Preparation method of physical-chemical double-crosslinking silk fibroin porous frozen gel

Abstract

The invention relates to the technical field of preparation of silk fibroin porous frozen gel, in particular to a preparation method of physical-chemical bi-crosslinked silk fibroin porous frozen gel, which comprises the following steps of: adopting an ethylene glycol diglycidyl ether / ammonium persulfate / N, N, N ', N'-tetramethylethylenediamine composite system, and preparing the physical-chemical bi-crosslinked silk fibroin porous frozen gel through N, N, N ', N'-tetramethylethylenediamine. Forming a redox initiation system by N, N '-tetramethylethylenediamine and ammonium persulfate, and rapidly generating free radicals at low temperature, so that the methacrylic acid silk fibroin is polymerized and crosslinked; the preparation method comprises the following steps: performing ring-opening reaction on ethylene glycol diglycidyl ether and amino on silk fibroin to form chemical crosslinking, and promoting the formation of a beta-folding structure of the silk fibroin in an environment with changed pH of N, N, N ', N'-tetramethylethylenediamine, so that physical crosslinking and chemical crosslinking can be simultaneously realized in a freezing process; the limitation that toxic reagents need to be used for treatment or the pore diameter is too small in a traditional method is overcome, and the obtained frozen gel has the characteristics of macroporous structure, shape memory, rapid water absorption and the like, has excellent biocompatibility and can be used as a porous scaffold material in tissue engineering.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of porous silk fibroin cryogel, and in particular to a preparation method of physically-chemically double-crosslinked porous silk fibroin cryogel. Background Art

[0002] As a natural protein material, silk fibroin has attracted extensive attention in the field of tissue engineering due to its excellent biocompatibility, controllable biodegradability and unique mechanical properties. In particular, silk fibroin cryogel has shown great application potential in soft tissue repair due to its porous interconnected structure and adjustable mechanical properties. However, the current methods for preparing silk fibroin cryogel still have some limitations.

[0003] There are two main methods for preparing silk fibroin cryogels. The first type is to use epoxy compounds, such as ethylene glycol diglycidyl ether (EGDE), etc. Its mechanism is to react with lysine and arginine residues on silk fibroin molecules through epoxy groups to form covalent crosslinks, while reducing the mobility of the molecular chain, inducing the formation of β-folded structure of silk fibroin, thereby producing physical crosslinks. This type of cryogel has excellent mechanical properties, but generally based on physical crosslinking. The resulting cryogel has a small pore size (<50μm), which is difficult to meet the needs of cell migration and tissue growth in tissue engineering. The other type is to use ammonium persulfate-N,N,N′,N′-tetramethylethylenediamine (APS / TEMED) initiation system to make methacrylated silk fibroin (SilMA) produce free radical polymerization to prepare cryogel. Although this method can form a cryogel with a macroporous structure, the mechanical properties are relatively poor, and methanol post-treatment is required to induce the formation of β-folded structure and improve its mechanical properties. Methanol has potential cytotoxicity and is difficult to completely remove in the final cryogel, which limits its application in the biomedical field.

[0004] In summary, when epoxy compounds are used to prepare silk fibroin cryogel, the pore size of the finished product is small, which is difficult to meet the needs of cell migration and tissue growth in tissue engineering. When ammonium persulfate-N,N,N′,N′-tetramethylethylenediamine (APS / TEMED) initiation system is used to prepare silk fibroin cryogel, methanol post-treatment is required. Methanol has potential cytotoxicity, which limits its application in the biomedical field. Therefore, it is of great significance to develop a method for preparing silk fibroin cryogel that does not require the use of toxic solvents, can achieve chemical crosslinking and physical crosslinking at the same time, and has an appropriate pore size. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a physically-chemically double-crosslinked porous silk fibroin cryogel, so as to solve the problem that when epoxy compounds are used to prepare silk fibroin cryogel, the pore size of the finished product is small, which is difficult to meet the needs of cell migration and tissue growth in tissue engineering; when an ammonium persulfate-N,N,N′,N′-tetramethylethylenediamine (APS / TEMED) initiation system is used to prepare silk fibroin cryogel, methanol post-treatment is required, and methanol has potential cytotoxicity, which limits its application in the biomedical field.

[0006] To achieve the above object, the present invention provides a method for preparing a physically-chemically double-crosslinked silk fibroin porous cryogel, the method for preparing the physically-chemically double-crosslinked silk fibroin porous cryogel comprising the following steps: The silk fibroin derived from silkworm Bombyx mori is modified by using glycidyl methacrylate to obtain the modified silk fibroin; Using the modified silk fibroin to prepare a prepolymer solution, and pretreating the prepolymer solution to obtain a treated polymer solution; Adding N,N,N′,N′-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant into the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold and cooling it to obtain a gel blank; The gel blank is thawed, and after thawing, the gel ingredients are soaked in deionized water, and the gel is taken out to obtain a physically and chemically double-crosslinked silk fibroin cryogel.

[0007] The specific content of the step of "modifying the silk fibroin derived from silkworm silk with glycidyl methacrylate to obtain the modified silk fibroin" is: 4 g of degummed silk was completely dissolved in 21 g of lithium bromide solution (25 mL of deionized water), placed in a 60°C thermostat until it became clear and transparent, then taken out and slowly added with 1.5 mL of glycidyl methacrylate solution, and the mixture was heated in a water bath and rotated for 3 hours. Subsequently, the obtained solution was filtered and poured into a dialysis bag with a molecular weight cut-off of 8-14 kDa for 4 days (deionized water was changed several times a day) to obtain modified silk fibroin; Finally, the modified silk fibroin was frozen at -20°C for 12 h, freeze-dried for 24 h, and sealed and stored in a -20°C refrigerator for further use.

[0008] Wherein, in the step of "slowly dropwise adding 1.5 mL of glycidyl methacrylate solution and rotating the reaction for 3 hours under water bath heating conditions", the water bath heating temperature is 60°C and the rotation speed of the rotation reaction is 300 rpm.

[0009] The specific content of the step of "preparing a prepolymer solution using the modified silk fibroin, and pretreating the prepolymer solution to obtain a treated polymer solution" is: Weigh 80 mg, 100 mg and 120 mg of the modified silk fibroin sponge respectively into 1 mL of phosphate buffer solution, vortex until completely dissolved, and let stand for 15 minutes to obtain the prepolymer solution; placing the prepolymer solution in a 37° C. water bath for 7 minutes to obtain the heated solution; The prepolymer solution in the water bath containing the heated solution was taken out and placed in an ice-water mixture for 1 minute to obtain the treated polymer solution.

[0010] The specific content of the step of "adding N,N,N',N'-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant to the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold and cooling it to obtain a gel blank" is: Taking out the prepolymer solution in the treated polymer solution, adding 265 microliters of ethylene glycol diglycidyl ether, 3 microliters of N,N,N′,N′-tetramethylethylenediamine and 50 microliters of ammonium persulfate reactant to obtain the mixed solution; The mixed solution was added into a polytetrafluoroethylene mold (6 mm in diameter and 7 mm in height) precooled at -20°C, and placed in a -20°C refrigerator for freezing polymerization for 24 hours to obtain the gel blank.

[0011] Among them, in the step of "adding N,N,N',N'-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant to the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold for refrigeration to obtain a gel blank", the ammonium persulfate reactant is an ammonium persulfate solution with a mass concentration of 10% using PBS buffer as a solvent.

[0012] The specific content of the step of "thawing the gel blank, soaking the gel ingredients in deionized water after thawing, taking out the gel, and obtaining the physically and chemically double cross-linked silk fibroin cryogel" is: Taking out the gel blank from the mold, and thawing it with deionized water to obtain the thawed gel blank; The thawed gel blank was immersed in deionized water for 24 hours, and the gel was taken out to obtain 8%, 10% and 12% silk fibroin cryogels, that is, physicochemical double-crosslinked porous silk fibroin cryogel materials with through-pore structures.

[0013] The invention discloses a method for preparing a porous frozen gel of silk fibroin with physical-chemical double crosslinking. The method adopts a composite system of ethylene glycol diglycidyl ether / ammonium persulfate / N,N,N′,N′-tetramethylethylenediamine, and forms a redox initiation system through N,N,N′,N′-tetramethylethylenediamine and ammonium persulfate to quickly generate free radicals at low temperature, so that methacrylated silk fibroin undergoes polymerization and crosslinking; at the same time, ethylene glycol diglycidyl ether undergoes a ring-opening reaction with amino groups on the silk fibroin to form chemical crosslinking, and promotes the formation of a β-folding structure of the silk fibroin under a pH environment changed by N,N,N′,N′-tetramethylethylenediamine. The method can simultaneously realize physical crosslinking and chemical crosslinking during the freezing process, and overcomes the limitations of the traditional method that toxic reagents need to be used for treatment or the pore size is too small. The obtained frozen gel has the characteristics of macroporous structure, shape memory, rapid water absorption, and excellent biocompatibility, and can be used as a porous scaffold material in tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 The present invention provides a flowchart of the steps of preparing the physical-chemical double cross-linked silk fibroin porous cryogel.

[0016] Figure 2 The invention provides a Fourier transform infrared spectrum (FTIR) and an X-ray diffraction (XRD) spectrum of a silk fibroin cryogel without adding ethylene glycol diglycidyl ether and a silk fibroin cryogel with adding ethylene glycol diglycidyl ether.

[0017] Figure 3 The following are scanning electron microscope photos of the surface part and cross section of the silk fibroin cryogel prepared according to the present invention.

[0018] Figure 4 Schematic diagram of stress-strain curves and elastic modulus of cryogels with different prepolymer concentrations.

[0019] Figure 5 These are water-responsive shape memory photos of silk fibroin cryogel without and with ethylene glycol diglycidyl ether added.

[0020] Figure 6 This is a photograph of the rapid blood absorption of the silk fibroin cryogel prepared according to the present invention. DETAILED DESCRIPTION

[0021] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0022] See also Figures 1 to 6 The present invention provides a method for preparing a physical-chemical double cross-linked silk fibroin porous cryogel, the method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel comprising the following steps: Step 1, modifying silk fibroin derived from silkworm silk with glycidyl methacrylate to obtain modified silk fibroin; Step 2: preparing a prepolymer solution using the modified silk fibroin, and pretreating the prepolymer solution to obtain a treated polymer solution; Step 3: Add N,N,N′,N′-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant into the treated polymer solution to obtain a mixed solution, and add the mixed solution into a mold for refrigeration to obtain a gel blank; Step 4: thawing the gel blank, soaking the gel ingredients in deionized water after thawing, taking out the gel, and obtaining a physically and chemically double cross-linked silk fibroin cryogel.

[0023] In this embodiment, the method adopts a composite system of ethylene glycol diglycidyl ether / ammonium persulfate / N,N,N′,N′-tetramethylethylenediamine, and forms a redox initiation system through N,N,N′,N′-tetramethylethylenediamine and ammonium persulfate to quickly generate free radicals at low temperature, so that the methacrylated silk protein undergoes polymerization and cross-linking; at the same time, ethylene glycol diglycidyl ether undergoes a ring-opening reaction with the amino groups on the silk protein to form chemical cross-linking, and promotes the formation of the silk protein β-folding structure under the pH environment changed by N,N,N′,N′-tetramethylethylenediamine. This method can achieve physical cross-linking and chemical cross-linking at the same time during the freezing process, overcoming the limitations of traditional methods that require the use of toxic reagents or too small pore size. The resulting cryogel has the characteristics of macroporous structure, shape memory, and rapid water absorption, and has excellent biocompatibility, and can be used as a porous scaffold material in tissue engineering.

[0024] Further, in step 1, the specific content of preparing the modified methacrylated silk fibroin (SilMA) is as follows: 4 g of degummed silk is completely dissolved in 21 g of lithium bromide solution (25 mL of deionized water), placed in a 60°C thermostat until it is clear and transparent, then taken out, 1.5 mL of glycidyl methacrylate solution is slowly added dropwise, and the reaction is carried out at 60°C (water bath heating) at a speed of 300 rpm for 3 hours. Subsequently, the obtained solution is filtered, poured into a dialysis bag with a molecular weight cutoff of 8-14 kDa, and dialyzed for 4 days, and the deionized water is replaced several times a day to obtain modified silk fibroin. Finally, the modified silk fibroin is frozen at -20°C for 12 h, then freeze-dried for 24 h, and sealed and stored in a -20°C refrigerator for further use.

[0025] Further, the specific content of step 2 is: weighing 80 mg, 100 mg and 120 mg of SilMA sponge respectively into 1 mL of phosphate buffer solution, vortexing until completely dissolved, standing for 15 minutes to obtain the prepolymer solution, placing the prepolymer solution in a 37°C water bath for 7 minutes to obtain the heated solution, taking out the prepolymer solution placed in a water bath and placing it in an ice-water mixture for 1 minute to obtain the treated polymer solution. .

[0026] Furthermore, the specific content of step three is: taking out the prepolymer solution in the treated polymer solution, adding 265 microliters of ethylene glycol diglycidyl ether (EGDE), 3 microliters of N,N,N′,N′-tetramethylethylenediamine (TEMED) and 50 microliters of ammonium persulfate (APS) reactant to obtain the mixed solution, adding the mixed solution into a -20°C pre-cooled polytetrafluoroethylene mold (diameter 6 mm, height 7 mm), and placing it in a -20°C refrigerator for frozen polymerization for 24 hours, the ammonium persulfate reactant uses PBS buffer as solvent, and the mass concentration is 10% ammonium persulfate solution.

[0027] Furthermore, the specific content of step four is: taking out the gel blank from the mold, thawing it with deionized water to obtain the gel blank, that is, the silk fibroin frozen cryogel, soaking the gel blank in deionized water for 24 hours to completely remove the cross-linking agent, taking out the cryogel, and obtaining 8%, 10% and 12% silk fibroin protein cryogels, that is, physicochemical double cross-linked porous silk fibroin cryogel materials with through-pore structure.

[0028] In summary, the technical scheme proposes a method for preparing a porous cryogel of silk fibroin with physical-chemical double crosslinking. In this preparation method, TEMED and APS form a redox initiation system, which quickly generates free radicals at low temperature, so that SilMA undergoes polymerization and crosslinking; at the same time, EGDE undergoes a ring-opening reaction with the amino groups on the silk fibroin to form chemical crosslinking. The presence of TEMED also changes the pH value of the reaction environment, which is beneficial to the crosslinking reaction of EGDE, reduces the mobility of the molecular chain, and promotes the formation of the β-folding structure of the silk fibroin. Through this method, physical crosslinking and chemical crosslinking can be achieved simultaneously during the freezing process, and a double-crosslinked silk fibroin cryogel with a macroporous structure, shape memory and rapid water absorption performance can be prepared, which can overcome the limitations of existing methods and obtain a porous scaffold material with excellent biocompatibility.

[0029] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing a physical-chemical double cross-linked silk fibroin porous cryogel, characterized in that: The steps include: The silk fibroin derived from silkworm Bombyx mori is modified by using glycidyl methacrylate to obtain the modified silk fibroin; Using the modified silk fibroin to prepare a prepolymer solution, and pretreating the prepolymer solution to obtain a treated polymer solution; Adding N,N,N′,N′-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant into the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold and cooling it to obtain a gel blank; The gel blank is thawed, and after thawing, the gel ingredients are soaked in deionized water, and the gel is taken out to obtain a physically and chemically double-crosslinked silk fibroin cryogel.

2. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 1, characterized in that: The specific content of the step "modifying the silk fibroin derived from silkworm silk with glycidyl methacrylate to obtain the modified silk fibroin" is: 4 g of degummed silk was completely dissolved in 21 g of lithium bromide solution, placed in a 60°C thermostat until it became clear and transparent, then taken out and slowly added with 1.5 mL of glycidyl methacrylate solution, and then heated in a water bath with rotation for 3 hours; Subsequently, the obtained solution was filtered and poured into a dialysis bag with a molecular weight cut-off of 8-14 kDa for 4 days to obtain modified silk fibroin; Finally, the modified silk fibroin was frozen at -20°C for 12 h, freeze-dried for 24 h, and sealed and stored in a -20°C refrigerator for further use.

3. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 2, characterized in that: In the step of "slowly dropwise adding 1.5 mL of the glycidyl methacrylate solution and performing a rotation reaction for 3 hours under heating in a water bath", the water bath heating temperature is 60°C and the rotation speed of the rotation reaction is 300 rpm.

4. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 3, characterized in that: The specific content of the step "preparing a prepolymer solution using the modified silk fibroin, and pretreating the prepolymer solution to obtain a treated polymer solution" is: Weigh 80 mg, 100 mg and 120 mg of the modified silk fibroin sponge respectively into 1 mL of phosphate buffer solution, vortex until completely dissolved, and let stand for 15 minutes to obtain the prepolymer solution; placing the prepolymer solution in a 37° C. water bath for 7 minutes to obtain the heated solution; The prepolymer solution in the water bath containing the heated solution was taken out and placed in an ice-water mixture for 1 minute to obtain the treated polymer solution.

5. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 4, characterized in that: The specific content of the step "adding N,N,N',N'-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant to the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold and cooling it to obtain a gel blank" is: Taking out the prepolymer solution in the treated polymer solution, adding 265 microliters of ethylene glycol diglycidyl ether, 3 microliters of N,N,N′,N′-tetramethylethylenediamine and 50 microliters of ammonium persulfate reactant to obtain the mixed solution; The mixed solution was added into a polytetrafluoroethylene mold precooled at -20°C, and placed in a -20°C refrigerator for freezing polymerization for 24 hours to obtain the gel blank.

6. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 5, characterized in that: In the step of "adding N,N,N',N'-tetramethylethylenediamine, ethylene glycol diglycidyl ether and ammonium persulfate reactant to the treated polymer solution to obtain a mixed solution, and adding the mixed solution into a mold and cooling it to obtain a gel blank", the ammonium persulfate reactant is an ammonium persulfate solution with a mass concentration of 10% using PBS buffer as a solvent.

7. The method for preparing the physical-chemical double cross-linked silk fibroin porous cryogel according to claim 6, characterized in that: The specific content of the step of "thawing the gel blank, soaking the gel ingredients in deionized water after thawing, taking out the gel, and obtaining the physicochemical double cross-linked silk fibroin cryogel" is: Taking out the gel blank from the mold, and thawing it with deionized water to obtain the thawed gel blank; The thawed gel blank was immersed in deionized water for 24 hours, and the gel was taken out to obtain 8%, 10% and 12% silk fibroin cryogels, that is, physicochemical double-crosslinked porous silk fibroin cryogel materials with through-pore structures.

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