Method for forming hydrogel by using hemoglobin to catalyze crosslinking of silk fibroin
By using hemoglobin as a catalyst, combining silk fibroin and hydrogen peroxide, the low-cost and efficient preparation of silk fibroin hydrogels is achieved, and the problems of high catalyst costs and harsh reaction conditions in the prior art are solved, and a hydrogel with mechanical strength and stability is obtained.
Patent Information
- Application Number
- CN202411791517.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-07
AI Technical Summary
In the prior art, when peroxidase is used to catalyze the cross-linking of silk fibroprotein to form hydrogels, the cost is high, the catalyst is prone to deactivate, and the reaction conditions are demanding, making it difficult to achieve low-cost and efficient biocatalytic preparation.
Hemoglobin is used as a biocatalyst, and through the joint action with silk fibroin and hydrogen peroxide, the self-crosslinking reaction of silk fibroin is initiated to form a hydrogel with mechanical strength and elasticity.
The use of low-cost, wide-source hemoglobin catalysts is achieved, which simplifies the preparation process, reduces production costs, and improves the mechanical properties and stability of silk fibroprotein hydrogels.
Smart Images
Figure CN119570068A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of silk fibroin hydrogel preparation, and in particular to a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst. Background Art
[0002] As a material with high water absorption and water retention, hydrogel is widely used in cosmetics, agriculture, chemical industry, medicine and other fields. In particular, it has broad application potential in the biomedical field, such as drug release, tissue engineering, and regenerative medicine. Silk fibroin is a natural biopolymer derived from silk. It has small side chains in the crystalline region and many non-polar groups. Its molecular conformation is β-folding, and the peptide chains are arranged neatly and tightly. The molecular chains are bound by hydrogen bonds and intermolecular forces, and the binding force is strong, which gives silk a high breaking strength.
[0003] Silk fibroin hydrogel is mainly formed by introducing alcohols, shearing, ultrasound or ultraviolet light to induce silk molecules to transform into a stable β-folded structure. However, the prepared silk fibroin gel is soft and fragile, and has no elasticity. At present, free radicals can be generated by catalytic induction of the active sites of tyrosine residues in the side chains of silk fibroin, which triggers the self-crosslinking gel reaction between silk fibroin molecular chains to obtain silk fibroin hydrogels with mechanical strength and elasticity. The most studied is enzymatic chemical crosslinking, especially peroxidase (such as horseradish peroxidase). However, peroxidases are expensive, easily inactivated, and have relatively harsh requirements for catalytic reaction conditions. Therefore, it is of great significance to develop low-cost and efficient biocatalysts to replace peroxidase. Summary of the invention
[0004] The purpose of the present invention is to provide a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin to address the deficiencies in the prior art.
[0005] To achieve the above object, the technical solution adopted by the present invention is:
[0006] Provided is a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, comprising the following steps:
[0007] Step 1, dissolving the degummed silk fibers with a neutral salt solution to obtain a regenerated silk fibroin salt solution; wherein the mass volume ratio of the degummed silk fibers to the neutral salt solution is 1 g: (8-30) mL; the neutral salt is one or more of lithium bromide, calcium chloride, and calcium nitrate tetrahydrate;
[0008] Step 2, dialysis and purification of the silk fibroin salt solution obtained in step 1 to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%;
[0009] Step 3, dialyzing and concentrating the silk fibroin aqueous solution obtained in step 2 to obtain a concentrated silk fibroin aqueous solution; wherein the mass concentration of the concentrated silk fibroin aqueous solution is 2 to 12 wt %;
[0010] Step 4, add hemoglobin to the concentrated silk fibroin aqueous solution obtained in step 3 and mix well to make the hemoglobin concentration therein 0.6-3.0 g / L, then add hydrogen peroxide solution with a mass concentration of 0.1-30 wt%, mix well, and initiate the cross-linking reaction of the silk fibroin gel, and keep the reaction at a constant temperature of 20-40°C for 0.5-2.0 hours.
[0011] Furthermore, in step 1, the neutral salt solution is a ternary solution of calcium chloride / ethanol / water, an aqueous lithium bromide solution, an aqueous calcium chloride solution, or an aqueous calcium nitrate tetrahydrate solution.
[0012] Furthermore, in step 1, the dissolution temperature of the degummed silk fiber is 50-100° C., and the complete dissolution time is 2-6 hours.
[0013] Furthermore, the dissolution temperature of the degummed silk fiber is 70-75° C., and the complete dissolution time is 2-3 hours.
[0014] Furthermore, in step 2, the molecular weight cutoff of the dialysis bag used for the dialysis purification is 8000-14000Da, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours.
[0015] Furthermore, in step three, the dialysis concentrate used in the dialysis concentration is a polyethylene glycol solution with a mass fraction of 10-15%, and the average molecular weight of the polyethylene glycol is 20,000.
[0016] Furthermore, the hemoglobin is bovine hemoglobin or human hemoglobin.
[0017] Furthermore, the source of silk fibroin is one or more of mulberry silk, silkworm silk, tussah silk and castor silk.
[0018] Furthermore, in step 4, the temperature of the gel cross-linking reaction is 37° C., and the time is 1.0 to 1.5 h.
[0019] Furthermore, in step 4, the mass concentration of the hydrogen peroxide solution is 0.2-0.6wt%; the volume ratio of the concentrated silk fibroin aqueous solution to the hydrogen peroxide solution is (33-100):1.
[0020] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:
[0021] The invention adopts cheap and readily available hemoglobin as a biocatalyst to prepare silk fibroin hydrogel, and the preparation method is simple and efficient. Compared with traditional enzyme catalysts such as horseradish peroxidase, hemoglobin has a wide source, low price, mild reaction conditions and good catalytic effect.
[0022] The preparation method of the present invention can achieve good mechanical properties and stability by utilizing low concentration (2.0-8.0wt%) of silk fibroin for catalytic cross-linking, which is beneficial to cost saving and thus has potential industrial application development value. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The present invention provides a synthetic route for the hemoglobin-catalyzed induction of silk fibroin cross-linking.
[0024] Figure 2 This is a physical picture of the cross-linking process of silk fibroin induced by hemoglobin catalysis in the present invention.
[0025] Figure 3 Circular dichroism spectra of silk fibroin gel before and after reaction in Example 1.
[0026] Figure 4 This is the fluorescence spectrum of the silk fibroin gel before and after the reaction in Example 1.
[0027] Figure 5 This is a scanning electron microscope microscopic morphology image of the silk fibroin gel before and after the reaction in Example 2. DETAILED DESCRIPTION
[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.
[0029] Example 1
[0030] refer to Figure 1-2 This embodiment provides a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, comprising the following steps:
[0031] Step 1, weighing 5 g of degummed silk fiber and dissolving it in 100 mL of a ternary solution of calcium chloride / ethanol / water, dissolving it at 70° C. for 2 hours to obtain a regenerated silk protein salt solution; wherein the molar ratio of calcium chloride / ethanol / water in the ternary solution of calcium chloride / ethanol / water is 1:2:8;
[0032] Step 2, placing the silk fibroin salt solution obtained in step 1 into a dialysis bag with a molecular weight cutoff of 8000-14000Da, and performing dialysis purification, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours, to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%;
[0033] Step 3, the silk fibroin aqueous solution obtained in step 2 is dialyzed and concentrated, wherein the dialyzed concentrated solution used in the above dialysis concentration is a 10% by mass polyethylene glycol (average molecular weight of 20,000) solution, to obtain concentrated silk fibroin aqueous solutions of different concentrations;
[0034] Step 4: Take 3 mL of 2 wt% concentrated silk fibroin aqueous solution, add 2 mg of bovine hemoglobin and 30 μL of 0.3 wt% hydrogen peroxide solution to initiate the cross-linking reaction of the silk fibroin gel, and keep it at a constant temperature of 37°C for 0.5 h to obtain the hemoglobin-catalyzed cross-linked silk fibroin hydrogel.
[0035] The silk fibroin before and after the cross-linking reaction was tested by circular dichroism (such as Figure 3 As shown in the figure, the transformation process of the secondary structure of the protein was tracked. Before the reaction, the silk system had no secondary structure peaks, only amorphous structure peaks. After adding hemoglobin, the system showed α-helix peaks (208 and 222nm). At the same time, the peak position change of the fluorescence spectrum showed that dityrosine bonds were formed between the silk protein gels after the reaction, proving that chemical self-crosslinking reaction occurred between silk proteins under the catalytic induction of hemoglobin (as shown in the figure). Figure 4 shown).
[0036] Example 2
[0037] This embodiment provides a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, comprising the following steps:
[0038] Step 1, weighing 5 g of degummed silk fiber and dissolving it in 120 mL of lithium bromide aqueous solution, dissolving it at 70° C. for 1.5 h to obtain a regenerated silk fibroin salt solution; wherein the concentration of the lithium bromide aqueous solution is 9.3 mol / L;
[0039] Step 2, placing the silk fibroin salt solution obtained in step 1 into a dialysis bag with a molecular weight cutoff of 8000-14000Da, and performing dialysis purification, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours, to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%;
[0040] Step 3, the silk fibroin aqueous solution obtained in step 2 is dialyzed and concentrated, wherein the dialyzed concentrated solution used in the above dialysis concentration is a 10% by mass polyethylene glycol (average molecular weight of 20,000) solution, to obtain concentrated silk fibroin aqueous solutions of different concentrations;
[0041] Step 4: Take 3 mL of 4 wt% concentrated silk fibroin aqueous solution, add 4 mg of bovine hemoglobin and 60 μL of 0.3 wt% hydrogen peroxide solution to initiate the cross-linking reaction of the silk fibroin gel, and keep it at a constant temperature of 37°C for 1 hour to obtain the hemoglobin-catalyzed cross-linked silk fibroin hydrogel.
[0042] The gel after the reaction is freeze-dried to retain the morphology of the silk protein after cross-linking, such as Figure 4 Scanning electron microscopy shows that after the silk fibroin is cross-linked, the internal through-hole structure and pore conditions can be seen, which provide support for cell planting and growth and can be used as a porous scaffold material for medical tissue engineering.
[0043] Example 3
[0044] This embodiment provides a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, comprising the following steps:
[0045] Step 1, weighing 5 g of degummed silk fiber and dissolving it in 130 mL of a ternary solution of calcium chloride / ethanol / water, dissolving it at 72° C. for 2 hours to obtain a regenerated silk protein salt solution; wherein the molar ratio of calcium chloride / ethanol / water in the ternary solution of calcium chloride / ethanol / water is 1:2:8;
[0046] Step 2, placing the silk fibroin salt solution obtained in step 1 into a dialysis bag with a molecular weight cutoff of 8000-14000Da, and performing dialysis purification, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours, to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%;
[0047] Step 3, the silk fibroin aqueous solution obtained in step 2 is dialyzed and concentrated, wherein the dialyzed concentrated solution used in the above dialysis concentration is a 10% by mass polyethylene glycol (average molecular weight of 20,000) solution, to obtain concentrated silk fibroin aqueous solutions of different concentrations;
[0048] Step 4: Take 3 mL of 6 wt% concentrated silk fibroin aqueous solution, add 6 mg of bovine hemoglobin and 90 μL of 0.3 wt% hydrogen peroxide solution to initiate the cross-linking reaction of the silk fibroin gel, and keep it at a constant temperature of 37°C for 1.5 hours to obtain the hemoglobin-catalyzed cross-linked silk fibroin hydrogel.
[0049] Example 4
[0050] This embodiment provides a method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, comprising the following steps:
[0051] Step 1, weighing 5 g of degummed silk fiber and dissolving it in 120 mL of lithium bromide aqueous solution, placing it at 72° C. for 2 hours to obtain a regenerated silk fibroin salt solution; wherein the concentration of the lithium bromide aqueous solution is 9.3 mol / L;
[0052] Step 2, placing the silk fibroin salt solution obtained in step 1 into a dialysis bag with a molecular weight cutoff of 8000-14000Da, and performing dialysis purification, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours, to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%;
[0053] Step 3, the silk fibroin aqueous solution obtained in step 2 is dialyzed and concentrated, wherein the dialyzed concentrated solution used in the above dialysis concentration is a 10% by mass polyethylene glycol (average molecular weight of 20,000) solution, to obtain concentrated silk fibroin aqueous solutions of different concentrations;
[0054] Step 4: Take 3 mL of 8 wt% concentrated silk fibroin aqueous solution, add 8 mg of bovine hemoglobin and 90 μL of 0.3 wt% hydrogen peroxide solution to initiate the cross-linking reaction of the silk fibroin gel, and keep it at a constant temperature of 37°C for 2 hours to obtain the hemoglobin-catalyzed cross-linked silk fibroin hydrogel.
[0055] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as a catalyst, characterized in that: The steps include: Step 1, dissolving the degummed silk fibers with a neutral salt solution to obtain a regenerated silk fibroin salt solution; wherein the mass volume ratio of the degummed silk fibers to the neutral salt solution is 1 g: (8-30) mL; the neutral salt is one or more of lithium bromide, calcium chloride, and calcium nitrate tetrahydrate; Step 2, dialysis and purification of the silk fibroin salt solution obtained in step 1 to obtain a pure silk fibroin aqueous solution; wherein the mass concentration of the silk fibroin aqueous solution obtained by dialysis is 0.8-1.0wt%; Step 3, dialyzing and concentrating the silk fibroin aqueous solution obtained in step 2 to obtain a concentrated silk fibroin aqueous solution; wherein the mass concentration of the concentrated silk fibroin aqueous solution is 2 to 12 wt %; Step 4, add hemoglobin to the concentrated silk fibroin aqueous solution obtained in step 3 and mix well to make the hemoglobin concentration therein 0.6-3.0 g / L, then add hydrogen peroxide solution with a mass concentration of 0.1-30 wt%, mix well, and initiate the cross-linking reaction of the silk fibroin gel, and keep the reaction at a constant temperature of 20-40°C for 0.5-2.0 hours.
2. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 1, the neutral salt solution is a ternary solution of calcium chloride / ethanol / water, an aqueous solution of lithium bromide, an aqueous solution of calcium chloride, or an aqueous solution of calcium nitrate tetrahydrate.
3. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 1, the dissolution temperature of the degummed silk fiber is 50-100° C., and the complete dissolution time is 2-6 hours.
4. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 3, characterized in that: The dissolution temperature of the degummed silk fiber is 70-75° C., and the complete dissolution time is 2-3 hours.
5. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 2, the molecular weight cutoff of the dialysis bag used for the dialysis purification is 8000-14000Da, the number of dialysis is 9-12 times, and the time of each dialysis is 3-6 hours.
6. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 3, the dialysis concentrate used in the dialysis concentration is a polyethylene glycol solution with a mass fraction of 10-15%, and the average molecular weight of the polyethylene glycol is 20,000.
7. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: The hemoglobin is bovine hemoglobin or human hemoglobin.
8. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: The source of silk fibroin is one or more of mulberry silk, silkworm silk, tussah silk and castor silk.
9. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 4, the temperature of the gel cross-linking reaction is 37° C. and the time is 1.0 to 1.5 h.
10. The method for forming a hydrogel by cross-linking silk fibroin using hemoglobin as catalyzed by claim 1, characterized in that: In step 4, the mass concentration of the hydrogen peroxide solution is 0.2-0.6wt%; the volume ratio of the concentrated silk fibroin aqueous solution to the hydrogen peroxide solution is (33-100):1.