A preparation method capable of adjusting surface roughness of silk fibroin film

Abstract

The application discloses a preparation method of a silk fibroin film with adjustable surface roughness. The degummed silk fibroin is dissolved in a LiBr solution, and a regenerated silk fibroin solution is prepared through dialysis; the silk fibroin solution is poured to obtain a silk fibroin film, and the silk fibroin film is treated through salting-out, so that a silk fibroin film with different surface roughness is obtained. The application utilizes the interaction between water / inorganic salt and silk fibroin molecules to induce the conformation rearrangement of the silk fibroin molecules, and then promotes the crystallization and aggregation of the silk fibroin molecules, so that the surface roughness of the silk fibroin film is changed. Meanwhile, similar other physical properties are maintained.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method capable of adjusting the surface roughness of silk fibroin film, and relates to the field of biomedical materials. BACKGROUND

[0002] Biological materials have a wide range of applications in the medical field, among which silk fibroin film as an important biological material has good biocompatibility and controllable characteristics, and has a wide application prospect in tissue engineering, drug delivery, medical devices, etc.

[0003] In the process of biological material and cell interaction, the physical properties of biological materials, especially the characteristics of surface morphology, are considered to be one of the important factors affecting cell behavior and state. Existing research shows that the micro-morphology of the surface of biological materials, such as roughness and topography, can regulate the physiological behaviors of cells such as differentiation, migration, proliferation, and secretion.

[0004] For polymer film biological materials, chemical etching method, surface deposition method and plasma treatment method are often used to adjust the surface roughness of polymer film. However, chemical etching method uses chemical reagents with corrosive properties, the operation is complex, and the selectivity of the material is limited; surface deposition method often causes changes in the chemical properties of the film surface while changing the surface roughness; the plasma equipment has high cost and complex operation, and will affect the physical and chemical properties of the film. For example, before the present application, a method for adjusting the surface roughness of silk fibroin film using plasma treatment was reported in the International Journal of Biological Macromolecules (2024, 257, 128352). However, this treatment method can only adjust the surface roughness within a small range. In addition, after plasma treatment, the mechanical properties of silk fibroin film also change. These changes in physical and chemical properties will affect the behavior of cells.

[0005] Therefore, it is necessary to propose a simple and effective method to realize the regulation of the surface roughness of thin film biological materials without affecting other material properties, so as to realize the regulation of cell function, to solve the problems existing in the prior art, and to further promote the application of silk fibroin film in the medical field. SUMMARY

[0006] The purpose of the present application is to provide a simple and effective method for preparing silk fibroin films with different surface roughness. The present application provides a preparation method for silk fibroin films with adjustable surface roughness by combining casting and salting-out technology. The silk fibroin films prepared by this method can be used to regulate the physiological behavior of cells.

[0007] The application is to dissolve the degummed silk fibroin in LiBr solution, and prepare regenerated silk fibroin solution through dialysis, and to obtain silk fibroin membrane through casting method, and to treat the silk fibroin membrane through salting-out method, and finally to obtain silk fibroin membrane with different surface roughness.

[0008] The application has the following advantages:

[0009] The application utilizes the interaction between water / inorganic salt and silk fibroin molecules to induce conformational rearrangement of silk fibroin molecules, and then to promote crystallization and aggregation of silk fibroin molecules, so as to change the surface roughness of silk fibroin membrane. Meanwhile, similar other physical properties, such as mechanical properties and wettability, are maintained, and the regulation of single physical property of silk fibroin membrane is realized.

[0010] The application has the advantages of simple preparation, convenient operation and controllable effect, and avoids the change of other properties due to the change of a material property. Roughness as a single variable is suitable for studying the mechanism of the change of roughness on the behavior and response of body cells.

[0011] In addition, the rough silk fibroin membrane promotes the differentiation of anti-inflammatory macrophages and enhances the ability of the macrophages to secrete anti-inflammatory cytokines. After implantation in vivo, the early inflammatory response is reduced, and a new way and possibility are provided for the application of biomaterials in the fields of tissue engineering and medical devices. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Figure 1 is a scanning electron microscope image of the silk fibroin membrane prepared in Example 1.

[0013] Figure 2 Figure 2 is a scanning electron microscope image of the silk fibroin membrane prepared in Example 2.

[0014] Figure 3 Figure 3 is a scanning electron microscope image of the silk fibroin membrane prepared in Example 3.

[0015] Figure 4 Figure 4 is a scanning electron microscope image of the silk fibroin membrane prepared in Example 4. DETAILED DESCRIPTION

[0016] The application will be further described in detail through specific examples, and the following examples are used to explain the application, but the application is not limited to the following examples.

[0017] The method of the application comprises the following steps:

[0018] Step (1), the cocoon layer is placed in a 0.5% mass fraction Na2CO3 boiling water solution for degumming twice, each time for 30 min, and the obtained silk fibroin is washed with deionized water, then wrung dry and dried in an oven.

[0019] Step (2), the silk fibroin fibers obtained in step (1) were dissolved in 9M LiBr solution at 60°C for 4h, the obtained liquid was filtered and then put into dialysis bag (MWCO 8000-14000) for dialysis in deionized water for 3d, and the water was changed constantly, finally the silk fibroin solution with concentration of 1-10% (w / v) was obtained.

[0020] Step (3), the silk fibroin solution obtained in step (2) was slowly poured into a suitable mold, the uniformity and thickness of the casting were controlled, then it was transferred to a clean bench, the environmental temperature and humidity were maintained, and the air flow rate was adjusted to obtain the silk fibroin membrane.

[0021] Step (4), the silk fibroin membrane obtained in step (3) was taken out of the mold and soaked in a salt solution for treatment.

[0022] Step (5), the silk fibroin membrane in step (4) was taken out of the salt solution, and the silk fibroin membrane with different surface roughness was obtained after removing the residual salt compounds on the surface.

[0023] As preferred, the cocoon layer of the present application is one of domestic silkworm, tussah silkworm and other wild silkworm silk or a mixture of two or more.

[0024] As preferred, the concentration of the silk fibroin solution of the present application is 5-10% (w / v).

[0025] As preferred, the mold of the present application is one of different specifications of polystyrene culture dishes or glass culture dishes. The volume of the silk fibroin solution added to the mold is 4-20 mL.

[0026] As preferred, the environmental temperature is 20-35°C, and the environmental relative humidity is 30-70%.

[0027] As preferred, the air flow rate is 0.3-0.6 m / s.

[0028] As preferred, the salt used for the salting-out treatment of the silk fibroin membrane is one of ammonium acetate, ammonium sulfate, potassium sulfate, sodium sulfate, potassium citrate and sodium citrate.

[0029] As preferred, the concentration of the salt used is 0.5-3M.

[0030] As preferred, the salting-out treatment time is 24-72h.

[0031] Example 1

[0032] Step (1), 10 g of cut cocoon layer was dipped in 0.5% Na2CO3 boiling water solution twice for 30 min each time, and the obtained silk fibroin fibers were washed with deionized water, wrung dry, and dried in an oven.

[0033] Step (2), the silk fibroin fibers obtained in step (1) were placed in 9M LiBr solution and dissolved at 60°C for 4h, and the obtained liquid was filtered and placed in a dialysis bag (molecular weight 8000-14000 MWCO) for dialysis in deionized water for 3d, with water being changed constantly, and finally an 8% (w / v) silk fibroin solution was obtained.

[0034] Step (3), the silk fibroin solution in step (2) was poured into a polystyrene culture dish, which was placed in a clean bench, and the air speed was adjusted to high at room temperature (25°C) and 60% RH, and a silk fibroin film was obtained after no visible liquid was observed in the culture dish.

[0035] The scanning electron microscope of the silk fibroin film is shown in Figure 1 It can be seen that the surface of the silk fibroin film is smooth and no micro-nano structure is visible. The atomic force microscope was used for surface roughness test, and the root mean square roughness of the silk fibroin film was 1.0 nm.

[0036] Example 2

[0037] Step (1), 10 g of cut cocoon layer was dipped in 0.5% Na2CO3 boiling water solution twice for 30 min each time, and the obtained silk fibroin fibers were washed with deionized water, wrung dry, and dried in an oven.

[0038] Step (2), the silk fibroin fibers obtained in step (1) were placed in 9M LiBr solution and dissolved at 60°C for 4h, and the obtained liquid was filtered and placed in a dialysis bag (molecular weight 8000-14000 MWCO) for dialysis in deionized water for 3d, with water being changed constantly, and finally an 8% (w / v) silk fibroin solution was obtained.

[0039] Step (3), the silk fibroin solution in step (2) was poured into a polystyrene culture dish, which was placed in a clean bench, and the air speed was adjusted to high at room temperature (25°C) and 60% RH, and a silk fibroin film was obtained after no visible liquid was observed in the culture dish.

[0040] Step (4), the silk fibroin film in step (3) was taken out of the culture dish, immersed in 2M ammonium sulfate solution for 48h, and then taken out, and the surface residual salt was washed off with pure water, and dried until no visible liquid was observed to obtain a silk fibroin film with a specific surface roughness.

[0041] The scanning electron microscope of the silk fibroin film is shown inFigure 2 It can be seen that the surface of the silk fibroin film has a certain micro-nano structure. The roughness of the film was measured by atomic force microscopy, and the root mean square roughness of the silk fibroin film was 56.1 nm.

[0042] Example 3

[0043] Step (1), 10 g of cut cocoon layer was weighed and degummed twice in a 0.5% Na2CO3 boiling water solution for 30 min each time. The obtained silk fibroin fibers were washed with deionized water, wrung dry, and dried in an oven.

[0044] Step (2), the silk fibroin fibers obtained in step (1) were placed in a 9M LiBr solution and dissolved at 60°C for 4h. The obtained liquid was filtered and placed in a dialysis bag (molecular weight 8000-14000 MWCO) and dialyzed in deionized water for 3d, with constant water change. Finally, a 5% (w / v) silk fibroin solution was obtained.

[0045] Step (3), the silk fibroin solution in step (2) was poured into a polystyrene culture dish and placed in a clean bench. The air speed was adjusted to low at room temperature (25°C) and 60% RH. After no visible liquid was observed in the culture dish, the silk fibroin film was obtained.

[0046] The scanning electron microscope of the silk fibroin film is shown in Figure 3 It can be seen that the surface of the silk fibroin film has a certain micro-nano structure. The roughness of the film was measured by atomic force microscopy, and the root mean square roughness of the silk fibroin film was 56.1 nm.

[0047] Example 4

[0048] Step (1), 10 g of cut cocoon layer was weighed and degummed twice in a 0.5% Na2CO3 boiling water solution for 30 min each time. The obtained silk fibroin fibers were washed with deionized water, wrung dry, and dried in an oven.

[0049] Step (2), the silk fibroin fibers obtained in step (1) were placed in a 9M LiBr solution and dissolved at 60°C for 4h. The obtained liquid was filtered and placed in a dialysis bag (molecular weight 8000-14000 MWCO) and dialyzed in deionized water for 3d, with constant water change. Finally, a 5% (w / v) silk fibroin solution was obtained.

[0050] Step (3), the silk fibroin solution in step (2) was poured into a polystyrene culture dish and placed in a clean bench. The air speed was adjusted to low at room temperature (25°C) and 60% RH. After no visible liquid was observed in the culture dish, the silk fibroin film was obtained.

[0051] Step (4), the silk fibroin film in step (3) is taken out from the culture dish, and is soaked in 2M ammonium sulfate solution for 48h, then is taken out, washed with pure water to remove the residual salt on the surface, and is dried to obtain a silk fibroin film with specific surface roughness.

[0052] The scanning electron microscope of the silk fibroin film is shown in Figure 4 As can be seen, there are uneven structures on the surface of the silk fibroin film. The atomic force microscope is used for surface roughness test, and the root mean square roughness of the silk fibroin film is 35.7nm.

[0053] Therefore, the present application provides a new silk fibroin film with different surface roughness, the preparation process is simple, the conditions are mild, the surface roughness can be adjusted at the micro-nano level, and other physical properties of the silk fibroin film such as wettability and mechanical properties remain similar.

[0054] The above only lists some specific embodiments of the present application, and obviously, the present application is not limited to the above embodiments, and there are many variations, and all the variations directly derived or thought from the disclosed content by those skilled in the art should be considered as the protection scope of the present application.

Claims

1. A method for preparing a silk fibroin film capable of adjusting surface roughness, characterized by The method comprises the following steps: Step (1), degumming cocoon layer twice in Na2CO3 boiling water solution to obtain silk fibroin fibers; rinsing with deionized water, wringing and drying; Step (2), dissolving the silk fibroin fibers obtained in step (1) in LiBr solution under a set temperature condition to obtain a liquid, which is filtered and then loaded into a dialysis bag, and dialyzed in deionized water to obtain a silk fibroin protein solution; Step (3), slowly pouring the silk fibroin protein solution obtained in step (2) into a mold, controlling the uniformity and thickness of the casting, and then transferring to an ultra-clean workbench, maintaining a certain environmental temperature and humidity, and adjusting the air flow speed to obtain a silk fibroin protein film; Step (4), taking the silk fibroin protein film obtained in step (3) out of the mold and soaking in a salt solution for treatment; Step (5), taking the silk fibroin protein film in step (4) out of the salt solution, and obtaining a silk fibroin protein film with different surface roughness after removing the residual salt compounds on the surface; The air flow speed in step (3) is 0.3-0.6 m / s; The environmental temperature in step (3) is 20-35℃, and the environmental relative humidity is 30-70%; Step (4) adopts salting-out method to treat the silk fibroin protein film, wherein the salting-out treatment time is 24-72 h.

2. The method of claim 1, wherein the surface roughness of the silk fibroin film is adjusted. The cocoon layer in step (1) is one or more than two kinds of cocoon layer of domestic silkworm, tussah and other wild silkworm silk.

3. The method of claim 1, wherein the surface roughness of the silk fibroin film is adjusted. The mass concentration of the silk fibroin protein solution in step (2) is 5-10%.

4. The method of claim 1, wherein the surface roughness of the silk fibroin film is adjusted. The mold in step (3) is one or both of different specifications of polystyrene culture dishes or glass culture dishes.

5. The method of claim 4, wherein the surface roughness of the silk fibroin film is adjusted. The volume of the silk fibroin protein solution added to the mold is 4-20 mL.

6. The method of claim 1, wherein the surface roughness of the silk fibroin film is adjusted. The salt solution in step (4) is one of ammonium acetate, ammonium sulfate, potassium sulfate, sodium sulfate, potassium citrate or sodium citrate.

7. The method of claim 6, wherein the surface roughness of the silk fibroin film is adjusted. The concentration of the salt solution is 0.5-3 M.

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