WO2022156100A1

WO2022156100A1 - High-strength silk protein nanofiber membrane and preparation method therefor

Info

Publication number: WO2022156100A1
Application number: PCT/CN2021/094756
Authority: WO
Inventors: 吕强; 张筱旖
Original assignee: 苏州大学
Priority date: 2021-01-20
Filing date: 2021-05-20
Publication date: 2022-07-28
Also published as: CN112876711A; CN112876711B

Abstract

A high-strength silk protein nanofiber membrane and a preparation method therefor. The preparation method comprises: freeze-drying a high-crystal silk protein nanofiber aqueous solution to obtain silk protein nanofiber freeze-dried powder; dissolving the silk protein nanofiber freeze-dried powder in a formic acid solution to obtain a silk protein nanofiber formic acid solution; and volatilizing the silk protein nanofiber formic acid solution into a membrane to obtain the silk protein nanofiber membrane. High-crystal silk protein nanofibers used in the method have a beta-sheet crystal structure and good stability, can still keep an original secondary structure and nanofiber morphology after being dissolved in formic acid, and allow the tensile mechanical properties of a prepared membrane material to be greatly improved due to the optimization of a non-covalent action. The preparation method implements the transformation of mechanical properties from fragile to tough by means of simple solvent conversion, and a high tensile mechanical strength is shown in both a dry state and a wet state, which respectively reaches 69.2-76.9 MPa and 13.5-14.6 MPa; moreover, the preparation method is performed at normal temperature and normal pressure, and is simple and easy to implement.

Description

A kind of high-strength silk protein nanofiber membrane and preparation method thereof

This application claims the priority of the Chinese patent application with the application number 202110074285.3 and the invention title "A high-strength silk protein nanofiber membrane and its preparation method" submitted to the China Patent Office on January 20, 2021, the entire contents of which are approved by Reference is incorporated in this application.

technical field

The invention belongs to the technical field of high-strength biological materials, and in particular relates to a high-strength silk protein nanofiber membrane and a preparation method thereof.

Background technique

High-strength materials have always been a research hotspot in the field of materials due to their excellent properties and wide applications. Natural high-strength materials self-assemble into complex micro-nano structures by optimizing the non-covalent interactions between components, making them have excellent properties. For example, cellulose in plants, chitin in arthropod bones, silk protein in animal silk, etc., all form nanofibers of different sizes, which are formed by optimizing non-covalent interactions such as hydrogen bonding, electrostatic, and hydrophobic interactions. Functional materials with high strength, stiffness and toughness. Therefore, regulating the supramolecular assembly of nanofibrous units at the micro- and nano-scale is a key step in the formation of high-strength materials.

As a typical natural high-strength material, silk has attracted widespread attention in the fields of textiles, regenerative medicine, and smart industries. Silk proteins can be prepared into various material forms, such as fibers, membranes, hydrogels, scaffolds, and microspheres, which can be widely used in tissue engineering, flexible electronic devices, and drug delivery. However, unlike natural silk fibers, regenerated silk protein materials usually suffer from poor mechanical properties due to the breakdown of their hierarchical structure. Although researchers have improved the properties of regenerated silk protein materials by adjusting the content of beta-sheet, optimizing the orientation structure, and preparing composite materials, and achieved a certain mechanical improvement effect, the lack of nanofiber primitives and the loss of assembly structure The further improvement of mechanical properties is still limited.

In recent years, researchers have achieved the remodeling of silk protein nanofibers through various strategies. However, complex and harsh preparation conditions, such as ethanol treatment and ultrasonic dispersion, limit their hierarchical structure assembly and further functional optimization. Based on the application requirements in the field of biomedicine, the formation of uniform and stable nanofibers under natural conditions such as normal temperature and pressure is an ideal choice for obtaining supramolecular structures with higher mechanical properties.

However, due to the high charge density of silk protein nanofibers, which inhibits the formation of effective non-covalent interactions between nanofibers, the formed regenerated silk nanofiber membranes are fragile and fragile, and cannot even be measured by traditional tensile models.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a high-strength silk protein nanofiber membrane and a preparation method thereof. The nanofiber membrane prepared by the method has high tensile strength.

The invention provides a preparation method of a high-strength silk protein nanofiber membrane, comprising the following steps:

S1: freeze-dry the high-crystalline fibroin nanofiber aqueous solution to obtain a lyophilized silk protein nanofiber powder; the crystallinity of the silk protein fibers in the high-crystalline fibroin nanofiber aqueous solution is ≥40%, the diameter is 10-30 nm, and the length is 200- 2000nm;

S2: Dissolving the silk protein nanofiber freeze-dried powder in a formic acid solution to obtain a silk protein nanofiber formic acid solution;

S3: volatilize the formic acid in the silk protein nanofiber formic acid solution to form a film to obtain a silk protein nanofiber film.

Preferably, in the step S1, the high-crystalline fibroin nanofiber aqueous solution is prepared according to the following method:

Concentrating the silk protein aqueous solution to the first silk protein solution with a concentration of 8-12 wt%;

Concentrating the first silk protein solution to a second silk protein solution with a concentration of 16-24%;

The second silk protein solution is diluted with water to a concentration of 0.01-4 wt %, sealed and incubated to obtain a high-crystalline silk protein nanofiber solution.

Preferably, the concentration of the high-crystalline fibroin nanofiber aqueous solution is 0.01-4 wt%.

Preferably, in the step S2, the concentration of the silk protein nanofiber formic acid solution is 1-20 wt%.

Preferably, in the step S2, the dissolution time is 0.1-24h.

Preferably, in the step S2, the dissolving temperature is 4-80°C.

Preferably, in the step S3, the temperature of the film formation is 4-60°C.

Preferably, in the step S3, the film forming time is 2-72 hours.

The present invention provides a high-strength silk protein nanofiber membrane, which is prepared by the preparation method described in the above technical solution.

The high-crystalline fibroin nanofibers used in the method provided by the invention have the crystal structure of beta-sheet and good stability, and can still maintain their original secondary structure and nanofiber morphology when dissolved in formic acid. The optimization of the action greatly improves the tensile mechanical properties of the prepared membrane material. The invention realizes the transformation of mechanical properties from fragile to tough through simple solvent conversion, and shows high tensile mechanical strength in both dry and wet states, reaching 69.2-76.9 MPa and 13.5-14.6 MPa respectively, and in It is carried out under normal temperature and normal pressure, and the preparation method is simple and easy.

Description of drawings

Figure 1 is an atomic force microscope picture of the silk protein nanofibers prepared in Example 1 of the present invention dissolved in water (a) and formic acid (b) respectively;

Figure 2 is a digital photograph of the silk protein nanofiber aqueous solution (a) and the silk protein nanofiber formic acid solution (b) prepared in Example 1 of the present invention cast into films respectively;

Fig. 3 is the infrared spectrogram of silk protein nanofiber aqueous solution film (SNF-H2O) and silk protein nanofiber formic acid film (SNF-FA) prepared in Example 1 of the present invention;

FIG. 4 is a stress-strain curve diagram of the silk protein nanofiber membrane prepared in Example 1 of the present invention in a dry state and a wet state.

Detailed ways

The high-crystalline fibroin nanofibers used in the method provided by the invention have the crystal structure of beta-sheet and good stability, and can still maintain their original secondary structure and nanofiber morphology when dissolved in formic acid. The optimization of the action greatly improves the tensile mechanical properties of the prepared membrane material. The present invention realizes the transformation of mechanical properties from fragile to tough through simple solvent conversion, and shows high tensile mechanical strength in both dry and wet states, which are 76.9 MPa and 14.6 MPa, respectively, and under normal temperature and pressure The preparation method is simple and easy. The method for reducing charge repulsion, optimizing non-covalent interaction and regulating mechanical properties through formic acid provided by the invention provides experimental basis for mechanical regulation of other materials, and can be widely used in the field of high-strength material preparation.

In the present invention, the high-crystalline silk protein nanofiber aqueous solution is freeze-dried to obtain silk protein nanofiber freeze-dried powder. In the present invention, the high-crystalline fibroin nanofiber aqueous solution is preferably prepared according to the following method:

In the present invention, the concentration of the high-crystalline fibroin nanofiber aqueous solution is 0.01-4%; in a specific embodiment, the concentration of the high-crystalline fibroin nanofiber aqueous solution is 0.5%, 1%, 2% or 4%.

After obtaining the silk protein nanofiber freeze-dried powder, the present invention dissolves the silk protein nanofiber freeze-dried powder in a formic acid solution to obtain a silk protein nanofiber formic acid solution. In the present invention, the concentration of the silk protein nanofiber formic acid solution is 1-20 wt%. In a specific embodiment, the concentration of the silk protein nanofiber formic acid solution is 4wt%, 1wt%, 2wt% or 10wt%. In the present invention, the amount of formic acid is used to regulate the concentration of the formic acid solution of the silk protein nanofibers, and then the non-covalent interaction between the silk protein nanofibers is regulated to realize the transformation of the mechanical properties of the material and obtain a high-strength silk protein membrane material. The method for reducing charge repulsion, optimizing non-covalent interaction and regulating mechanical properties through formic acid provided by the invention provides experimental basis for mechanical regulation of other materials, and can be widely used in the field of high-strength material preparation.

In the present invention, the dissolving temperature is 4-80°C, and the dissolving time is 0.1-24h; in a specific embodiment, the dissolving temperature is 60°C, 20°C, 4°C, 40°C or 10°C; The time is 0.5h, 24h, 1h, 4h or 12h. The present application realizes the transformation of mechanical properties from brittleness to toughness through solvent conversion, and can be carried out at normal temperature and pressure, and the preparation method is simple and easy.

After obtaining the silk protein nanofiber formic acid solution, the present invention volatilizes the formic acid in the silk protein nanofiber formic acid solution to form a film to obtain a silk protein nanofiber film.

In the present invention, the film-forming temperature is 4-60°C; the film-forming time is 2-72h; in a specific embodiment, the film-forming temperature is 60°C, 40°C, 30°C or 20°C; The time is 2h, 4h, 24h, 48 or 72h.

The thickness of the high-strength silk protein nanofiber membrane is 33 μm, 35 μm, 40 μm, 38 μm or 30 μm.

The present invention adopts the following method to test the tensile strength of the high-strength silk protein nanometer:

The test was carried out using a universal testing machine (Instron 5967, sample length: 20 mm; tensile speed: 10 mm/min) at 25±0.5° C., 60±5% relative humidity. In the dry state, the samples were equilibrated in a constant temperature and humidity room (25±0.5°C, 60±5% humidity) for 24 hours. In the wet state, the samples were soaked in 0.01M phosphate buffered saline (PBS) for 1 hour, and then the mechanical properties were measured.

In order to further illustrate the present invention, a high-strength silk protein nanofiber membrane provided by the present invention and its preparation method are described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

(1) freeze-drying 0.5% silk protein nanofiber aqueous solution to obtain silk protein nanofiber freeze-dried powder;

(2) Dissolving silk protein nanofiber freeze-dried powder in formic acid solution at 60°C for 0.5 h to obtain a 10% silk protein nanofiber formic acid solution;

(3) 10% silk protein nanofiber formic acid solution was evaporated at 60° C. for 2 h to obtain a silk protein nanofiber membrane with a thickness of 30 μm.

Figure 1 is an atomic force microscope picture of the silk fibroin nanofibers prepared in Example 1 of the present invention dissolved in water (a) and formic acid (b) respectively; it can be seen from Figure 1: in the aqueous solution of formic acid, the silk fibroin is all The morphology of nanofibers is presented; the length of nanofibers in aqueous solution is distributed in 200-1000 nm, while the length of nanofibers in formic acid is reduced to 50-200 nm, and the shorter fiber length provides more opportunities for fiber-to-fiber interaction.

Figure 2 is a digital photo of the silk protein nanofiber aqueous solution (a) and the silk protein nanofiber formic acid solution (b) prepared in Example 1 of the present invention, which were cast into films respectively. It can be seen in the figure that the silk protein films prepared under the aqueous system are very good. The fibroin films prepared under the formic acid system exhibited intact morphology, indicating that the enhancement of the non-covalent effect between nanofibers reversed the film-forming properties of the films.

Figure 3 is the infrared spectrum of the silk protein nanofiber aqueous solution film (SNF-H ₂ O) and the silk protein nanofiber formic acid film (SNF-FA) prepared in Example 1 of the present invention; it can be seen from Figure 3 that the two films Both have beta-sheet secondary structure, providing stable fiber units for supramolecular assembly;

Figure 4 is the stress-strain curve of the silk protein nanofiber membrane prepared in Example 1 of the present invention in dry and wet states. The control group is the traditional silk protein membrane (SF-MA) treated with methanol. For the preparation method, see Flexibility Regeneration of Silk Fibroin in Vitro (Biomacromolecules 2012,13,2148～2153); it can be seen from Figure 4 that the silk protein nanofiber membrane provided by the present invention shows high tensile mechanical strength in both dry state and wet state, which are 76.9 MPa and 14.6MPa.

Table 1 Zeta potential value of the membrane prepared in Example 1

It can be seen from Table 1 that after the nanofibers are dissolved in formic acid, the zeta potential changes from -35.1mV to 9.0mV, and the decrease in the absolute value of the potential indicates a decrease in charge repulsion, which is beneficial to the improvement of non-covalent interactions between nanofibers. .

Example 2:

(1) freeze-drying 2% silk protein nanofiber aqueous solution to obtain silk protein nanofiber freeze-dried powder;

(2) Dissolving silk protein nanofiber freeze-dried powder in formic acid solution at 20°C for 1 h to obtain a 2% silk protein nanofiber formic acid solution;

(3) The 2% silk protein nanofiber formic acid solution was evaporated at 20° C. for 72 h to obtain a silk protein nanofiber membrane with a thickness of 38 μm.

The tensile mechanical strengths of the silk protein nanofiber membrane prepared in Example 2 in dry state and wet state were 69.2 MPa and 13.5 MPa, respectively.

Example 3:

(2) Dissolving the silk protein nanofiber freeze-dried powder in formic acid solution at 4°C for 24 hours to obtain a 2% silk protein nanofiber formic acid solution;

(3) The 2% silk protein nanofiber formic acid solution was evaporated at 30° C. for 48 h to obtain a silk protein nanofiber membrane with a thickness of 40 μm.

The tensile mechanical strengths of the silk protein nanofiber membrane prepared in Example 3 in dry and wet states were 72.5 MPa and 14.0 MPa, respectively.

Example 4:

(1) freeze-drying 4% silk protein nanofiber aqueous solution to obtain silk protein nanofiber freeze-dried powder;

(2) Dissolving silk protein nanofiber freeze-dried powder in formic acid solution at 40°C for 4 hours to obtain 4% silk protein nanofiber formic acid solution;

(3) A 4% silk protein nanofiber formic acid solution was evaporated at 40°C for 24 h to obtain a silk protein nanofiber membrane with a thickness of 35 μm.

The tensile mechanical strength of the silk protein nanofiber membrane prepared in Example 4 was 76.5 MPa and 13.7 MPa in dry state and wet state, respectively.

Example 5:

(1) freeze-drying 1% silk protein nanofiber aqueous solution to obtain silk protein nanofiber freeze-dried powder;

(2) Dissolving the silk protein nanofiber freeze-dried powder in formic acid solution at 10°C for 12 hours to obtain a 1% silk protein nanofiber formic acid solution;

(3) The 1% silk protein nanofiber formic acid solution was evaporated at 60° C. for 4 h to obtain a silk protein nanofiber membrane with a thickness of 33 μm.

The tensile mechanical strength of the silk protein nanofiber membrane prepared in Example 5 was 75.8 MPa and 14.0 MPa in dry state and wet state, respectively.

As can be seen from the above embodiments, the present invention provides a method for preparing a high-strength silk protein nanofiber membrane, comprising the following steps: S1: freeze-drying an aqueous solution of high-crystalline silk protein nanofibers to obtain silk protein nanofiber freeze-dried powder; The crystallinity of the fibroin nanofibers in the fibroin nanofiber aqueous solution is ≥40%, the diameter is 10-30 nm, and the length is 200-2000 nm; S2: Dissolving the fibroin nanofiber freeze-dried powder in a formic acid solution to obtain fibroin Nanofiber formic acid solution; S3: volatilizing the formic acid in the silk protein nanofiber formic acid solution to form a film to obtain a silk protein nanofiber film. The high-crystalline fibroin nanofibers used in the method provided by the invention have the crystal structure of beta-sheet and good stability, and can still maintain their original secondary structure and nanofiber morphology when dissolved in formic acid. The optimization of the action greatly improves the tensile mechanical properties of the prepared membrane material. The present invention realizes the transformation of mechanical properties from fragile to tough through simple solvent conversion, and shows high tensile mechanical strength in both dry and wet states, reaching 69.2-76.9 MPa and 13.5-14.6 MPa, respectively, and in It is carried out under normal temperature and normal pressure, and the preparation method is simple and easy. The method for reducing charge repulsion, optimizing non-covalent interaction and regulating mechanical properties by formic acid provided by the present invention provides experimental basis for mechanical regulation of other materials, and can be widely used in the field of high-strength material preparation.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims

A preparation method of high-strength silk protein nanofiber membrane, comprising the following steps:

S1: freeze-dry the high-crystalline fibroin nanofiber aqueous solution to obtain a lyophilized silk protein nanofiber powder; the crystallinity of the silk protein fibers in the high-crystalline fibroin nanofiber aqueous solution is ≥40%, the diameter is 10-30 nm, and the length is 200- 2000nm;

S2: Dissolving the silk protein nanofiber freeze-dried powder in a formic acid solution to obtain a silk protein nanofiber formic acid solution;

S3: volatilize the formic acid in the silk protein nanofiber formic acid solution to form a film to obtain a silk protein nanofiber film.
The preparation method according to claim 1, wherein, in the step S1, the high-crystalline fibroin nanofiber aqueous solution is prepared according to the following method:

Concentrating the silk protein aqueous solution to a first silk protein solution with a concentration of 8-12 wt%;

Concentrating the first silk protein solution to a second silk protein solution with a concentration of 16-24%;

The second silk protein solution is diluted with water to a concentration of 0.01-4 wt %, sealed and incubated to obtain a high-crystalline silk protein nanofiber solution.
The preparation method according to claim 1, characterized in that, in the step S1, the concentration of the high-crystalline fibroin nanofiber aqueous solution is 0.01-4 wt%.
The preparation method according to claim 1, wherein in the step S2, the concentration of the silk protein nanofiber formic acid solution is 1-20 wt%.
The preparation method according to claim 1, characterized in that, in the step S2, the dissolving time is 0.1-24h.
The preparation method according to claim 1, characterized in that, in the step S2, the dissolving temperature is 4-80°C.
The preparation method according to claim 1, wherein, in the step S3, the temperature of the film formation is 4-60°C.
The preparation method according to claim 1, wherein, in the step S3, the film forming time is 2-72 hours.
A high-strength silk protein nanofiber membrane, prepared by the preparation method of any one of claims 1-8.