CN106310380B - A kind of nanofiber Silk fibroin gel and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of nanofiber Silk fibroin gels and preparation method thereof, and salt-formic acid is dissolved in after natural silk degumming and obtains silk fibroin protein solution；It will be in fibroin albumen-salt-formic acid solution injection mold；Mold equipped with silk fibroin protein solution is immersed into organic solvent or aqueous solution, standing a period of time is to form Silk fibroin gel.Preparation method of the present invention is simple, process is short, facilitates operation, is easy to produce in batches.Silk fibroin gel prepared by the present invention has natural bionic nano fibrillar structure, adjustable mechanical property and good biocompatibility.

Description

A kind of nanofibrillated silk fibroin gel and preparation method thereof

technical field

The invention relates to a nanofibrillated silk fibroin gel and a preparation method thereof. The prepared material can be applied to the fields of regenerative medicine such as tissue engineering and drug sustained release.

Background technique

There are millions of patients with organ or tissue damage and functional loss due to diseases and accidents every year. In the United States alone, more than 8 million operations are needed to treat such patients every year, and the economic cost is more than 400 billion US dollars. With the development of modern medicine and surgical techniques, tissue or organ transplantation to repair functional loss has been widely accepted, however, it faces a huge donor gap. The formation of tissues or organs in vivo or in vitro by means of regenerative medicine provides a new treatment option for the repair of damaged functions. Among them, the selection and construction of tissue engineering scaffold materials become one of the keys to this treatment method. Gels (hydrogels) formed with water as a medium exist widely in both nature and artificial synthesis. In organisms, ranging from cells, tissues to organs, they can be regarded as complex hydrogel systems. In the field of biomedical materials, polymer hydrogels with good biocompatibility and degradability have extremely high application value and prospects, and can be widely used in tissue repair scaffold materials, drug controlled release carriers, cells and biological activities. Molecule loading and delivery, etc.

Hydrogels used for loading and delivering cells must have biocompatibility, suitable substance delivery capacity, adequate mechanical stability, and controllable biodegradability. The advantage of using synthetic polymers to prepare hydrogels is that the gelation process and hydrogel properties can be precisely controlled by selecting specific molecular weights, molecular structures, and cross-linking methods, but the biocompatibility of hydrogels is poor; Hydrogels prepared from natural polymer materials have good biocompatibility with tissues and cells, but poor controllability and repeatability.

Silk protein is a natural polymer biomaterial derived from nature. It has excellent mechanical properties, controllable biodegradability, and easy processing, especially its biocompatibility with collagen, making it an ideal raw material for regenerative medical scaffolds. . Silk protein aqueous solution self-assembles to form a gel in a natural state, but it takes a long time and takes several days to complete. In order to quickly prepare silk fibroin hydrogel, it can be adjusted by adjusting solution parameters (such as solution concentration, pH value, temperature), applying exogenous factors (such as stirring, ultrasonic treatment), and adding other materials (such as poloxamer). accomplish. Although silk fibroin hydrogels can be easily prepared, there are still some problems to be solved. One of the existing key problems is that the existing silk fibroin dissolution process causes the silk fibroin to degrade greatly, which seriously affects the mechanical properties of the silk fibroin hydrogel and limits its application; the second problem is the preparation of the silk fibroin hydrogel. The extraction process is relatively complicated, including the following three main steps: 1. High-concentration neutral salt dissolution, 2. Long-term dialysis to obtain pure silk fibroin aqueous solution, 3. Inducing silk fibroin aqueous solution to coagulate to form a gel through internal and external factors; Third, the current silk fibroin hydrogel still has a sponge-like porous structure inside and does not have a biomimetic nanofiber structure. Therefore, its biological properties still need to be further improved, which cannot meet the needs of biological applications.

Therefore, to overcome the above-mentioned problems in the prior art, a simple and easy-to-operate preparation method is developed, and a silk fibroin nanofiber hydrogel with bionic structure and controllable mechanical properties is constructed to play a role in the application of silk fibroin in the field of biomedical materials. Application is very important.

Contents of the invention

The purpose of the present invention is to provide a simple and easy-to-operate method for preparing nanofibrillated silk fibroin hydrogel, and the silk fibroin hydrogel prepared by the method has a biomimetic nanofibrillated structure and excellent mechanical properties.

In order to achieve the above object, the technical solution adopted in the present invention is: a preparation method of nanofibrillated silk fibroin hydrogel, comprising the following steps:

(1) Silk fibroin solution is obtained by dissolving silk in salt-formic acid solution after degumming;

(2) inject the silk fibroin solution in step (1) into the mould;

(3) Dip the mold filled with the silk fibroin solution in step (2) into an aqueous solution of an organic solvent or an aqueous solution, and let stand to form a nanofibrillated silk fibroin hydrogel.

In the above technical solution, the silk in step (1) is mulberry silk, tussah silk, castor silk or celestial silk.

In the above technical scheme, the salt in the salt-formic acid solution in step (1) is lithium bromide, calcium chloride, zinc chloride, magnesium chloride, strontium chloride, lithium thiocyanate, sodium thiocyanate, magnesium thiocyanate or calcium nitrate.

In the above technical solution, in the salt-formic acid solution described in step (1), the salt concentration is 1-20 w/v%; the formic acid concentration is 98wt%; the silk fibroin protein concentration in the silk fibroin solution is 5-50 w/v% /v%.

In the above technical scheme, limiting formic acid can not only ensure the dissolution of silk, protect the fibril structure of silk, but also avoid excessive degradation of silk, so that the regenerated silk protein material has good structure and performance; it avoids serious degradation of other acids such as phosphoric acid and hydrochloric acid Silk, thereby losing the problem of use value; It also avoids the defect that other acids such as acetic acid cannot dissolve silk, and cannot realize the regeneration and preparation of silk protein. Therefore, the formic acid of the present invention is beneficial to the uniformity of the silk fibroin solution, ensures the solubility, and provides a good foundation for the formation of nanofibrillated silk fibroin hydrogel, especially excellent mechanical properties and bionic nano-fibril structure.

In the above technical solution, the organic solvent in step (2) is methanol, ethanol, isopropanol; the concentration of the aqueous solution of the organic solvent is 1-99wt%, that is, the mass content of the organic solvent is 1-99wt%.

In the above technical solution, in step (2), the mold is designed according to the shape of the product to be prepared.

The invention also discloses the nanofibrillated silk fibroin hydrogel prepared according to the above technical scheme; the gel is mainly composed of fibers of 10-100nm, and the wet compression modulus is 10kPa-100MPa; it can be used in the preparation of tissue engineering in the bracket.

The excellent mechanical properties of silk are directly related to its ultra-high molecular weight and multi-level self-assembled structure, especially the nano-fibril structure. In the present invention, the salt-formic acid solution can dissolve silk at the level of nano-fibrils to obtain a silk fibroin nano-fibril solution. Because the silk still retains its fibril structure, the solution does not miscible with water and organic solvents; When the silk fibroin nanofibril solution is immersed in water and organic solvents, the concentration of formic acid decreases and the pH of the solution decreases, thereby inducing the silk fibroin molecule to change from amorphous to β-sheet crystal structure, and at the same time, cross-linking occurs between nanofibrils , and finally condense to form a gel; the original fibril structure in the solution endows the silk fibroin gel with excellent mechanical properties and biomimetic nano-fibril structure. The gel is mainly composed of 10~100nm fibers, and the wet compressive modulus is 10kPa~100MPa , achieved unexpected technical effects.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

(1) The preparation method of the present invention is simple and highly operable. After the silk is dissolved in salt-formic acid, it can be directly immersed in an organic solvent or water to form a gel, which is mainly composed of 10-100nm fibers and has a wet compression modulus of 10kPa. ~100MPa, achieved unexpected technical effects.

(2) In the preparation method of the present invention, pure water can be selected as an inducer to promote the one-step dissolved silk fibroin solution to directly form a gel to form a scaffold, which can avoid post-treatment of organic solvents and the resulting toxicity of the scaffold; The fibroin gel is mainly composed of nanofibrils, and the diameter of the fibrils is between 10 and 100 nanometers; the compression modulus of the prepared silk fibroin gel is between 10kPa and 100MPa, which can not only be used for the repair of soft tissue damage such as skin and cornea , and can be used for the repair of hard tissue damage, such as bone; it provides a good basis for the application of silk as a regenerative medical material.

Description of drawings

Fig. 1 is the camera and the scanning electron micrograph of the silk fibroin egg nanofiber hydrogel that embodiment one makes;

Fig. 2 is the X-ray diffraction figure of the silk fibroin egg nanofiber hydrogel that embodiment two makes;

Fig. 3 is the scanning electron micrograph of the silk fibroin egg nanofiber hydrogel freeze-dried that embodiment two makes;

Figure 4 is the camera and scanning electron micrographs of the silk fibroin egg nanofiber hydrogel prepared in Example 3.

Detailed ways

The present invention will be further described below in conjunction with embodiment:

Embodiment one

(1) Natural mulberry silk was degummed by boiling 0.5wt% sodium bicarbonate solution for 30 minutes, and repeated 3 times to obtain degummed mulberry silk;

(2) Dissolve degummed silk in 2w/v calcium chloride-98wt% formic acid solution to obtain 10 w/v% silk protein solution;

(3) The above-mentioned silk fibroin solution was directly injected into a plastic tube, then immersed in deionized water, and nanofibrillated silk fibroin hydrogel was formed within 8 hours.

Accompanying drawing 1 is the photograph of the camera of above-mentioned prepared silk fibroin egg nanofiber hydrogel and the scanning electron microscope picture after freeze-drying. It can be seen from the figure that the gel is milky white with a smooth surface, and the interior of the gel is mainly composed of silk fibroin nanofibrils. After mechanical compression test, the compressive modulus of the gel is 523.2KPa.

Embodiment two:

(1) Natural mulberry silk was degummed by boiling 0.05wt% sodium bicarbonate solution for 30 minutes, and repeated 3 times to obtain degummed mulberry silk;

(2) Dissolve degummed silk in 4w/v% calcium chloride-98wt% formic acid solution to obtain 25 w/v% silk protein solution;

(3) The above-mentioned silk fibroin solution was injected into a 24-well plate, and then immersed in deionized water, and nanofibrillated silk fibroin hydrogel was formed within 6 hours.

Accompanying drawing 2, accompanying drawing 3 are respectively the X-ray diffraction spectrogram and the scanning electron microscope picture after freeze-drying of the silk fibroin egg nanofiber hydrogel prepared above, it can be seen from the figure that the secondary structure of silk protein is mainly β-sheet Crystalline structure, the interior of the scaffold is composed of nanofibers. The compressive modulus of the gel was 16.2 MPa after mechanical compression test.

Embodiment three:

(1) Natural tussah silk was degummed by boiling with 0.5wt% sodium bicarbonate solution for 30 minutes, and the degummed tussah silk was obtained after repeating 3 times;

(2) Dissolving degummed tussah silk in 10w/v% lithium bromide-98wt% formic acid solution to obtain a silk protein solution with a concentration of 50w/v%;

(3) The above-mentioned silk fibroin solution was injected into a 6-well plate, then immersed in an aqueous solution, and tussah silk nanofibrillated silk fibroin hydrogel was formed within 20 hours.

Accompanying drawing 4 is the scanning electron micrograph of the silk fibroin egg nanofiber hydrogel prepared above after freeze-drying. It can be seen from the figure that the interior of the hydrogel is also a nano-fibril structure. The compressive modulus of the gel was 98.2MPa after mechanical compression test.

Embodiment four:

(1) Natural mulberry silk was degummed by boiling 0.05wt% sodium bicarbonate solution for 30 minutes, and repeated 3 times to obtain degummed mulberry silk;

(2) Dissolving degummed silk in 10w/v% calcium chloride-98wt% formic acid solution to obtain a silk protein solution with a concentration of 6 w/v%;

(3) The above-mentioned silk fibroin solution was injected into a 6-well plate, then immersed in 50 wt% deionized water, and nanofibrillated silk fibroin hydrogel was formed within 12 hours.

The compressive modulus of the gel is 213.2KPa through the mechanical compression test.

Claims (3)

1. a kind of preparation method of nanofiber silk fibroin hydrogel, which comprises the following steps:

(1) it is dissolved in after natural silk degumming in salt-formic acid lysate and obtains silk fibroin protein solution；In the salt-formic acid lysate, salt Concentration is 2~10 w/v%；Formic acid concn is 98wt%；The concentration of fibroin albumen is 5~50 w/ in the silk fibroin protein solution v%；

It (2) will be in the silk fibroin protein solution injection mold of step (1)；

(3) step (2) are immersed in the water equipped with the mold of silk fibroin protein solution, standing forms nanofiber fibroin albumen water-setting Glue；The nanofiber silk fibroin hydrogel includes the fiber of 10 ~ 100nm of diameter.

2. the preparation method of nanofiber silk fibroin hydrogel according to claim 1, it is characterised in that: the silk For one or more of mulberry silk, tussah silk, ricinus silk, wild silk yarn.

3. the preparation method of nanofiber silk fibroin hydrogel according to claim 1, it is characterised in that: the salt- Salt in formic acid lysate is lithium bromide, calcium chloride, zinc chloride, magnesium chloride, strontium chloride, lithium rhodanate, sodium sulfocyanate, thiocyanic acid One or more of magnesium, calcium nitrate.