CN106146865A - A kind of dual network high intensity fibroin hydrogel and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of natural polymer materials, in particular to a double-network high-strength silk protein hydrogel and a preparation method thereof. In the present invention, the silk protein hydrogel chemically cross-linked by horseradish peroxidase (the first network) is soaked in a solution that can induce silk protein β- sheet transformation to form a physical cross-link (second network), that is A double-network high-strength silk protein hydrogel with a solid content of 2-20 wt% can be prepared. The hydrogel has good elasticity, high strength and is easy to control, biocompatibility and stable performance under simulated in vivo environmental conditions, and can be widely used in many biomedical and regenerative medicine projects such as drug sustained release carriers, cartilage tissue and ligament tissue. field.

Description

A double-network high-strength silk protein hydrogel and its preparation method

technical field

The invention belongs to the technical field of natural macromolecular materials, and in particular relates to a double-network high-strength natural biological macromolecular hydrogel and a preparation method thereof.

Background technique

With the development of the field of tissue engineering, the demand for biomedical materials and their preparation technologies with excellent performance and suitable for use in a biologically friendly environment is increasing. Hydrogel is a cross-linked network rich in a large number of water molecules, has strong permeability, and is similar to extracellular matrix in structure and function, so hydrogel is not only an important class of cell scaffolds that can be used to rebuild tissue engineering It is also a kind of promising drug sustained-release carrier material.

As a natural polymer material, silk protein has abundant sources, excellent mechanical properties, controllable degradation properties and good biocompatibility, so it has broad application prospects in the biomedical field. But at the same time, for hydrogel materials prepared from regenerated silk protein solutions, their poor mechanical properties usually limit their practical application in some tissue engineering fields, such as cartilage or ligaments that need to bear high pressure Restoration materials. In order to solve this problem, it has been reported that silk protein hydrogels were obtained by chemical crosslinking with horseradish peroxidase, but the storage modulus is usually only 0.1-10 KPa (CN 104017374 A), and the hydrogel structure is not Stable, the transition from gel to solution will occur when placed at 37 ^o C for a long time. In the research on the preparation of high-strength silk protein hydrogel, it has been reported that the characteristics of surfactants are used to prepare high-strength silk protein hydrogel (silk protein/water: 15/85 (w/w)), and its compression mold However, the hydrogel prepared by silk protein at a low concentration does not have good mechanical properties, and the surfactant will have certain cytotoxicity; some studies have also reported the use of silk protein and cellulose derivatives A high-strength composite hydrogel (CN 104845382 A) is prepared from the material, which also has certain restrictions on the concentration of silk protein.

The present invention obtains a chemical cross-link network by chemically cross-linking silk protein with horseradish peroxidase, and then puts it in a solution that can induce silk protein to undergo β- sheet transformation to form a physical cross-link network, and obtains a class of high modulus and toughness Strong and biocompatible double-network high-strength silk protein hydrogel, the solid content of this type of hydrogel can be adjusted between 2-20 wt% to control its corresponding mechanical properties. In addition, its preparation method is simple and repeatable, and cells can spread and grow on its surface.

Contents of the invention

The purpose of the present invention is to provide a dual-network high-strength silk protein hydrogel with adjustable mechanical properties and good stability and a preparation method thereof.

The double-network high-strength silk protein hydrogel provided by the present invention is formed by chemically cross-linking silk protein hydrogel with a mass fraction of 2-20 wt% silk protein aqueous solution through horseradish peroxidase, and then soaking it in Physical cross-linking is formed in the solution that can induce the transformation of silk protein β sheet, and the double-network high-strength silk protein hydrogel is obtained. The aqueous solution capable of inducing β sheet is any one containing organic solvents, surfactants, or acidic solutions.

The preparation method of the double-network high-strength silk protein hydrogel provided by the invention, the specific steps are:

(1) Prepare silk protein aqueous solution so that its final concentration is 2-20 wt%;

(2) Add horseradish peroxidase (0.01-1 mg/mL) and hydrogen peroxide (0.01-1% v/v) solution to the silk protein solution obtained in step (1), the solvent is water, 25-50 Chemical cross-linking at ℃ to obtain silk protein gel network;

(3) Soak the silk protein gel network obtained in step (2) in a solution capable of inducing silk protein β- sheet transition for 6-48 hours to obtain a double-network high-strength silk protein hydrogel.

In the present invention, the silk protein is one or a mixture of silkworm or tussah silk protein.

In the present invention, the mass ratio range of the horseradish peroxidase to hydrogen peroxide is preferably 10:1-1:1.

In the present invention, the solution capable of inducing β sheet is any one of organic solvents, surfactants, or acidic solutions, or a mixture of several of them.

In the present invention, the organic solvent is (but not limited to) any one of methanol, ethanol, propanol, butanol, pentanol, pyridine, acetone, chloroform, or a mixture of several of them, with a concentration of 50 -95% v/v.

In the present invention, the surfactants are (but not limited to) any one of sodium lauryl sulfate, sodium dodecylbenzenesulfonate or polyethylene glycol octylphenyl ether, and the concentration is 5 -40 mM.

In the present invention, the acidic solution is any one that can reduce the pH of the solution to 2-5, and the concentration is 0.05-5% w/w.

In the present invention, the silk protein aqueous solution in step (1) can be a pure silk protein solution, or a mixed solution/suspension containing other components, such as water-soluble polymers, functional inorganic materials or drugs molecules etc.

The method of the invention can also be used to prepare high-strength silk protein membranes and silk protein scaffold materials.

The double-network high-strength silk protein hydrogel prepared by the invention can be used in the fields of biomedical engineering and nano functional materials.

Adopting the method of the present invention to prepare double-network high-strength silk protein hydrogel has the following advantages:

(1) The raw material of the hydrogel described in the present invention is low-cost silk protein certified by the US Food and Drug Administration, and the resulting hydrogel has good biocompatibility and biodegradability;

(2) The horseradish peroxidase used in the present invention is mainly extracted from the roots of wasabi plants, and has been commercially produced; The oxidase-crosslinked silk protein hydrogel has no cytotoxicity;

(3) The solution used in the present invention that can induce the transformation of silk protein β sheet, and the residue of the enzyme-catalyzed reaction can be removed by replacing with deionized water;

(4) The double-network high-strength hydrogel prepared by the present invention is composed of chemical cross-linking network and physical cross-linking network, and its performance is better than that of silk protein hydrogel chemically cross-linked by horseradish peroxidase alone, and The hydrogel is stable under simulated in vivo environmental conditions;

(5) The present invention can still prepare hydrogels with good mechanical properties for low concentrations of silk protein (such as 2-5 wt%), which is different from the limitation of silk protein concentration by the reported high-strength silk protein hydrogel preparation method ;

(6) The silk protein solution of the present invention can be a mixed solution of water-soluble polymers, functional inorganic nanoparticles, drug molecules, etc., and can prepare multifunctional high-strength hydrogels;

(7) In addition, the preparation conditions of the present invention are simple and efficient, environmentally friendly, and reproducible, and have the value of popularization and application.

Description of drawings

Figure 1 is a schematic diagram of the bending deformation of the dual-network high-strength silk protein hydrogel (4 wt%).

Figure 2 is the tensile and compressive mechanical property curves of NaHCO ₃ degummed silk protein (10 wt%) hydrogel.

detailed description

Below in conjunction with specific embodiment, to further illustrate the present invention. It is necessary to point out here that embodiment is only used to further illustrate the present invention, can not be interpreted as the limitation of protection scope of the present invention, those skilled in the art can make some non-essential improvements according to the content of the present invention above and adjust.

Example 1

Take 2 mL of mulberry silk protein solution with a mass fraction of 10 wt%, add 40 μL of 0.5 mg/mL horseradish peroxidase, mix evenly, add 40 μL of 0.05% hydrogen peroxide aqueous solution, and form a gel at 37 °C network; then soak it in 75% ethanol solution for 24 h to obtain a high-strength silk protein hydrogel with a compressive modulus of 1.37 MPa and a tensile modulus of 3.35 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 2

Take 4 mL of tussah silk protein solution with a mass fraction of 2 wt%, add 80 μL of 1 mg/mL horseradish peroxidase, mix evenly, add 80 μL of 0.2% hydrogen peroxide aqueous solution, and form a gel at 40 °C network; then soak it in 70% isopropanol solution for 12 h to obtain a high-strength silk protein hydrogel with a compressive modulus of 0.28 MPa and a tensile modulus of 0.2 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 3

Take 2 mL of tussah silk protein solution with a mass fraction of 20 wt%, add 40 μL of 0.1 mg/mL horseradish peroxidase, mix evenly, add 40 μL of 0.01% hydrogen peroxide aqueous solution, and form a gel at 25 °C network; then soak it in 0.5M sodium dodecyl sulfate solution for 48 h to obtain a silk protein hydrogel with good mechanical properties, with a compressive modulus of 3.2 MPa and a tensile modulus of 3.5 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 4

Take 2 mL of mulberry silk protein solution with a mass fraction of 10 wt%, add 40 μL of 0.8 mg/mL horseradish peroxidase, mix evenly, add 40 μL of 0.2% hydrogen peroxide aqueous solution, and form a gel at 37 °C network; then soak it in an aqueous solution of acetic acid with a pH of 2.5 for 24 h to obtain a high-strength silk protein hydrogel with a compressive modulus of 1.0 MPa and a tensile modulus of 2.1 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 5

Take 5 mL of a mixed solution of tussah silk protein with a mass fraction of 8 wt% and 8 wt% gelatin, add 100 μL of 0.5 mg/mL horseradish peroxidase, mix evenly and add 100 μL of 1% hydrogen peroxide aqueous solution at 25 ℃ to form a gel network; then soak it in 0.1M sodium dodecylsulfonate solution for 10 h to obtain a high-strength silk protein hydrogel with a compressive modulus of 1.25 MPa and a tensile modulus of 2.0 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 6

Take 2 mL of a mixed solution of silkworm silk protein with a mass fraction of 12 wt% and 2 wt% graphene oxide, add 40 μL of 0.2 mg/mL horseradish peroxidase, mix well and add 40 μL of 0.05% peroxidase Hydrogen oxide aqueous solution, 37 ℃ to form a gel network; then soak it in 75% n-butanol solution for 24 h to obtain a high-strength functionalized silk protein hydrogel, the compressive modulus reached 2.52 MPa, and the tensile modulus 2.35 MPa. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Example 7

Take 5 mL of mulberry silk protein solution with a mass fraction of 4 wt%, add 50 μL of 1 mg/mL horseradish peroxidase, mix evenly, add 50 μL of 0.3% hydrogen peroxide aqueous solution, and pour the mixed solution into In a 10cm*10cm mold, the film-forming chamber forms a film for 24 hours; then it is soaked in 75% ethanol solution for 24 hours to obtain a high-strength silk protein film, with a tensile modulus of 103Mpa in a wet state and a tensile strain of 300 %. Tests show that it has stable performance and good cell compatibility under simulated in vivo environmental conditions.

Claims (10)

1. the preparation method of a dual network high intensity fibroin hydrogel, it is characterised in that concretely comprise the following steps:

(1) silk protein aqueous solution is prepared so that it is ultimate density is 2-20 wt%;

(2) in step (1) gained silk protein solution, add horseradish peroxidase and the 0.01-1% v/v of 0.01-1 mg/mL Hydrogenperoxide steam generator, solvent is to be chemically crosslinked at water, 25-50 DEG C, obtains fibroin gel network；

(3) step (2) gained fibroin gel network is soaked in can induce fibroinβFold in the solution changed, during immersion Between be 6-48 h, obtain dual network high intensity fibroin hydrogel.

Preparation method the most according to claim 1, it is characterised in that described fibroin is Bombyxmori Linnaeus or tussah silk albumen One or both mixing.

Preparation method the most according to claim 1, it is characterised in that described horseradish peroxidase and hydrogen peroxide Mass ratio is 10:1-1:1.

4. according to the preparation method described in claim 1,2 or 3, it is characterised in that described can induceβThe solution folded is Containing any one of organic solvent, surfactant-based or acid solution, or the most several mixing.

Preparation method the most according to claim 4, it is characterised in that described organic solvent class be methanol, ethanol, third Any one of alcohol, butanol, amylalcohol, pyridine, acetone, chloroform, or the most several mixing, concentration is 50-95% v/v.

Preparation method the most according to claim 4, it is characterised in that described surfactant-based for lauryl sulphate acid Any one of sodium, dodecylbenzene sodium sulfonate or Triton X-100, concentration is 5-40 mM.

Preparation method the most according to claim 4, it is characterised in that described acid solution is for can be reduced to pH value of solution Any one of 2-5, concentration is 0.05-5% w/w.

Preparation method the most according to claim 4, it is characterised in that described in step (1), silk protein aqueous solution is pure silk Protein solution, or be also containing the mixed solution/suspension of other compositions, other compositions include water soluble polymer, functional Inorganic material or drug molecule.

9. the dual network high intensity fibroin hydrogel that a preparation method as described in one of claim 1-8 prepares.

10. dual network high intensity fibroin hydrogel as claimed in claim 9 is at biomedical engineering and nano-functional material The application in field.