis, the olefin chitosan is obtained by freeze-drying method.
The preparation method according to claim 1, characterized in that: the specific operation of the preparation of the gelatin-chitosan hydrogel in step (3) is as follows: dissolve the olefin chitosan in 1.0% acetic acid solution, and add it after being completely dissolved The sulfhydryl gelatin is stirred and dissolved at 50°C to obtain a pre-gluing solution; then the pre-gluing solution is ultrasonically removed to remove bubbles, then poured into a mold, and placed at 4°C for 2 hours to form a gel to obtain a gelatin-chitosan hydrogel.
The preparation method according to claim 1, wherein the mass fractions of mercapto gelatin and olefin chitosan in the pre-gluing solution of step (3) are 5.0-20.0% and 0.2-3.0%, respectively.
The preparation method according to claim 1, wherein the specific operation of the preparation of the high-strength gelatin-chitosan hydrogel in step (4) is as follows: weigh a certain amount of sulfate, add deionized water and stir to dissolve , Adjust the pH to 10.0 with NaOH solution, soak the gelatin-chitosan hydrogel in step (3) in a salt solution at 25°C for 12 hours, take it out, and continuously rinse with 0.1M pH 7.4 phosphate buffer to remove water Sulfate in the gel is a high-strength gelatin hydrogel with a physical/chemical double-crosslinked network.
A high-strength gelatin hydrogel with a physical/chemical double cross-linked network prepared by the preparation method according to any one of claims 1-6.