CN106729981B - A kind of silk fibroin nanofiber and its preparation method and application - Google Patents

Abstract

The present invention relates to high-molecular biologic medical material tech fields, and in particular to a kind of silk fibroin nano-fiber and its preparation method and application: S1. takes the silk fibroin protein solution of purifying, its mass concentration is adjusted to 0.1-5%；S2. the silk fibroin protein solution of purifying is obtained into fibroin albumen Frozen Body in -10 ~ 0 DEG C of progress freezing processing 2 ~ for 24 hours；S3. fibroin albumen Frozen Body is melted into defrosting, centrifugation removal insoluble matter, the silk fibroin protein solution reconstructed at room temperature；S4. the silk fibroin protein solution of reconstruct is diluted to mass concentration is 0.01-0.5%, stands 4-12h at 25-37 DEG C, is subsequently placed in liquid nitrogen and is frozen into solid, be then freeze-dried solid, obtains silk fibroin nano-fiber.The nanofiber technical process of fibroin albumen of the present invention is simple, effective, easily-controllable；Preparation condition is green, mild, will not reduce the biocompatibility of material；It can be realized the nanofiber in the different brackets with pore structure simultaneously.

Description

A kind of silk fibroin nanofiber and its preparation method and application

technical field

The invention relates to the technical field of polymer biomedical materials, in particular to a silk fibroin nanofiber and its preparation method and application.

Background technique

It is an urgent need for tissue engineering and regenerative medicine to develop biomaterial scaffolds that can effectively promote tissue regeneration. The natural extracellular matrix structure is the best template for us to design tissue engineering scaffolds. The natural extracellular matrix is assembled by a large amount of collagen, elastin and other fibrous proteins, and has a rich micro-nano fiber structure on the scale, which provides physical support and guidance signals for cell adhesion and migration. From a bionic point of view, biomaterial scaffold materials for tissue repair can effectively regulate cell behavior, promote cell adhesion, growth and proliferation, and guide cell migration, thereby promoting tissue regeneration. . Therefore, the nanofibrillation of polymer materials and its application in tissue engineering scaffolds are of great significance to promote the functionalization of scaffold materials.

Silk fibroin is a high-purity protein secreted by the endothelial cells of the silk glands of silkworm silk. It has good biocompatibility, low immunogenicity, excellent mechanical properties, and can be degraded into polypeptides and free amino acids that are absorbed and metabolized by the body. Ideal raw material for engineering supports. At present, extensive research has been carried out on silkworm silk fibroin as a tissue engineering scaffold at home and abroad, and various techniques including phase separation, salt extraction, and freeze-drying have been used to prepare silk fibroin porous scaffolds. However, the porous scaffold obtained by the above method lacks micro-nanofiber guidance signals to promote and guide cell growth. Currently, silk fibroin nanofibrillation methods mainly include electrospinning and induced self-assembly. The electrospinning of silk fibroin usually requires the use of organic solvents, which reduces the biocompatibility of the material, and the silk fibroin nanofibers prepared by electrospinning are also difficult to prepare a three-dimensional porous scaffold with a suitable pore structure. Silk fibroin can also form nanofibers through self-assembly. The invention patent with the Chinese authorized announcement number CN 102008756 B proposes that the mixed solution of silkworm silk fibroin collagen is slowly self-assembled in advance to form a nanofiber structure, and then a porous material containing a nanofiber structure is prepared on the pore wall. The invention patent of Chinese Patent Publication No. CN102357264 A proposes that the silk fibroin solution is repeatedly dried-dissolved, slowly self-assembled into nanofibers, and then used to prepare a porous material with a nanofiber structure on the pore wall. However, these silk fibroin self-assembly methods have a slow self-assembly process and complicated processes, and the obtained nanofibers are short in size and cannot form a nanofiber network structure similar to extracellular matrix.

In order to overcome the above-mentioned problems in the prior art, the inventors designed a novel silk fibroin nanofibrillation method to promote the application of silk fibroin as a tissue engineering scaffold.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a silk fibroin nanofiber and its preparation method and application, to reconstruct the secondary structure of silk fibroin by freezing and thawing, combined with freeze-drying technology, can effectively induce The nanofibrillation of silk fibroin has a simple, effective and easy-to-control process.

The purpose of the present invention is achieved through the following technical solutions:

A method for silk fibroin nanofibrillation, comprising the following steps:

S1. Take the purified silk fibroin solution and adjust its mass concentration to 0.1-5%;

S2. Freezing the purified silk fibroin solution at -10-0°C for 2-24 hours to obtain a silk fibroin frozen body;

S3. thawing the frozen silk fibroin at room temperature, centrifuging to remove insoluble matter, and obtaining a reconstituted silk fibroin solution;

S4. Dilute the reconstituted silk fibroin solution to a mass concentration of 0.01-0.5%, let it stand at 25-37°C for 4-12h, then place it in liquid nitrogen to freeze into a solid, and then freeze-dry the solid, Obtain nanofibrillated silk fibroin.

Further, the silk fibroin is Bombyx mori silk fibroin.

Further, the freezing treatment is freezing at -4°C for 24 hours.

Further, the centrifugation in the step S3 is performed at a speed of 5000-15000r/min for 3-10min.

Further, the freeze-drying treatment time in the step S4 is 24 hours.

A silk fibroin nanofiber prepared by the above method.

Further, its diameter is 10-200nm.

A nanofiberized porous scaffold, the scaffold has a pore structure or a channel structure, and the above-mentioned silk fibroin nanofiber is filled in the pore structure or the channel structure.

Further, the specific method of filling is:

S1. Take the purified silk fibroin solution and adjust its mass concentration to 0.1-5%;

S2. Freezing the purified silk fibroin solution at -10-0°C for 2-24 hours to obtain a silk fibroin frozen body;

S3. thawing the frozen silk fibroin at room temperature, centrifuging to remove insoluble matter, and obtaining a reconstituted silk fibroin solution;

S4. Dilute the mass concentration of the reconstituted silk fibroin solution to 0.01-0.4%, pour it into the scaffold material, let it stand at 25-37°C for 4-12h, then place it in liquid nitrogen to freeze into a solid, and then freeze the solid freeze-drying to obtain a nanofiberized porous scaffold; the scaffold material is a scaffold material with a pore structure or a channel structure.

The principle of the present invention is: during the freezing and thawing process of silk fibroin, the secondary structure transforms to a metastable structure to form reconstituted silk fibroin; the reconstituted silk fibroin solution can maintain a stable structure under low temperature conditions, When the concentration reaches 0.01-0.5%, it is easy to undergo structural transformation when it is above 25°C, forming β-folded silk fibroin; it can be effectively assembled into a dispersed nanofiber structure under liquid nitrogen rapid freezing; at the same time, the present invention provides The application of silk fibroin nanofibrillation, by perfusing the diluted reconstituted silk fibroin solution into the void with porous or tubular channel structure, successfully obtained nanofibrillation in the scaffold, effectively promoting the biological function of the scaffold sex.

The beneficial effects of the present invention are:

(1) The invention reconstructs the secondary structure of silk fibroin by freeze-thawing method, combined with freeze-drying technology, can effectively induce the nanofibrillation of silk fibroin, and the process is simple, effective and easy to control;

(2) The preparation process of the present invention is completed at a temperature lower than the physiological environment and in a water environment, so the preparation process is green and mild, and will not reduce the biocompatibility of the material;

(3) The silk fibroin solution used in the present invention has a low concentration, so the viscosity is low, and it is easy to pour into the pores of different porous scaffolds and the channels of tubular materials to realize nanofibrillation in the scaffold.

Description of drawings

Fig. 1 is the 10 micron scanning electron microscope picture of the silk fibroin nanofiber prepared in embodiment 1;

Fig. 2 is the 1 micron scanning electron microscope picture of the silk fibroin nanofiber prepared in embodiment 1;

FIG. 3 is a 500-micron scanning electron microscope image of the nanofibrillated silk fibroin porous scaffold prepared in Example 4. FIG.

Detailed ways

The technical solution of the present invention will be further described in detail below in conjunction with specific examples, but the protection scope of the present invention is not limited to the following description.

Example 1

Immerse 50 g of silkworm raw silk in 2.5 L of 0.05% NaCO ₃ solution, boil it at 98-100°C for 30 min, repeat 3 times to degumming the silk, wash and dry it thoroughly to obtain pure silk fiber; add silk fiber to into a 9.3 mol/L LiBr solution, stirred and dissolved at 60°C for 1 hour to obtain a silk fibroin mixed solution; put the obtained silk fibroin mixed solution into a dialysis bag, and dialyze with deionized water for 3 days to obtain purified silk fibroin solution;

Adjust the mass concentration of silk fibroin solution to 1%, freeze at -7°C for 24 hours and then thaw at room temperature to obtain a reconstituted silk fibroin solution;

Centrifuge the reconstituted silk fibroin solution at 13000 r/min for 5 min to remove insoluble matter;

Dilute the mass concentration of the above-mentioned reconstituted silk fibroin solution to 0.1%, place it at 37°C, and let it stand for 8 hours;

The above solution was placed in liquid nitrogen and frozen into a solid;

Put the frozen solid into a freeze drier, freeze and dry for 24 hours to obtain silk fibroin nanofibers.

The nanofibers of silk fibroin obtained in the preparation process of Example 1 were observed with a scanning electron microscope, and the results are shown in Fig. 1 and Fig. 2 .

Example 2

Add the degummed silk fibroin fiber to the CaCl ₂ /CH ₃ CH ₂ OH/H ₂ O ternary solution with a molar ratio of 1:2:8, stir and dissolve at 70°C for 1 hour to obtain a silk fibroin mixed solution; The obtained silk fibroin mixed solution was put into a dialysis bag, and dialyzed with deionized water for 3 days to obtain a purified silk fibroin solution;

Adjust the mass concentration of silk fibroin solution to 5%, freeze at -10°C for 24 hours and then thaw at room temperature to obtain a reconstituted silk fibroin solution;

Centrifuge the reconstituted silk fibroin solution at 15000 r/min for 10 min to remove insoluble matter;

Dilute the mass concentration of the reconstituted silk fibroin solution to 0.01%, place it at 30°C, and let it stand for 12 hours;

The above solution was placed in liquid nitrogen and frozen into a solid;

Put the frozen solid into a freeze drier, freeze and dry for 24 hours to obtain silk fibroin nanofibers.

Example 3

Add the degummed silk fibroin fiber to 9 mol/L LiBr solution, stir and dissolve at 60°C for 2 hours to obtain a silk fibroin protein mixed solution; put the obtained silk fibroin protein mixed solution into a dialysis bag, and use deionized Water dialysis for 3 days to obtain a purified silk fibroin solution;

Adjust the mass concentration of silk fibroin solution to 0.1%, freeze at -4°C for 24 hours and then thaw at room temperature to obtain a reconstituted silk fibroin solution;

Centrifuge the reconstituted silk fibroin solution at 10,000 r/min for 3 min to remove insoluble matter;

Dilute the mass concentration of the above-mentioned reconstituted silk fibroin solution to 0.5%, place it at 35°C, and let it stand for 4 hours;

The above solution was placed in liquid nitrogen and frozen into a solid;

Put the frozen solid into a freeze drier, freeze and dry for 24 hours to obtain silk fibroin nanofibers.

Example 4

Add the degummed silk fibroin fiber to 9.3 mol/L LiBr solution, stir and dissolve at 60°C for 1 hour to obtain a silk fibroin protein mixed solution; put the obtained silk fibroin protein mixed solution into a dialysis bag, and use deionized Water dialysis for 3 days to obtain a purified silk fibroin solution;

Adjust the mass concentration of silk fibroin solution to 1%, freeze at -7°C for 24 hours and then thaw at room temperature to obtain a reconstituted silk fibroin solution;

Centrifuge the reconstituted silk fibroin solution at 13000 r/min for 5 min to remove insoluble matter;

Dilute the mass concentration of the reconstituted silk fibroin solution to 0.1%, perfuse it into the EDC-crosslinked silk fibroin porous scaffold at 250 μL/cm ³ , place it at 25°C, and let it stand for 6 hours;

Freezing the above-mentioned porous scaffold in liquid nitrogen, so that the solution perfused into the interior is frozen into a solid;

Put the frozen scaffold into a freeze dryer, and freeze-dry for 24 hours to obtain a silk fibroin porous scaffold made of silk fibroin nanofibers.

The nanofibrillated silk fibroin scaffold obtained in the preparation process of Example 4 was observed under a scanning electron microscope, and the results are shown in FIG. 3 .

The above descriptions are only preferred embodiments of the present invention, and it should be understood that the present invention is not limited to the forms disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and Modifications can be made within the scope of the ideas described herein, by virtue of the above teachings or skill or knowledge in the relevant art. However, changes and changes made by those skilled in the art do not depart from the spirit and scope of the present invention, and should all be within the protection scope of the appended claims of the present invention.

Claims (8)

1. a kind of method of silk fibroin nano-fiber, which comprises the following steps:

Its mass concentration is adjusted to 0.1-5% by the silk fibroin protein solution for S1. taking purifying；

S2. the silk fibroin protein solution of purifying is obtained into fibroin albumen Frozen Body in -10 ~ 0 DEG C of progress freezing processing 2 ~ for 24 hours；

S3. fibroin albumen Frozen Body is melted into defrosting at room temperature, centrifugation removes insoluble matter, and the fibroin albumen reconstructed is molten Liquid；

S4. the silk fibroin protein solution of reconstruct is diluted to mass concentration is 0.01-0.5%, 4-12h is stood at 25-37 DEG C, so It is placed in liquid nitrogen and is frozen into solid, solid is then done into freeze-drying process, obtain the fibroin albumen of nanofiber.

2. a kind of method of silk fibroin nano-fiber according to claim 1, which is characterized in that the freezing processing It is to be freezed for 24 hours at -4 DEG C.

3. a kind of method of silk fibroin nano-fiber according to claim 1, which is characterized in that in the step S3 Centrifugation is to be centrifuged 3-10min with the revolving speed of 5000-15000r/min.

4. a kind of method of silk fibroin nano-fiber according to claim 1, which is characterized in that in the step S4 The freeze-drying process time is for 24 hours.

5. a kind of silk fibroin nano-fiber, which is characterized in that be prepared by claim 1-4 any one the method.

6. a kind of silk fibroin nano-fiber according to claim 5, which is characterized in that a diameter of 10-200nm.

7. a kind of porous support of nanofiber, which is characterized in that the bracket has pore structure or channel design, and hole Filled with silk fibroin nano-fiber described in claim 5 in structure or channel design.

8. a kind of porous support of nanofiber according to claim 7, which is characterized in that the specific method of the filling Are as follows:

The silk fibroin protein solution mass concentration of reconstruct is diluted to 0.01-0.4%, is filled into timbering material, at 25-37 DEG C 4-12h is stood, is subsequently placed in liquid nitrogen and is frozen into solid, be then freeze-dried solid, obtain the porous branch of nanofiber Frame；The timbering material is the timbering material with pore structure or channel design.