Silk fibroin hydrogel material with stable mechanical properties and preparation process thereof

Abstract

This invention discloses a mechanically stable silk fibroin hydrogel material and its preparation process. First, degummed silk fibroin is used as raw material to prepare methacryloyl fibroin (SFMA) and silk fibroin nanofibers (BSNF). Finally, the SFMA solution, BSNF solution, and photoinitiator aqueous solution are mixed and photocrosslinked. This invention, by incorporating BSNF with a stable β-sheet structure into SFMA, prepares an SFMA / BSNF hybrid hydrogel with stable mechanical properties, overcoming the mechanical instability of pure SFMA hydrogels and providing a more stable material basis for its application in 3D printing and tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of tissue repair materials technology, and provides a mechanically stable silk fibroin hydrogel material and its preparation process. Background Technology

[0002] Silk fibroin (SF) is a natural bio-protein fiber extracted from silkworm silk. It exhibits good biocompatibility and a wide range of adjustable mechanical properties (Pa-MPa), making it widely used in regenerative medicine research. Methacrylated silk fibroin (SFMA), obtained through chemical modification of SF, possesses photocrosslinking properties and is widely used in 3D printing and tissue repair research.

[0003] Currently, the mechanical properties of SFMA hydrogels can be controlled by adjusting the intensity and time of photo-initiated crosslinking, as well as the catalyst concentration. However, hydrogels prepared from pure SFMA often exhibit unstable mechanical properties, as the β-sheet secondary structures in the protein gradually undergo self-assembly over time. With the increase in the number of β-sheet structures, the overall mechanical properties of the SFMA hydrogel also continuously improve over time. Mechanics plays a significant regulatory role in tissue repair, and the unstable mechanical properties of pure SFMA hydrogels can affect the effectiveness of tissue regeneration and repair. Therefore, how to prepare mechanically stable SFMA hydrogels is a key issue for their application in tissue repair. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a mechanically stable silk fibroin hydrogel material and its preparation process, aiming to solve the problem of unstable mechanical properties of existing SFMA hydrogels.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a preparation process for a mechanically stable silk fibroin hydrogel material, the specific steps of which are as follows:

[0007] (1) Degumming raw silk to obtain degummed silk fibroin;

[0008] (2) Preparation of methacrylamide silk fibroin SFMA: Degummed silk fibroin was dissolved in lithium bromide solution, glycidyl methacrylate (GMA) was added, and the reaction was carried out to obtain SFMA;

[0009] (3) Preparation of silk fibroin nanofiber solution, i.e., BSNF solution: Degummed silk fibroin is dissolved in lithium bromide solution.

[0010] Dialysis and centrifugation yielded an aqueous solution of silk fibroin, which was concentrated and self-assembled into metastable silk fibroin particles. The particles were then diluted with deionized water.

[0011] Incubation yields a BSNF solution;

[0012] (4) Prepare SFMA solution using deionized water, mix SFMA solution, BSNF solution and photoinitiator aqueous solution evenly to obtain a mixed solution, pour into a mold, perform photocrosslinking reaction, and then perform post-treatment to obtain the silk fibroin hydrogel material.

[0013] Preferably, the specific method of step (1) is as follows: add 2.5g of raw silkworm silk to 1L of boiling 2.12g / L sodium carbonate solution, continue to boil and stir for 30-60 minutes, take it out and wash it with deionized water 3-5 times, and dry it in an oven at 60℃.

[0014] Preferably, in step (2), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and stirred to dissolve it to a concentration of 200 g / L. GMA is then added to make the concentration 424 mmol / L.

[0015] Preferably, in step (2), the reaction conditions are: 60℃ for 3 to 6 hours.

[0016] Preferably, in step (2), after the reaction is completed, the mixture is dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide and unreacted GMA. The mixture is then centrifuged 1 to 2 times to remove impurities and freeze-dried to obtain SFMA.

[0017] Further preferred centrifugation conditions are: 4℃, 9000rpm, 20 minutes.

[0018] Preferably, in step (3), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and the solution is stirred to dissolve it. The solution is then dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide. The solution is then centrifuged 1 to 2 times to remove impurities, resulting in a silk fibroin aqueous solution with a mass concentration of 4-6%.

[0019] Preferably, in step (3), the concentration is increased to 20% by mass at 60°C to obtain a concentrated solution, which is then self-assembled into metastable silk fibroin particles. The concentrated solution is then diluted to 2% by deionized water and incubated to obtain a BSNF solution.

[0020] Preferably, in step (3), the incubation conditions are: 60°C sealed incubation until a gel is formed.

[0021] Preferably, in step (4), the photoinitiator aqueous solution is obtained by dissolving lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in water; when preparing the mixture, the mass concentration of the SFMA solution is 4-10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the photoinitiator aqueous solution is 0.5%; the mixing volume ratio of the SFMA solution, BSNF solution, and photoinitiator aqueous solution is 1:1:1.

[0022] More preferably, when preparing the mixture, the SFMA solution has a mass concentration of 4% and the BSNF solution has a mass concentration of 2%.

[0023] More preferably, when preparing the mixture, the SFMA solution used has a mass concentration of 10% and the BSNF solution has a mass concentration of 2%.

[0024] Preferably, in step (4), the photocrosslinking conditions are: 405nm light source, 10–60 mW / cm². 2 The time is 0.5 to 4 minutes.

[0025] Preferably, in step (4), the post-treatment method is to soak in phosphate buffer (2.0mM KH2PO4, 137mM NaCl, 10.0mM Na2HPO4, 2.7mM KCl, pH 7.4) for 12 to 24 hours.

[0026] The present invention also provides a mechanically stable silk fibroin hydrogel material, which is obtained by the aforementioned preparation method.

[0027] This invention also provides the application of the aforementioned mechanically stable silk fibroin hydrogel material in the preparation of tissue repair materials.

[0028] The beneficial effects of this invention are:

[0029] This invention provides a mechanically stable silk fibroin hydrogel material and its preparation process. First, degummed silk fibroin is used as raw material to prepare methacrylamide-treated silk fibroin and silk fibroin nanofibers, followed by SFMA and BSNF solutions. Finally, the SFMA, BSNF, and photoinitiator aqueous solutions are mixed and photocrosslinked. This invention, by incorporating BSNF with a stable β-sheet structure into SFMA, prepares an SFMA / BSNF hybrid hydrogel with stable mechanical properties, overcoming the mechanical instability of pure SFMA hydrogels and providing a more stable material basis for its application in 3D printing and tissue repair.

[0030] Existing SFMA alone cannot form a hydrogel, while 10% and 20% SFMA can, but their mechanical properties are unstable, with their Young's modulus increasing continuously with standing time. BSNF alone cannot form a gel under photocrosslinking initiation. However, by introducing BSNF into SFMA for modification, a low concentration of 4% SFMA / 2% BSNF can form a hydrogel, and the Young's modulus does not change with standing time (Tables 1-3), significantly improving the mechanical stability of the SFMA / BSNF mixed hydrogel. Further comparison of the effects of different SFMA and BSNF ratios on the mechanical properties of the hydrogel shows that 4% SFMA / 2% BSNF and 10% SFMA / 2% BSNF ratios can both form mechanically stable mixed hydrogels, while 20% SFMA / 2% BSNF, due to its lower BSNF content, cannot form a mechanically stable mixed hydrogel, and its Young's modulus shows a trend of first increasing and then decreasing (Tables 1-3). In summary, by introducing sufficient BSNF into SFMA, a mechanically stable silk fibroin hydrogel can be obtained. This not only successfully solves the problem that 4% SFMA alone cannot form a hydrogel, but also addresses the drawback of existing high-concentration SFMA hydrogels gradually hardening over time. Furthermore, since both SFMA and BSNF are silk fibroin proteins, the resulting hydrogel has a single composition, which is beneficial for clinical translation.

[0031] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0033] Figure 1 This diagram illustrates the preparation of mechanically stable silk fibroin hydrogel materials, comprising four parts: raw silk degumming process, SFMA preparation process, BSNF preparation process, and preparation process of mechanically stable SFMA / BSNF mixed silk fibroin hydrogels. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments.

[0035] like Figure 1 As shown, a preparation process for a mechanically stable silk fibroin hydrogel material is described, with the following specific steps:

[0036] Example 1

[0037] (1) Degumming of raw silk: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring to dissolve it completely. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed silk fibroin.

[0038] (2) Preparation of SFMA: 20.19 g of lithium bromide was dissolved in 25 ml of deionized water and fully dissolved to obtain a 9.3 mol / L lithium bromide solution. The solution was heated to 60 °C, and 5 g of degummed silk fibroin was added. The solution was kept warm and stirred for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added (to make the concentration 424 mmol / L). The reaction was carried out for 6 hours. The solution was dialyzed with a 12 kDa dialysis membrane in deionized water for 7 days to remove lithium bromide and unreacted GMA, and methacrylamide silk fibroin SFMA was obtained.

[0039] (3) Preparation of BSNF: 20.19 g of lithium bromide was dissolved in deionized water and dissolved completely to obtain a 9.3 mol / L lithium bromide solution. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of about 6 wt%. The solution was then slowly concentrated to 20% at 60 °C. The concentrated solution was diluted to 2% with deionized water and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.

[0040] (4) Prepare an SFMA solution using deionized water. Mix the SFMA solution, BSNF solution, and photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Pour the mixture into a 3cm dish and initiate the photocrosslinking reaction (405nm light source, 25mw / cm). 2 The silk fibroin hydrogel material was obtained by soaking the material in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours (0.5 minutes).

[0041] When preparing the mixture in step (4), adjust the mass concentration of SFMA solution, BSNF solution and photoinitiator aqueous solution to obtain the corresponding silk fibroin hydrogel material. Young's modulus was tested using a bio-nanoindenter (Optics11, Netherlands). The results are shown in Table 1.

[0042] Table 1. Young's modulus of hydrogel materials after 0.5 minutes of photocrosslinking reaction.

[0043] In Table 1, "-" indicates that a hydrogel cannot be formed.

[0044] Example 2

[0045] (1) Degumming of raw silk: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring to dissolve it completely. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed silk fibroin.

[0046] (2) Preparation of SFMA: 20.19 g of lithium bromide was dissolved in 25 ml of deionized water and fully dissolved to obtain a 9.3 mol / L lithium bromide solution. The solution was heated to 60 °C, and 5 g of degummed silk fibroin was added. The solution was kept warm and stirred for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added (to make the concentration 424 mmol / L). The reaction was carried out for 6 hours. The solution was dialyzed with a 12 kDa dialysis membrane in deionized water for 7 days to remove lithium bromide and unreacted GMA, and methacrylamide silk fibroin SFMA was obtained.

[0047] (3) Preparation of BSNF: 20.19 g of lithium bromide was dissolved in deionized water and dissolved completely to obtain a 9.3 mol / L lithium bromide solution. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of about 6 wt%. The solution was then slowly concentrated to 20% at 60 °C. The concentrated solution was diluted to 2% with deionized water and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.

[0048] (4) Prepare an SFMA solution using deionized water. Mix the SFMA solution, BSNF solution, and photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Pour the mixture into a 3cm dish and initiate the photocrosslinking reaction (405nm light source, 25mw / cm). 2 The silk fibroin hydrogel material was obtained by soaking the sample in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours (1 minute) for 1 hour.

[0049] When preparing the mixture in step (4), adjust the mass concentration of SFMA solution, BSNF solution and photoinitiator aqueous solution to obtain the corresponding silk fibroin hydrogel material. Young's modulus was tested using a bio-nanoindenter (Optics11, Netherlands). The results are shown in Table 2.

[0050] Table 2 Young's modulus of hydrogel materials after 1 minute of photocrosslinking reaction.

[0051] In Table 2, "-" indicates that a hydrogel cannot be formed.

[0052] Example 3

[0053] (1) Degumming of raw silk: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring to dissolve it completely. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed silk fibroin.

[0054] (2) Preparation of SFMA: 20.19 g of lithium bromide was dissolved in 25 ml of deionized water and fully dissolved to obtain a 9.3 mol / L lithium bromide solution. The solution was heated to 60 °C, and 5 g of degummed silk fibroin was added. The solution was kept warm and stirred for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added (to make the concentration 424 mmol / L). The reaction was carried out for 6 hours. The solution was dialyzed with a 12 kDa dialysis membrane in deionized water for 7 days to remove lithium bromide and unreacted GMA, and methacrylamide silk fibroin SFMA was obtained.

[0055] (3) Preparation of BSNF: 20.19 g of lithium bromide was dissolved in deionized water and dissolved completely to obtain a 9.3 mol / L lithium bromide solution. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of about 6 wt%. The solution was then slowly concentrated to 20% at 60 °C. The concentrated solution was diluted to 2% with deionized water and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.

[0056] (4) Prepare an SFMA solution using deionized water. Mix the SFMA solution, BSNF solution, and photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Pour the mixture into a 3cm dish and initiate the photocrosslinking reaction (405nm light source, 25mw / cm). 2The silk fibroin hydrogel material was obtained by soaking the sample in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours (4 minutes).

[0057] Adjust the mass concentrations of SFMA solution, BSNF solution and photoinitiator aqueous solution used in step (4) to prepare the mixture, and obtain the corresponding silk fibroin hydrogel material. Young's modulus was tested using a bio-nanoindenter (Optics11, Netherlands). The results are shown in Table 3.

[0058] Table 3 Young's modulus of hydrogel materials after 4 minutes of photocrosslinking reaction.

[0059] In Table 3, "-" indicates that a hydrogel cannot be formed.

[0060] Example 4

[0061] (1) Degumming of raw silk: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring to dissolve it completely. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed silk fibroin.

[0062] (2) Preparation of SFMA: 20.19 g of lithium bromide was dissolved in 25 ml of deionized water and fully dissolved to obtain a 9.3 mol / L lithium bromide solution. The solution was heated to 60 °C, and 5 g of degummed silk fibroin was added. The solution was kept warm and stirred for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added (to make the concentration 424 mmol / L). The reaction was carried out for 6 hours. The solution was dialyzed with a 12 kDa dialysis membrane in deionized water for 7 days to remove lithium bromide and unreacted GMA, and methacrylamide silk fibroin SFMA was obtained.

[0063] (3) Preparation of BSNF: 20.19 g of lithium bromide was dissolved in deionized water and dissolved completely to obtain a 9.3 mol / L lithium bromide solution. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of about 6 wt%. The solution was then slowly concentrated to 20% at 60 °C. The concentrated solution was diluted to 2% with deionized water and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.

[0064] (4) Prepare a 10% (w / w) SFMA solution using deionized water. Mix the SFMA solution, BSNF solution, and a 0.5% (w / w) photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Pour the mixture into a 3cm dish and initiate the photocrosslinking reaction (405nm light source, 60mw / cm). 2 The silk fibroin hydrogel material was obtained by soaking the sample in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours (1 minute). The Young's modulus was tested using a bio-nanoindenter (Optics11, Netherlands), and the results are shown in Table 4.

[0065] Table 4 Young's modulus of pure SFMA hydrogels and SFMA / BSNF hybrid hydrogels

[0066]

[0067] As can be seen from Tables 1, 2, 3 and 4 above, when the mass concentration of SFMA solution used in the preparation of the mixture is 4-10%, the mass concentration of BSNF solution is 2%, and the mass concentration of photoinitiator aqueous solution is 0.5%, the resulting hydrogel material has stable mechanical properties.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A preparation process for a mechanically stable silk fibroin hydrogel material, characterized in that, The specific steps are as follows: (1) Degumming of raw silk to obtain degummed silk fibroin; (2) Preparation of methacrylamide silk fibroin SFMA: Degummed silk fibroin was dissolved in lithium bromide solution, glycidyl methacrylate was added, and the reaction was carried out to obtain SFMA; (3) Preparation of silk fibroin nanofiber solution, i.e. BSNF solution: Degummed silk fibroin was dissolved in lithium bromide solution, dialyzed, centrifuged to obtain silk fibroin aqueous solution, concentrated, self-assembled into metastable silk fibroin particles, diluted with deionized water, and incubated to obtain BSNF solution. (4) Prepare SFMA solution using deionized water, mix SFMA solution, BSNF solution and photoinitiator aqueous solution evenly to obtain a mixture, pour into a mold, perform photocrosslinking reaction, and then perform post-treatment to obtain the silk fibroin hydrogel material. In step (4), the photoinitiator aqueous solution is obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water; when preparing the mixture, the mass concentration of SFMA solution is 4-10%, the mass concentration of BSNF solution is 2%, and the mass concentration of photoinitiator aqueous solution is 0.5%; the mixing volume ratio of SFMA solution, BSNF solution and photoinitiator aqueous solution is 1:1:1; The specific method of step (1) is as follows: Add 2.5g of raw silkworm silk to 1L of boiling 2.12g / L sodium carbonate solution, continue to boil and stir for 30-60 minutes, take it out and wash it with deionized water 3-5 times, and dry it in a 60℃ oven. In step (2), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and it is stirred to dissolve it to a concentration of 200 g / L. GMA is then added to make the concentration 424 mmol / L. In step (2), the reaction conditions are: 60℃ for 3 to 6 hours; In step (2), after the reaction is completed, the mixture is dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide and unreacted GMA. The mixture is then centrifuged 1 to 2 times to remove impurities and freeze-dried to obtain SFMA. In step (3), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and it is stirred to dissolve. The solution is then dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide. The solution is then centrifuged 1 to 2 times to remove impurities, and a silk fibroin aqueous solution with a mass concentration of 4-6% is obtained. In step (3), the concentration is increased to 20% by mass at 60°C to obtain a concentrated solution, which is then self-assembled into metastable silk fibroin particles. The concentrated solution is then diluted to 2% by deionized water and incubated to obtain a BSNF solution. The incubation conditions are: 60°C in a sealed environment until a gel is formed; In step (4), the photocrosslinking conditions are: 405nm light source, 10–60 mw / cm². 2 The time is 0.5 to 4 minutes; The post-treatment method is to soak in phosphate buffer for 12–24 hours.

2. A mechanically stable silk fibroin hydrogel material, characterized in that, It is obtained through the preparation process described in claim 1.

3. The application of the mechanically stable silk fibroin hydrogel material as described in claim 2 in the preparation of tissue repair materials.

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