CN107080860B

CN107080860B - Silk protein sponge and preparation method thereof

Info

Publication number: CN107080860B
Application number: CN201710301039.0A
Authority: CN
Inventors: 祝文莉; 吕强
Original assignee: Sinate Suzhou Biological Science & Technology Co ltd
Current assignee: Sinate Suzhou Biological Science & Technology Co ltd
Priority date: 2017-05-02
Filing date: 2017-05-02
Publication date: 2020-08-14
Anticipated expiration: 2037-05-02
Also published as: CN107080860A

Abstract

The invention provides a fibroin sponge and a preparation method thereof, wherein the fibroin sponge is of an amorphous structure with a nanofiber-micropore composite structure and is insoluble in water. The application also provides a preparation method of the silk protein sponge, which comprises the following steps: A) adjusting the concentration of the amorphous silk protein nanofiber aqueous solution; B) freezing the amorphous silk protein nanofiber solution obtained in the step A); C) and D) freeze-drying the amorphous silk protein nano-fiber obtained in the step B) to obtain silk protein sponge. According to the preparation method, the porous silk protein sponge which is completely composed of the nano fibers and is of an amorphous structure is prepared through the steps. Different from the original method for preparing the fibroin sponge, the preparation method of the sponge material does not need other treatment modes to induce crystallization to realize the stability of the sponge material in water, avoids the use of other inducers such as methanol, acid and the like, and has the advantages of simple process, environmental protection and excellent biocompatibility.

Description

Silk protein sponge and preparation method thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a fibroin sponge and a preparation method thereof.

Background

Silk protein has attracted much attention in the field of biomedical materials due to its excellent biocompatibility, mechanical properties, degradability, ease of forming, and ability to be processed in aqueous solutions. The silk protein sponge with a porous structure is widely applied to laboratory research of different tissue repair, and a plurality of products are in clinical test, so that the silk protein sponge material is the most important component of silk protein-based biological materials and has wide application prospect.

Researchers have developed various methods for preparing fibroin sponges, mainly including salting out and freeze-drying; the porous sponge prepared by the salting-out method has the main conformation of beta-sheet crystallization, has high mechanical property and is not suitable for being applied to soft tissue repair; although the silk protein prepared by the freeze drying method is generally in an amorphous structure, the silk protein can be dissolved in water, so that the silk protein cannot be applied, and beta-sheet can be induced to form only by alcohol treatment and vacuum water treatment, and finally the water-insoluble sponge material is obtained. Therefore, although there are various methods such as adding highly crystalline fibroin for induction, using acid for inhibiting crystallization, etc. to reduce the crystallinity of the sponge, and reducing the hardness of the sponge on the basis of maintaining the stability of the sponge in water, there is still no solution to the inherent contradiction between the water insolubility, mechanical properties, and crystal structure of the fibroin porous sponge. On the other hand, in recent years, the introduction of the extracellular matrix-like nanofiber structure has become an effective means for improving the biocompatibility of the material, and the research has been carried out to improve the proportion of the nanofiber structure in the fibroin sponge by adding a nanofiber inducing method, but the fibroin sponge is completely composed of nanofibers, and the fibroin sponge with a composite structure still needs to be continuously researched.

Recently, the applicant researches and develops a fibroin nanofiber with an amorphous structure, the diameter of the fibroin nanofiber is 10-20 nm, the length of the fibroin nanofiber is 100 nm-1 mu m, the fibroin nanofiber has good hydrophilicity, and a high-concentration fibroin solution can be formed, so that the fibroin sponge completely consisting of the nanofiber and having a composite structure can be prepared on the basis of the fibroin nanofiber. However, the above-mentioned silk fibroin nanofibers having an amorphous structure are metastable and easily transform into highly crystalline silk fibroin fibers with changes in temperature, time, and the like, thereby forming a gel. How to avoid the formation of beta-sheet in the preparation process is a key technical problem which needs to be solved urgently for preparing the silk protein sponge material with a nanofiber-porous composite structure.

Therefore, based on the above-mentioned progress, it is necessary to enhance the interaction between molecules while suppressing the formation of a crystalline state of silk protein by finely controlling the molecular mobility of silk protein during the production process, and finally to produce a silk protein sponge material composed of an amorphous structure and insoluble in water.

Disclosure of Invention

The technical problem to be solved by the invention is to provide the fibroin sponge and the preparation method thereof.

In view of the above, the present application provides a fibroin sponge, which is an amorphous structure of a nanofiber-micropore composite structure, and is insoluble in water.

Preferably, the diameter of the nanofiber of the silk fibroin sponge is 10-20 nm, the length of the nanofiber is 100 nm-1 μm, and the pore diameter of the silk fibroin sponge is 50-800 μm.

The application also provides a preparation method of the silk protein sponge, which comprises the following steps:

A) adjusting the concentration of the amorphous silk protein nanofiber aqueous solution;

B) freezing the amorphous silk protein nanofiber solution obtained in the step A);

C) and D), freeze-drying the amorphous silk protein nano-fiber obtained in the step B) to obtain silk protein sponge.

Preferably, in the step a), the concentration of the amorphous silk protein nanofiber aqueous solution is adjusted by adding water for dilution or concentration.

Preferably, the concentration is by volatilization concentration or PEO displacement concentration.

Preferably, the concentration temperature is 1-15 ℃, and the concentration time is 0-48 h.

Preferably, in the step A), the diameter of the amorphous silk protein nanofiber in the amorphous silk protein nanofiber aqueous solution is 10-20 nm, and the length of the amorphous silk protein nanofiber is 100 nm-1 μm.

Preferably, in the step A), the concentration of the amorphous silk protein nanofiber aqueous solution after adjustment is 0.1-12 wt%.

Preferably, in the step B), the freezing temperature is-2 to-15 ℃, and the freezing time is 8 to 48 hours.

Preferably, in the step C), the freeze-drying temperature is-20 to-90 ℃, and the freeze-drying time is 12 to 48 hours.

The application provides a fibroin sponge which is an amorphous structure with a nanofiber-micropore composite structure and is insoluble in water. Therefore, the application provides a preparation method of the fibroin sponge, which comprises the steps of firstly adjusting the concentration of the amorphous fibroin nanofiber aqueous solution, and then sequentially freezing and freeze-drying the amorphous fibroin nanofiber aqueous solution with the adjusted concentration to finally obtain the fibroin sponge. In the process, the adjustment of the concentration of the amorphous silk fibroin nanofiber aqueous solution enables the silk fibroin nanofiber to be gradually transformed from a random amorphous state to an intermediate state, the conformation of the silk fibroin is further transformed to the intermediate state in the freezing process and gradually approaches to a crystalline state, crystalline particles are formed in the silk fibroin at the same time, finally, in the freeze-drying stage, the silk fibroin nanofiber mainly comprising the amorphous conformation is formed, the crystalline particles are freeze-dried in the silk fibroin nanofiber to form a porous structure, and finally, the amorphous structure of the nanofiber-microporous composite structure is obtained, and the water-insoluble silk fibroin sponge is obtained.

Furthermore, the temperature and time for adjusting the concentration determine the degree of conversion from a random structure to an intermediate state of the silk fibroin nanofiber in the solution, and subsequent freezing and freeze-drying further regulate and control the conversion of the random state to finally obtain a proper intermediate state composition, so that the porous silk fibroin sponge has stability in water.

Drawings

FIG. 1 is a macroscopic view of an amorphous silk protein nanofiber as employed in the present invention;

FIG. 2 is a microscopic view of an amorphous silk protein nanofiber employed in the present invention;

FIG. 3 is an infrared spectrum of an amorphous silk protein nanofiber employed in the present invention;

FIG. 4 is an electron micrograph (high magnification) of a porous silk protein sponge material prepared in example 2 of the present invention;

FIG. 5 is an electron micrograph (lower magnification) of a porous silk protein sponge material prepared in example 2 of the present invention;

FIG. 6 is an XRD photograph of a porous silk protein sponge material prepared in example 2 of the present invention.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

The embodiment of the invention discloses a silk fibroin sponge which is an amorphous structure with a nanofiber-micropore composite structure and is insoluble in water.

The application provides a silk protein sponge, which mainly comprises silk protein nano fibers in amorphous conformation and has a nano fiber-micropore composite structure. The fibroin sponge is insoluble in water, and has good stability in water and better biocompatibility; the diameter of the nanofiber of the silk fibroin sponge is 10-20 nm, the length of the nanofiber is 100 nm-1 mu m, and the pore diameter of the nanofiber is adjustable within 50-800 mu m.

In order to obtain the silk protein sponge with the structure, the application provides a preparation method of the silk protein sponge, which comprises the following steps:

The method comprises the steps of utilizing metastable amorphous fibroin nanofiber to prepare amorphous fibroin sponge with a nanofiber-micropore composite structure through concentration adjustment, freezing and freeze-drying.

In the process of preparing the silk fibroin sponge, the concentration of the amorphous silk fibroin nanofiber aqueous solution is firstly adjusted, in the process of adjusting the amorphous silk fibroin nanofiber aqueous solution, the amorphous silk fibroin nanofibers are gradually transformed from a random amorphous state to an intermediate state, and the transformation degree determines whether the subsequent freeze-drying has stability and crystallization degree in water. The concentration of the amorphous silk protein nanofiber solution is not more than 1 wt%, otherwise, gelation occurs during dialysis. The method for adjusting the concentration of the amorphous silk protein nanofiber aqueous solution can be carried out by adding water for dilution or different concentration modes; the concentration mode can be open volatilization concentration or PEO displacement concentration. Specifically, when the concentration of the amorphous silk protein nanofiber aqueous solution is concentrated and adjusted, the solution is placed into an open container, the concentration temperature is kept at 1-15 ℃, and the concentration time is 0-48 hours; when the amorphous silk protein nanofiber aqueous solution is concentrated and adjusted, a PEO displacement concentration method is utilized, the concentration temperature is kept at 1-15 ℃, and the concentration time is 0-48 h; both too high a concentration temperature and too long a concentration time lead to gel formation and preparation failure.

The raw material amorphous silk protein nanofiber adopted by the application can firstly provide a nanofiber structure imitating extracellular matrix, so that the porous silk protein sponge has better biocompatibility, and meanwhile, the amorphous silk protein nanofiber can exist in a solution state instead of gel, so that the preparation of the porous sponge becomes possible. The diameter of the amorphous silk fibroin nanofiber in the amorphous silk fibroin nanofiber solution is 10-20 nm, and the length of the amorphous silk fibroin nanofiber is 100 nm-1 mu m. The preparation method of the amorphous silk protein fiber can be prepared according to a manner well known to those skilled in the art, and the preparation of the amorphous silk protein nanofiber aqueous solution specifically comprises the following steps:

(1) dissolving silk fibers in a lithium bromide-formic acid mixed solvent, and regulating the concentration and the proportion of formic acid and lithium bromide in the lithium bromide-formic acid mixed solvent to influence the dissolution degree of the silk fibers and keep the nano fibril structure of silk fibroin in the silk;

(2) placing the silk fiber solution dissolved by the mixed solvent into a dialysis bag, and controlling the assembly rate of silk protein in the dialysis process by regulating and controlling the concentration and temperature of the silk protein so as to prevent the silk protein from gelling;

(3) dialyzing for about 72 hours, and removing lithium bromide and formic acid to obtain the aqueous solution of the amorphous silk protein nanofiber.

This application then freezes the amorphous silk protein nanofiber after will adjusting concentration to make silk protein's conformation further to intermediate state transformation, and be close to the crystalline state gradually and not form the crystalline state, thereby be favorable to silk protein freeze-drying after can have stability in aqueous, form crystalline particle in this in-process amorphous silk protein nanofiber simultaneously, so as to form the porous structure in silk protein nanofiber in the later stage. The conversion degree determines whether the silk fibroin sponge has stability and the content of the crystal structure of the silk fibroin sponge, so that the silk fibroin sponge has corresponding mechanical properties; the degree of silk protein turnover is related to molecular motility and time, while the freezing temperature determines molecular motility and freezing time. Therefore, the freezing temperature is-2 to-15 ℃, and the freezing time is 8 to 48 hours; in a specific embodiment, the freezing temperature is-4 to-12 ℃, and the freezing time is 8 to 24 hours. If the freezing temperature is too low, the obtained porous silk protein sponge can be dissolved in water, and if the freezing temperature is too high, the silk protein nano-fiber can be difficult to freeze or gel.

The amorphous silk protein nanofiber obtained by the method is freeze-dried to obtain silk protein sponge. The freeze-drying temperature is-20 to-90 ℃, and the freeze-drying time is 8 to 48 hours; in a specific embodiment, the freeze-drying temperature is-40 to-80 ℃, and the freeze-drying time is 24 to 48 hours. The freeze-drying temperature and time can influence the molecular mobility of the silk fibroin nanofiber, so that the mobility and the mechanical property of the finally obtained silk fibroin sponge are influenced.

In the process of preparing the fibroin sponge, the fibroin sponge with an amorphous conformation and a nanofiber-micropore composite structure is prepared through three processes of concentration adjustment, freezing and freeze-drying, and is insoluble in water and has better stability in water. The three processes are a synergistic action process, the concentration temperature and time determine the degree of conversion of the amorphous silk protein nanofiber from a random structure to an intermediate state in a solution, and subsequent freezing and freeze-drying further regulate and control the conversion to finally obtain a proper intermediate state composition, so that the silk protein porous sponge has stability in water. The three can be adjusted together according to the state of the solution to achieve the aim, and the water-insoluble silk protein porous sponge can not be obtained by considering one factor independently and simultaneously.

The key point of the invention is to change the interaction of silk protein fibers by finely regulating and controlling the temperature and the time, and realize the performance breakthrough that the amorphous structure silk protein has stability in water. The amorphous silk protein nanofiber is in a metastable state and is easy to be converted into a beta-sheet crystalline state, so in order to obtain a proper concentration to regulate and control the pore diameter of the prepared sponge, the concentration of the amorphous silk protein nanofiber solution needs to be regulated in a relatively low-temperature environment of 1-15 ℃, the time is controlled within 48h, and gel is formed due to overhigh temperature and overlong time, so that the preparation fails; meanwhile, the freezing temperature is also the key for regulating and controlling the interaction of silk proteins, and the interaction of the silk proteins can be inhibited by the excessively low freezing temperature, so that the prepared silk protein sponge is dissolved in water, and the water-insoluble sponge can be directly prepared from the water solution only by finely regulating and controlling the freezing temperature.

Although the key conditions seem to be conventional technologies, the key points are that the deep understanding of the state of the fibroin nanofiber and the assembly process of the fibroin and the synergistic effect of different parameters are realized, the different parameters have extremely high correlation, the influence of the state of the fibroin and different factors on the assembly of the fibroin is not considered, and the insoluble amorphous fibroin sponge cannot be prepared by mechanically applying the parameters.

Compared with the existing method for preparing the spongy silk fibroin, the metastable amorphous silk fibroin nanofiber is used as a base material, the silk fibroin sponge which is completely composed of the nanofiber and has a nanofiber-micropore composite structure is obtained by a traditional freeze drying method, the biocompatibility is better, the pore diameter of the silk fibroin sponge can be effectively regulated and controlled through concentration, and different application requirements are met; through fine regulation and control of temperature and time in the concentration adjustment and freezing process, silk protein nanofiber is inhibited from being converted to beta-sheet, interaction between molecules is improved, and finally, the water-insoluble silk protein sponge with an amorphous structure is obtained, so that the inherent contradiction between water stability and crystallinity of the original silk protein material is solved; the whole process mainly induces the interaction of silk protein molecules through the temperature control, directly prepares the water-insoluble silk protein sponge from the aqueous solution without adding any other substance or carrying out any post-treatment, and has mild condition, simple process and better producibility.

For further understanding of the present invention, the following examples are provided to illustrate the preparation method of the porous silk protein sponge provided by the present invention, and the scope of the present invention is not limited by the following examples.

Example 1 preparation of an aqueous solution of amorphous silk protein nanofibers

(1) Dissolving silk fibers in a lithium bromide-formic acid mixed solvent, wherein the mass ratio of formic acid to lithium bromide in the lithium bromide-formic acid mixed solvent is 1:13, and the concentration of lithium bromide is 9 mol/L;

(2) placing the silk fiber solution dissolved by the mixed solvent into a dialysis bag for dialysis for 72 hours to obtain an amorphous silk protein nanofiber aqueous solution; the concentration of silk fibers in the silk fiber solution is 0.4-1 wt%, and the dialysis temperature is 4-25 ℃.

The amorphous silk protein nanofiber solutions in the following examples were prepared from example 1.

Example 2

(1) 50ml of amorphous silk protein nanofiber solution with the concentration of 0.8 percent is concentrated for 36 hours at the temperature of 2 ℃ by using a PEO displacement concentration method, the concentration is adjusted to 4 percent, and the solution is injected into a mold;

(2) freezing the silk protein nanofiber solution injected into the mold at-4 ℃ for 24h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-63 ℃, and freeze-drying for 48 hours to obtain the water-insoluble fibroin sponge.

Fig. 1 and 2 are a macroscopic view and a microscopic view of the amorphous silk protein nanofiber adopted in the step (1), and fig. 3 is an infrared spectrum of the amorphous silk protein nanofiber adopted in the step (1), and it can be seen from the drawings that the amorphous silk protein nanofiber adopted in the step (1) is in a solution state, the fiber diameter is 10-20 nm, the length is 100 nm-1 μm, and the infrared spectrum shows that the conformation is an amorphous structure.

Fig. 4 and 5 are respectively a high power electron microscope image and a low power electron microscope image of the porous silk fibroin sponge prepared in this embodiment, and it can be seen from the images that the pore diameter of the silk fibroin sponge prepared in this embodiment is 100-200 μm, and the pore wall is composed of nanofibers; fig. 6 is an XRD photograph of the fibroin sponge prepared in this example, which shows that the scaffold of the fibroin sponge prepared in this example has an amorphous structure.

Placing the silk protein sponge prepared in the embodiment in water, culturing for 24h, and drying; the mass of the silk protein sponge before and after the silk protein sponge is weighed, and after the silk protein sponge is soaked in water for 24 hours, the mass loss of the silk protein sponge is less than 5%, so that the silk protein sponge prepared in the embodiment has better stability in water. The mechanical strength of the silk fibroin sponge in a wet state is about 4 kPa.

Example 3

(1) Adding purified water into 20ml of amorphous silk protein nanofiber solution with the concentration of 0.8% to dilute the solution to 0.3%, and injecting the solution into a mold;

(2) freezing the silk protein nanofiber solution injected into the mold at-2 ℃ for 24h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-20 ℃, and freeze-drying for 48 hours to obtain the water-insoluble fibroin sponge. The mechanical strength of the silk fibroin sponge in a wet state is about 1 kPa.

Example 4

(1) Concentrating 30ml of amorphous silk protein nanofiber solution with the concentration of 0.9% at 4 ℃ for 20h by using an open volatilization method, concentrating to 2%, and injecting into a mold;

(2) freezing the silk protein nanofiber solution injected into the mold at-12 ℃ for 8h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-90 ℃, and freeze-drying for 24h to obtain the water-insoluble fibroin sponge. The mechanical strength of the silk fibroin sponge in a wet state is about 3 kPa.

Example 5

(1) Concentrating 30ml of amorphous silk protein nanofiber solution with the concentration of 1.2% at 10 ℃ for 12h by using an open volatilization method, concentrating to 6%, and injecting into a mold;

(2) freezing the silk protein nanofiber solution injected into the mold at-5 ℃ for 12h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-40 ℃, and freeze-drying for 36h to obtain the water-insoluble sponge material. The mechanical strength of the silk fibroin sponge in a wet state is about 2 kPa.

Example 6

(1) Concentrating 80ml of amorphous silk protein nanofiber solution with the concentration of 1% at 1 ℃ for 46h by using a PEO displacement concentration method to 10%, and injecting into a mold;

(2) freezing the silk protein nanofiber solution injected into the mold at-9 ℃ for 24h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-60 ℃, and freeze-drying for 48 hours to obtain the water-insoluble sponge material. The mechanical strength of the silk fibroin sponge in a wet state is about 5 kPa.

Comparative example 1

(1) Concentrating 30ml of amorphous silk protein nanofiber solution with the concentration of 0.9% at 50 ℃ for 12h by using an open volatilization method, concentrating to 2%, and injecting into a mold;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-90 ℃, and freeze-drying for 24h to obtain the water-insoluble fibroin sponge. However, the silk protein assembly rate is too high in the early heat treatment, and the silk protein is gelled in the freezing process, so that the sponge after freeze-drying is fragile and has no mechanical property.

Comparative example 2

(2) freezing the silk protein nanofiber solution injected into the mold at-20 ℃ for 24h to form an ice-like object;

(3) and (3) placing the frozen material in a freeze dryer, wherein the temperature of a cold trap is-60 ℃, and freeze-drying for 48h to obtain the sponge material. However, the prepared sponge material has no stability in water and can be dissolved in water.

Comparative examples 1 and 2 were performed by changing one of the parameters of examples 4 and 6, respectively, while keeping the other parameters unchanged, but none of the silk protein sponges prepared had the desired properties of the present invention, indicating a correlation between the different factors.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of a silk protein sponge comprises the following steps:

C) freeze-drying the amorphous silk protein nanofiber obtained in the step B) to obtain silk protein sponge;

the concentration of the amorphous silk protein nanofiber aqueous solution is adjusted in a concentration mode;

the concentration temperature is 1-15 ℃, and the concentration time is 0-48 h;

the freezing temperature is-2 to-15 ℃, and the freezing time is 8 to 48 hours;

the freeze-drying temperature is-20 to-90 ℃, and the freeze-drying time is 12 to 48 hours;

the fibroin sponge is of an amorphous structure with a nanofiber-micropore composite structure, and is insoluble in water.

2. The preparation method according to claim 1, wherein the diameter of the nanofiber of the silk fibroin sponge is 10 to 20nm, the length is 100nm to 1 μm, and the pore size of the silk fibroin sponge is 50 to 800 μm.

3. The method of claim 1, wherein the concentrating is by volatilization concentration or PEO displacement concentration.

4. The preparation method according to claim 1, wherein in the step A), the diameter of the amorphous silk protein nanofiber in the amorphous silk protein nanofiber aqueous solution is 10-20 nm, and the length of the amorphous silk protein nanofiber is 100 nm-1 μm.

5. The preparation method according to claim 1, wherein in the step A), the concentration of the aqueous solution of the amorphous silk protein nanofibers after adjustment is 0.1-12 wt%.