A porous sericin scaffold material, a preparation method and application thereof

Abstract

The application discloses a sericin porous scaffold material and a preparation method and application thereof, and relates to the technical field of biological materials. The method comprises the following steps: dissolving sericin in water, adding carbodiimide and stirring uniformly, adding N-hydroxysuccinimide and stirring to perform a cross-linking reaction; obtaining a sericin scaffold precursor through a mechanochemical cross-linking strategy; precooling the sericin scaffold precursor, completely freeze-drying, and obtaining a sericin scaffold; dialyzing the sericin scaffold in water for 2-3 days, precooling again and freeze-drying, and obtaining a porous sericin scaffold material. The porous scaffold material containing only sericin is prepared through a carbodiimide cross-linking method combined with a mechanochemical reaction. The scaffold has the performances of high compression strength, high liquid absorption rate, fast shape recovery, aggregation of red blood cells and fast hemostasis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a silk fibroin porous scaffold material and a preparation method and application thereof. BACKGROUND

[0002] Silk fibroin is a water-soluble globulin. It can inhibit the release of pro-inflammatory factors, reduce the occurrence of inflammation, has low immunogenicity; can scavenge intracellular reactive oxygen species, inhibit apoptosis and thus has antioxidant effect; has in situ fluorescence characteristics, provides convenience for tracking the changes of silk fibroin material in the body, and has good biocompatibility, is a high-quality natural medical material. However, in industrial production, silk fibroin is mostly discharged in the form of wastewater, which not only wastes resources, but also destroys the aquatic ecosystem. Therefore, the development of silk fibroin-based biomaterials has important significance for energy saving and emission reduction.

[0003] Silk fibroin has a high dissolution rate in water, and the silk fibroin porous scaffold directly freeze-dried has very poor mechanical properties. The current solution is to prepare a composite biological scaffold by combining silk fibroin with other substances to exert the unique biological function of silk fibroin. Although this strategy masks the poor mechanical properties of silk fibroin, the doping of other substances, especially some synthetic polymers, results in poor biocompatibility of the composite material. This seriously affects the application of silk fibroin-based biological scaffolds. Therefore, it is of great significance to synthesize a porous scaffold containing only silk fibroin through a specific preparation strategy to fully exert the specific biological function of silk fibroin and expand its application value as a biomaterial. SUMMARY

[0004] In view of the deficiencies in the above background art, the present application provides a silk fibroin porous scaffold material and a preparation method and application thereof. The method prepares a porous scaffold material containing only silk fibroin through a carbodiimide cross-linking method and a mechanochemical synthesis strategy. The silk fibroin scaffold prepared by the method not only solves the problems of high dissolution rate and poor mechanical properties of silk fibroin, but also has high liquid absorption rate, rapid shape recovery, aggregation of red blood cells and rapid hemostasis. In addition, the silk fibroin porous scaffold material prepared by the present application is low in cost and green and non-toxic.

[0005] The first object of the present application is to provide a preparation method of a silk fibroin porous scaffold material, comprising the following steps:

[0006] Dissolve silk fibroin in water, add carbodiimide and stir uniformly, then add N-hydroxysuccinimide and stir for cross-linking reaction to obtain a cross-linked solution;

[0007] Apply external force to the cross-linked solution to make the activated proteins in the cross-linked solution aggregate and undergo cross-linking reaction, and collect the aggregates to obtain a silk fibroin scaffold precursor;

[0008] The sericin scaffold precursor is pre-cooled and completely freeze-dried to obtain the silk fibroin scaffold;

[0009] The silk fibroin scaffold is dialyzed in water for 2-3 days, pre-cooled again and freeze-dried to obtain the porous silk fibroin scaffold material.

[0010] Preferably, when the silk fibroin is dissolved in water, it is heated to complete dissolution by a boiling water bath, and then cooled to room temperature.

[0011] Preferably, the mass ratio of the silk fibroin, the carbodiimide and the N-hydroxysuccinimide is 5:1-10:1-5.

[0012] Preferably, the way of applying external force includes centrifugation or pressurization.

[0013] When the protein in the cross-linking solution is aggregated by centrifugation, the centrifugation speed is 1000-10000 rpm, and the centrifugation time is 1-20 min.

[0014] Preferably, the pre-cooling temperature is -80--20℃, and the pre-cooling time is 12-24 h.

[0015] Preferably, the molecular weight of the silk fibroin is ≥8000 MW.

[0016] Preferably, during the dialysis of the silk fibroin scaffold in water, the dialysis bag used has a molecular weight cut-off of 3500 MW.

[0017] Preferably, when the silk fibroin is dissolved in water, it is heated to complete dissolution by a boiling water bath, and then cooled to room temperature.

[0018] The temperature of the cross-linking reaction is 0-50℃, and the reaction time is 2 h or more.

[0019] Preferably, the freeze-drying time is 10-24 h.

[0020] The second object of the present application is to provide a silk fibroin porous scaffold material.

[0021] The third object of the present application is to provide the application of the silk fibroin porous scaffold material in the preparation of antibacterial or hemostatic dressings, and in the treatment of photoimaging or heavy sewage.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application provides a silk fibroin porous scaffold material, a preparation method and an application. The method provided by the present application has low cost, requires few reagents, and the product contains only silk fibroin and no small molecule reagents, is green and non-toxic, has a simple preparation process, and does not require high-precision equipment assistance.

[0024] The silk fibroin porous scaffold material obtained by the application has good biocompatibility, high compression strength, high liquid absorption rate, fast shape recovery, aggregation of red blood cells and fast hemostasis performance. The activated silk fibroin is covalently bonded to form a solid interconnected pore through shear destruction, centrifugal compaction and pre-cooling secondary compaction and force chemical cross-linking reaction. Compared with the silk fibroin before cross-linking, the silk fibroin scaffold has high compression strength and is scattered when meeting water. At the same time, the scaffold with high compression strength has stronger resistance to deformation, so it has the performance of fast shape recovery. The interconnected pore structure makes it have high liquid absorption rate. In addition, the silk fibroin itself has the effect of promoting coagulation, and the porous structure is beneficial to the aggregation of red blood cells. Fast shape recovery and high liquid absorption rate enable the silk fibroin scaffold to quickly absorb a large amount of bleeding when applied to deep tissue, and the excellent mechanical strength enables it to form a solid coagulation barrier after absorbing blood at the bleeding site, which is beneficial to the formation of the fibrin network in the coagulation process, thereby realizing fast hemostasis.

[0025] The application adjusts the stirring speed and centrifugal force in the cross-linking reaction process, so as to control the pore size, water absorption and mechanical properties of the silk fibroin porous scaffold.

[0026] From the preparation method, structure and performance, the application has high practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the photo of the silk fibroin before force chemical cross-linking and the water absorption of the silk fibroin scaffold after cross-linking.

[0028] Figure 2 is the scanning electron microscope graph of the porous scaffold prepared by the force chemical cross-linking method and the porous scaffold prepared by the force chemical cross-linking method.

[0029] Figure 3 is the scanning electron microscope graph of the silk fibroin porous scaffold material obtained in examples 1-3 before compression, after compression and after water absorption.

[0030] Figure 4 is the compression stress statistical result of the silk fibroin porous scaffold material obtained in examples 1-3.

[0031] Figure 5 is the shape recovery time statistical result of the silk fibroin porous scaffold material obtained in examples 1-3.

[0032] Figure 6 is the liquid absorption rate (absorption of deionized water, physiological saline and blood) statistical result of the silk fibroin porous scaffold material obtained in examples 1-3.

[0033] Figure 7 is the scanning electron microscope picture of the aggregation of red blood cells of the silk fibroin porous scaffold material obtained in examples 1-3. DETAILED DESCRIPTION

[0034] In order to make the skilled in the art better understand the technical solutions of the present application can be implemented, the following specific embodiments and the present application is further described with reference to the drawings, but the examples are not as a limitation of the present application.

[0035] The purpose of the present application is to provide a preparation method and application of silk fibroin porous scaffold material, and the composition is only silk fibroin. The silk fibroin porous scaffold material is prepared by carbodiimide crosslinking method combined with chemical reaction. The scaffold has the properties of high compressive strength, high liquid absorption rate, rapid shape recovery, aggregation of red blood cells and rapid hemostasis. In the prior art, silk fibroin can only play a role by adhering to other scaffold materials, and cannot be made into a biological scaffold as the only component. The present application prepares a porous scaffold material with silk fibroin as the only component, solves the problems of high dissolution rate and poor mechanical properties of silk fibroin, and enriches the natural source biological scaffold system.

[0036] In order to achieve the above purpose, the first aspect of the present application provides a preparation method of silk fibroin porous scaffold material, comprising the following steps:

[0037] Dissolve silk fibroin in water, add carbodiimide and stir uniformly, then add N-hydroxysuccinimide and stir for crosslinking reaction to obtain a crosslinking solution;

[0038] Apply external force to the crosslinking solution to make the activated proteins in the crosslinking solution aggregate and crosslinking reaction occurs, and collect the aggregates to obtain a silk fibroin scaffold precursor;

[0039] Precool the silk fibroin scaffold precursor, and completely freeze-dry to obtain a silk fibroin scaffold;

[0040] Dialyze the silk fibroin scaffold in water for 2-3 days, precool and freeze-dry again to obtain a porous silk fibroin scaffold material.

[0041] The silk fibroin scaffold prepared by the present application has the properties of high compressive strength, high liquid absorption rate, rapid shape recovery, aggregation of red blood cells and rapid hemostasis, and the pores are adjusted by adjusting the shear force and centrifugal force in the reaction process.

[0042] The mass ratio of the silk fibroin, carbodiimide and N-hydroxysuccinimide is 5:1-10:1-5.

[0043] The precooling temperature is-80--20℃, and the precooling time is 12-24 h.

[0044] The molecular weight of the silk fibroin is ≥8000 MW.

[0045] The dialysis bag used in the dialysis process in water has a molecular weight cut-off of 3500 MW.

[0046] The external force can be applied by centrifugation or pressurization.

[0047] When the protein in the cross-linking solution is aggregated by centrifugation, the centrifugation speed is 1000-10000 rpm, and the centrifugation time is 1-20 min.

[0048] When the sericin is dissolved in water, it is heated to complete dissolution by a boiling water bath, and then cooled to room temperature.

[0049] The temperature of the cross-linking reaction is 0-50°C, and the reaction time is 2 h or more.

[0050] The duration of the freeze-drying is 10-24 h.

[0051] In one embodiment, a method for preparing a sericin porous scaffold material includes the following steps:

[0052] (1) Dissolve sericin in water and heat to complete dissolution, then cool to room temperature, add carbodiimide and stir for 15 min, then add N-hydroxysuccinimide for cross-linking reaction to obtain a flocculent sericin dispersion.

[0053] The process is a self-cross-linking reaction of sericin using carbodiimide protein coupling method, and the reaction time is 2 h or more. The purpose of stirring is to control the spontaneous disorder cross-linking of sericin by shear force.

[0054] The molecular weight of the sericin in step (1) is 8000 MW or more; the mass ratio of sericin: carbodiimide: N-hydroxysuccinimide in step (1) is 5:1-10:1-5. The reaction time in step (1) is 2 h or more, and the reaction temperature is 0-50°C. The concentration of sericin in step (1) is 6% or less.

[0055] Carbodiimide can activate the carboxyl groups on sericin to obtain an intermediate O-acyl isourea, which further reacts with the amino groups on sericin to form stable amide bonds between the peptide chains of sericin. The role of N-hydroxysuccinimide is to improve the yield.

[0056] (2) Centrifuge the dispersion obtained in step (1) to remove the supernatant to obtain a sericin scaffold precursor.

[0057] The purpose of centrifugation is to increase the probability of collision and cross-linking of activated carboxyl and amino groups in sericin protein, and to control the water content in the sericin scaffold precursor, so as to affect the nucleation and growth of ice crystals in the subsequent precooling process. By adjusting the centrifugal force, the pore size, compression strength, liquid absorption and shape recovery performance of the porous material can be controlled.

[0058] (3) The sericin scaffold precursor obtained in step (2) is pre-cooled and completely freeze-dried to obtain a sericin protein scaffold;

[0059] By adjusting the precooling temperature, the nucleation and growth of ice crystals are controlled, so that a porous structure is obtained after freeze-drying.

[0060] (4) The sericin protein scaffold obtained in step (3) is dialyzed in water for 2-3 days, pre-cooled again and completely freeze-dried to obtain a porous sericin protein scaffold. The purpose of dialysis is to remove excess cross-linking agent.

[0061] The purpose of dialysis is to remove residual carbodiimide and N-hydroxysuccinimide in the product of step (3), so as to finally obtain a porous scaffold material containing only sericin.

[0062] In step (2), the centrifugal speed is above 1000 rpm, and the centrifugal time is above 1 min.

[0063] The precooling temperature is above 20℃, and the precooling time is above 1 h.

[0064] In step (4), the molecular weight cut-off of the dialysis bag used is 3500 MW.

[0065] The water used in the reaction system is deionized water.

[0066] The second aspect of the present application provides a sericin protein porous scaffold material.

[0067] The third aspect of the present application provides the use of a sericin protein porous scaffold material in the preparation of antibacterial or hemostatic dressings, and in the treatment of photoimaging or heavy sewage.

[0068] It should be noted that the experimental methods used in the present application are conventional methods unless otherwise specified; the reagents and materials used are commercially available unless otherwise specified.

[0069] The sericin protein (abbreviated as sericin) used in the following examples is from the laboratory. The sericin is extracted by a conventional high-temperature aqueous method.

[0070] Example 1:

[0071] The 500 mg silk fibroin was dissolved in 50 mL deionized water and heated to complete dissolution by a boiling water bath, and then cooled to room temperature. Then 500 mg carbodiimide was added and stirred uniformly, and 300 mg N-hydroxysuccinimide was added and stirred at room temperature for 2 h. The crosslinking solution was centrifuged (3000 rpm, 10 min), and the supernatant was removed to obtain the silk fibroin porous scaffold precursor. The precursor was pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the silk scaffold. The silk scaffold was dialyzed in deionized water for 2-3 days by using a dialysis bag with a molecular weight of 3500 MW, and then pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the porous biological scaffold of silk fibroin.

[0072] The product obtained has the properties of high compression strength, high liquid absorption rate, fast shape recovery, aggregation of red blood cells and fast hemostasis.

[0073] Example 2:

[0074] The 500 mg silk fibroin was dissolved in 50 mL deionized water and heated to complete dissolution, and then cooled to room temperature. Then 500 mg carbodiimide was added and stirred uniformly, and 300 mg N-hydroxysuccinimide was added and stirred at room temperature for 2 h. The crosslinking solution was centrifuged (6000 rpm, 10 min), and the supernatant was removed to obtain the silk fibroin porous scaffold precursor. The precursor was pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the silk scaffold. The silk scaffold was dialyzed in deionized water for 2-3 days by using a dialysis bag with a molecular weight of 3500 MW, and then pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the porous biological scaffold of silk fibroin.

[0075] The product obtained has the properties of high compression strength, high liquid absorption rate, fast shape recovery, aggregation of red blood cells and fast hemostasis.

[0076] Example 3:

[0077] The 500 mg silk fibroin was dissolved in 50 mL deionized water and heated to complete dissolution, and then cooled to room temperature. Then 500 mg carbodiimide was added and stirred uniformly, and 300 mg N-hydroxysuccinimide was added and stirred at room temperature for 2 h. The crosslinking solution was centrifuged (9000 rpm, 10 min), and the supernatant was removed to obtain the silk fibroin porous scaffold precursor. The precursor was pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the silk scaffold. The silk scaffold was dialyzed in deionized water for 2-3 days by using a dialysis bag with a molecular weight of 3500 MW, and then pre-cooled at -80℃ for 12 h, and then freeze-dried for more than 10 h to obtain the porous biological scaffold of silk fibroin.

[0078] The obtained product has the properties of high compression strength, high liquid absorption rate, fast shape recovery, aggregated red blood cells and fast hemostasis.

[0079] Comparative Example 1

[0080] 500 mg of silk fibroin was dissolved in 50 mL of deionized water and heated to complete dissolution, and cooled to room temperature. 500 mg of carbodiimide was added and stirred uniformly, then 300 mg of N-hydroxysuccinimide was added and stirred uniformly, and then placed at room temperature for 2 h. The gelatinous cross-linked material floating on the upper layer was pre-cooled at -80 ℃ for 12 h, and then freeze-dried to obtain a silk fibroin scaffold. The silk fibroin scaffold was dialyzed in a 3500 MW dialysis bag in deionized water for 2-3 days, and then pre-cooled at -80 ℃ for 12 h, and then freeze-dried for more than 10 h to obtain a porous silk fibroin scaffold.

[0081] In order to illustrate the related properties of the silk fibroin porous scaffold material prepared by the preparation method of the silk fibroin porous scaffold material provided by the present application, the related properties of the silk fibroin porous scaffold material prepared by the preparation method of the silk fibroin porous scaffold material provided by the present application are illustrated in combination with the accompanying drawings.

[0082] Figure 1 is a photo of the water absorption of the silk fibroin before the mechanochemical cross-linking and the silk fibroin scaffold provided by Example 1 after the cross-linking. The silk fibroin without cross-linking will immediately spread out after contacting water. The silk fibroin scaffold after mechanochemical cross-linking can still maintain the original shape after absorbing water. This proves that the mechanochemical cross-linking strategy is of great significance for improving the dissolution rate and mechanical properties of silk.

[0083] Figure 2 is a scanning electron microscope image of the porous scaffold prepared by the mechanochemical cross-linking method provided by Comparative Example 1 and the porous scaffold prepared by the mechanochemical cross-linking method provided by Example 1. Compared with the silk fibroin scaffold obtained by mechanochemical cross-linking, the scaffold obtained by not applying stirring and centrifugation during the reaction process is full of blind holes. In the case of a large amount of bleeding, the liquid absorption capacity of the hemostatic agent is one of the important properties to ensure efficient hemostasis. A large number of blind holes are not conducive to the absorption of blood, and the scaffold after mechanochemical cross-linking is an interconnected porous structure (such as Figure 3 ), so as to have a high blood absorption rate. This proves the necessity of stirring in the cross-linking reaction, and therefore, without the mechanochemical cross-linking involving stirring, an interconnected porous silk fibroin scaffold cannot be obtained.

[0084] Figure 3SEM images of the silk fibroin porous scaffold materials obtained in Examples 1-3 before compression, after compression, and after water absorption. The results show that the silk fibroin scaffolds prepared at different centrifugal speeds still maintain the original structure after being compressed and absorbing water. In addition, high-speed centrifugation leads to an increase in the density of the sponge precursor, and the subsequent space compression caused by the growth of large ice crystals makes the internal framework of the sponge thicker, which is manifested in the SEM results as the internal lamellar thickness of the scaffold obtained at 9000 rpm being thicker.

[0085] Figure 4 Statistical results of the compression stress of the silk fibroin porous scaffold materials obtained in Examples 1-3. The results show that the greater the centrifugal force, the greater the compression stress of the silk fibroin porous scaffold. The maximum compression stress is 29.21±3.16 kPa, and the minimum compression stress is 10.07±0.85 kPa. Compression stress represents the ability of the sample to resist compression, and a sponge with enhanced resistance to deformation can quickly recover deformation after being compressed and contacting liquid. This result proves that the mechanical properties of the silk fibroin porous scaffold can be controlled by adjusting the centrifugal speed.

[0086] Figure 5 Statistical results of the shape recovery time of the silk fibroin porous scaffold materials obtained in Examples 1-3. As the centrifugal speed increases, the speed of shape recovery accelerates, with the fastest being 1.07±0.17 s and the slowest being 2.11±0.90 s. This indicates that the silk fibroin scaffold has superfast water-triggered shape recovery performance. In addition, the shape recovery time of the silk fibroin porous scaffold after water absorption can be controlled by adjusting the centrifugal speed.

[0087] Figure 6 Statistical results of the liquid absorption rate of the silk fibroin porous scaffold materials obtained in Examples 1-3 (absorption of deionized water, physiological saline, and blood). The silk fibroin porous scaffold can absorb the lowest 22.07±0.46 times its own weight of deionized water and the highest 27.16±1.34 times. The lowest absorption of physiological saline is 19.22±0.84 times, and the highest is 23.95±2.57 times. The lowest absorption of blood by the silk fibroin porous scaffold is 18.42±0.86 times, and the highest is 20.74±1.70 times. These results demonstrate that the silk fibroin porous scaffold has a high absorption rate for deionized water, physiological saline, and blood. Therefore, the liquid absorption rate of the silk fibroin porous scaffold after water absorption can be controlled by adjusting the centrifugal speed.

[0088] Figure 7 SEM images of the silk fibroin porous scaffold materials obtained in Examples 1-3 aggregating red blood cells. The results show that the silk fibroin porous scaffold has the ability to aggregate red blood cells.

[0089] In summary, the above results prove that the interconnected porous silk fibroin scaffold with high mechanical properties, high liquid absorption rate and fast shape recovery can be prepared by the mechanochemical cross-linking strategy. The porous scaffold also has the ability to aggregate red blood cells. Since the final product is only silk fibroin and does not contain other substances, it has excellent biocompatibility. It can not only be applied in biological medical scenes such as hemostasis, but also can be applied in other scenes such as catalysis and sewage treatment according to the diversity of protein functions.

[0090] The preferred embodiments and their effects are described in the present application. However, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all the changes and modifications falling within the scope of the present application.

[0091] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a porous silk sericin scaffold material, characterized by, The method comprises the following steps: Dissolving sericin in water, adding carbodiimide and stirring uniformly, then adding N-hydroxysuccinimide and stirring to perform cross-linking reaction, to obtain a cross-linking solution; Applying external force to the cross-linking solution to make the activated proteins in the cross-linking solution aggregate and perform cross-linking reaction, collecting the aggregates to obtain a sericin scaffold precursor; Precooling the sericin scaffold precursor, and completely freeze-drying to obtain a sericin protein scaffold; Dialyzing the sericin protein scaffold in water for 2-3 days, precooling again and freeze-drying to obtain a porous sericin protein scaffold material; The mass ratio of the sericin, the carbodiimide and the N-hydroxysuccinimide is 5:1-10:1-5; The external force is applied in the form of centrifugation, the centrifugal speed is 1000-10000 rpm, and the centrifugal time is 1-20 min; When the sericin is dissolved in water, it is heated to complete dissolution by a boiling water bath, and then cooled to room temperature; The cross-linking reaction is performed at a temperature of 0-50℃ for 2 h or more; The molecular weight of the sericin is ≥8000 MW.

2. The method of claim 1, wherein the silk fibroin porous scaffold material is prepared by the steps of: The precooling temperature is-80--20℃, and the precooling time is 12-24 h.

3. The method for preparing the sericin porous scaffold material according to claim 1, characterized in that, During the dialysis of the sericin protein scaffold in water, a dialysis bag with a molecular weight cut-off of 3500 MW is used.

4. The method for preparing the sericin porous scaffold material according to claim 1, characterized in that, The freeze-drying time is 10-24 h. 5.A porous sericin protein scaffold material prepared by the method of any one of claims 1-4. 6.Use of the porous sericin protein scaffold material of claim 5 in preparation of antibacterial or hemostatic dressings, and in photoimaging or heavy sewage treatment.

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