Silk protein nano microfiber hydrogel and preparation method thereof

Abstract

The invention relates to the field of biomedical materials, and discloses fibroin nano microfiber hydrogel and a preparation method thereof. The preparation method comprises the following steps: preparing a regenerated silk protein aqueous solution; adding horse radish peroxidase and hydrogen peroxide into the obtained silk protein solution, and carrying out enzyme crosslinking at 37 DEG C; performing ultrasonic treatment on the obtained silk protein solution in an ice-water bath environment to induce transformation of a beta folding structure; and centrifuging the silk protein nano microfiber ultrafiltration tube to obtain the silk protein nano microfiber hydrogel. The preparation method is simple and efficient in preparation condition, green and environment-friendly, good in repeatability and high in popularization and application value, and the prepared fibroin nano microfiber hydrogel can be widely applied to biomedical materials.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, and more specifically, to a silk fibroin nanofibril hydrogel and a preparation method thereof. Background Art

[0002] With the development of biological tissue materials, the demand potential for biomedical materials with excellent performance, efficient preparation technology and easy popularization is huge. Hydrogel is a three-dimensional cross-linked network rich in a large number of water molecules, with strong permeability, and is similar to the extracellular matrix in structure and function. Among them, injectable hydrogel as a biomedical material for minimally invasive surgery has always been a research hotspot. Hydrogel is not only an important material for reconstructing tissue engineering cell scaffolds, but also a very promising delivery and sustained release carrier material.

[0003] As a natural polymer material, silk fibroin is widely used in biomedical materials due to its rich source, excellent mechanical properties, excellent biocompatibility, degradability, self-assembly, adhesiveness and hemostasis. The preparation methods of injectable hydrogels usually mainly induce gelation by physical and chemical methods. Among them, chemical cross-linking includes photocatalytic cross-linking and introducing chemical reagents for cross-linking, and the process is cumbersome or has potential biological toxicity; physical cross-linking usually requires relatively harsh conditions, such as pH, high temperature, mechanical stirring and ethanol treatment, etc., and its mechanical strength is usually uncontrollable and the gelation time is long, which limits its wide application. In the patent (CN 104017374 A), a method for preparing silk fibroin injectable hydrogel through silk fibroin nanofibers is described, but the method has a long time period for preparing silk fibroin nanofibrils, and sodium chloride needs to be added for centrifugation during the concentration process, and the process is relatively complex. The method for preparing silk fibroin nanofibrils referred to, that is, a method for preparing silk fibroin nanofibrils described in the patent (CN 103757729 A), prepared nanofibrils by adjusting high pH (6-12) and adding ethanol (2-30% v / v) or heating conditions. It takes 1-3 days to form nanofibrils at room temperature, and nanofibrils can be obtained by culturing at high temperature of 50-90 °C for 3-24 hours. Its control conditions are complex and the time for preparing microfibrils is long, which is not conducive to large-scale popularization.

[0004] Ultrasound-induced treatment is an effective and controllable method. During the ultrasound treatment process, mechanical vibration causes the formation and rupture of bubbles, thereby inducing the formation of β-sheets. Based on the above situation, if the ultrasound-induced treatment technology can be combined to provide a simple and rapid method for preparing silk fibroin nanofibril hydrogel, it can effectively solve the problems such as complex preparation conditions and long time period of current silk fibroin nanofibrils. Summary of the Invention

[0005] The object of the present invention is to provide a simple and rapid method for preparing a silk fibroin nanofibril hydrogel in view of the disadvantages of complex preparation conditions and long time periods for current silk fibroin nanofibrils. The hydrogel has good biocompatibility, can controllably load drug molecules or other nano-delivery carriers, and can be widely applied to biomedical tissue materials.

[0006] In the present invention, pre-crosslinking in a dilute silk fibroin solution is combined with ultrasonic induction to regulate the time for the transformation of silk fibroin molecules from random coils to β-sheets, and silk fibroin nanofibrils are rapidly prepared; then, separation is carried out through an ultrafiltration tube to rapidly obtain a silk fibroin nanofibril hydrogel. The preparation process of the present invention is simple, efficient, environmentally friendly, highly repeatable, and has good popularization. The obtained silk fibroin nanofibrils can load different types of active drugs or nano-delivery carriers.

[0007] In order to achieve the above object of the invention, the following technical solutions are adopted in this application:

[0008] In the first aspect, this application provides a method for rapidly preparing a silk fibroin nanofibril hydrogel. The preparation method is as follows: diluting a silk fibroin solution, pre-crosslinking, ultrasonic induction, ultrafiltration and centrifugation. The specific steps are as follows:

[0009] Step 1: Prepare an aqueous solution of regenerated silk fibroin;

[0010] Step 2: Add horseradish peroxidase and hydrogen peroxide to the silk fibroin solution obtained in Step 1, and perform enzyme crosslinking at 37 °C;

[0011] Step 3: Ultrasonically treat the silk fibroin solution obtained in Step 2 in an ice-water bath environment to induce the transformation of the β-sheet structure;

[0012] Step 4: Centrifuge the silk fibroin nanofibrils in Step 3 through an ultrafiltration tube to obtain a silk fibroin nanofibril hydrogel.

[0013] Further, in Step 1, the mass fraction of the aqueous solution of regenerated silk fibroin is 0.05% - 1%.

[0014] Further, in Step 2, the addition amount of horseradish peroxidase is 100 - 2000 Units / mL; the addition amount of hydrogen peroxide is 0.001 - 1.0% v / v; the solvent is pure water.

[0015] Further, in Step 2, the enzyme crosslinking time is 5 - 60 min.

[0016] Further, in Step 3, the ultrasonic power is 300 - 1000 W, the ultrasonic process is to turn on for 0.5 seconds and turn off for 0.5 seconds and repeat the cycle, and the cycle time is 5 - 30 min.

[0017] Further, in the step 4, the centrifugation conditions are: 5000 - 10000 rpm, 30 - 90 min.

[0018] In a second aspect, the present application provides a silk fibroin nanofibril hydrogel obtained by the preparation method described in the first aspect.

[0019] Further, the silk fibroin nanofibril hydrogel may further comprise drug molecules to be loaded or other inorganic or organic nano-delivery carriers.

[0020] In a third aspect, the present application provides the use of the silk fibroin nanofibril hydrogel described in the second aspect in the preparation of a biological tissue repair material.

[0021] In summary, the present application has the following beneficial effects:

[0022] 1. The hydrogel raw material of the present invention is silk fibroin which is low-cost and certified by the US Food and Drug Administration. The obtained hydrogel has good biocompatibility and biodegradability.

[0023] 2. The horseradish peroxidase used in the present invention is mainly extracted from the roots of wasabi plants and has been commercially produced; the peroxide is hydrogen peroxide at a low concentration commonly used in surgical operations, and experiments have shown that the silk fibroin hydrogel crosslinked with horseradish peroxidase has no cytotoxicity.

[0024] 3. The silk fibroin solution described in the present invention can be a mixed solution of water-soluble natural polymers, functional organic or inorganic nanoparticles, drug molecules, etc., and can be used to prepare a multifunctional silk fibroin nanofibril hydrogel.

[0025] 4. In addition, the preparation conditions of the present invention are simple, efficient, green, environmentally friendly, and have good repeatability, and have the value of popularization and application.

[0026] 5. The silk fibroin nanofibril hydrogel prepared by the present invention can be widely used in biomedical materials. Description of the Drawings

[0027] Figure 1 : SEM image of regenerated silk fibroin nanofibrils.

[0028] Figure 2 : Co-culture results of the drug thrombin / silk fibroin nanofibril hydrogel and cells. Detailed Description of the Embodiments

[0029] The following further describes the structure and effects of the present application in detail with reference to the embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. In addition, it should be noted that only parts related to the present invention are shown in the drawings for the convenience of description, rather than all the structures.

[0030] Example 1:

[0031] A method for preparing a silk fibroin nanofibril hydrogel, the process comprising the following steps:

[0032] Take a silk fibroin solution with a mass fraction of 0.75 wt%, add horseradish peroxidase 900 Units / ml and hydrogen peroxide 0.5 wt%, and react at 37 °C for 10 min. Subsequently, perform an ultrasonic (output power 300 W) experiment in an ice-water bath at a ratio of 50% (repeated cycle of 0.5 s on and 0.5 s off) for 15 min to obtain a silk fibroin nanofibril aqueous solution. Centrifuge the silk fibroin nanofibril solution at 5000 rpm for 60 min to prepare a silk fibroin nanofibril hydrogel. The experimental results show that the silk fibroin nanofibril hydrogel has injectability, and the internal structure is silk fibroin nanofibrils (such as Figure 1 ), and at the same time, the results of co-culture with cells show good biocompatibility.

[0033] Example 2:

[0034] A method for preparing a silk fibroin nanofibril hydrogel, the process comprising the following steps:

[0035] Take a silk fibroin solution with a mass fraction of 0.5 wt%, add horseradish peroxidase 900 U / ml and hydrogen peroxide 0.5 wt%, and react at 37 °C for 15 min. Subsequently, perform an ultrasonic (output power 300 W) experiment in an ice-water bath at a ratio of 50% (repeated cycle of 0.5 s on and 0.5 s off) for 30 min to obtain a silk fibroin solution containing nanofibers. Centrifuge the silk fibroin nanofibril solution at 10000 rpm for 30 min to prepare a silk fibroin nanofibril hydrogel. The experimental results show that the silk fibroin nanofibril hydrogel has injectability, and at the same time, the results of co-culture with cells show good biocompatibility.

[0036] Example 3:

[0037] A method for preparing a silk fibroin nanofibril hydrogel, the process comprising the following steps:

[0038] Take a silk fibroin solution with a mass fraction of 0.5 wt%, add horseradish peroxidase at 1200 U / ml and hydrogen peroxide at 0.3 wt%, and react at 37 °C for 5 min. Subsequently, perform an ultrasonic (output power 300 W) experiment in an ice-water bath at a ratio of 50% (repeated cycles of 0.5 s on and 0.5 s off) for 30 min to obtain a silk fibroin solution containing nanofibers. Centrifuge the silk fibroin nanofibril solution at 3000 rpm for 90 min to prepare a silk fibroin nanofibril hydrogel. The experimental results show that the silk fibroin nanofibril hydrogel has injectability, and the results of co-culture with cells indicate good biocompatibility.

[0039] Example 4:

[0040] A preparation method of a silk fibroin nanofibril hydrogel, the process comprising the following steps:

[0041] Take a silk fibroin solution with a mass fraction of 0.75 wt%, add horseradish peroxidase at 900 U / ml and hydrogen peroxide at 0.3 wt%, and react at 37 °C for 10 min. Subsequently, perform an ultrasonic (output power 300 W) experiment in an ice-water bath at a ratio of 50% (repeated cycles of 0.5 s on and 0.5 s off) for 30 min to obtain a silk fibroin solution containing nanofibers. Centrifuge the mixed solution of silk fibroin nanofibrils and nanorobot carriers at 10000 rpm for 30 min to prepare a silk fibroin nanofibril-based nanorobot hydrogel. The experimental results show that the silk fibroin nanofibril-based nanorobot hydrogel has injectability, and the results of co-culture with cells indicate good biocompatibility.

[0042] Example 5:

[0043] A preparation method of a silk fibroin nanofibril hydrogel, the process comprising the following steps:

[0044] Take a silk fibroin solution with a mass fraction of 0.25 wt%, add 90 nM thrombin to be loaded, and add horseradish peroxidase at 1200 U / ml and hydrogen peroxide at 0.3 wt%, and react at 37 °C for 10 min. Subsequently, perform an ultrasonic (output power 600 W) experiment in an ice-water bath at a ratio of 50% (repeated cycles of 0.5 s on and 0.5 s off) for 20 min to obtain a drug-loaded thrombin / silk fibroin nanofibril solution. Centrifuge the mixed solution of silk fibroin nanofibrils and drug thrombin at 5000 rpm for 60 min to prepare a drug-loaded thrombin silk fibroin nanofibril hydrogel. The experimental results show that the drug-loaded thrombin / silk fibroin nanofibril hydrogel has injectability, and the results of co-culture with cells indicate good biocompatibility ( Figure 2 ).

[0045] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment that do not contribute creatively as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing a silk protein nanofiber hydrogel, characterized in that: The following steps are involved: Step 1: preparing a regenerated silk protein aqueous solution; Step 2: adding horseradish peroxidase and hydrogen peroxide to the silk protein solution obtained in step 1, and performing enzyme cross-linking at 37° C.; Step 3: ultrasonically treating the silk protein solution obtained in step 2 in an ice-water bath to induce β-folding structure transformation; Step 4: Centrifuge the silk protein nanofiber ultrafiltration tube in step 3 to obtain the silk protein nanofiber hydrogel.

2. The preparation method according to claim 1, characterized in that: In the step 1, the mass fraction of the regenerated silk protein aqueous solution is 0.05% to 1%.

3. The preparation method according to claim 1, characterized in that: In the step 2, the amount of horseradish peroxidase added is 100-2000 Units / mL; the amount of hydrogen peroxide added is 0.001-1.0% v / v; and the solvent is pure water.

4. The preparation method according to claim 1, characterized in that: In the step 2, the enzyme cross-linking time is 5 to 60 minutes.

5. The preparation method according to claim 1, characterized in that: In step 3, the ultrasonic power is 300-1000W, the ultrasonic process is a cycle of on for 0.5 seconds and off for 0.5 seconds, and the cycle time is 5-30 minutes.

6. The preparation method according to claim 1, characterized in that: In step 4, the centrifugation conditions are: 5000-10000 rpm, 30-90 min.

7. The silk protein nanofiber hydrogel obtained by the preparation method according to any one of claims 1 to 6.

8. The silk protein nanofiber hydrogel according to claim 7, characterized in that: The silk protein nanofiber hydrogel may also contain drug molecules or other inorganic or organic nano delivery carriers to be loaded.

9. Use of the silk protein nanofiber hydrogel according to claim 7 or 8 in preparing biological tissue repair materials.

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