CN114395141A - Preparation method of high-strength silk protein nanofiber hydrogel - Google Patents
Preparation method of high-strength silk protein nanofiber hydrogel Download PDFInfo
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- CN114395141A CN114395141A CN202210054185.9A CN202210054185A CN114395141A CN 114395141 A CN114395141 A CN 114395141A CN 202210054185 A CN202210054185 A CN 202210054185A CN 114395141 A CN114395141 A CN 114395141A
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- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
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- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/09—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids
- C08J3/091—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in organic liquids characterised by the chemical constitution of the organic liquid
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- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention provides a preparation method of high-strength silk protein nanofiber gel, which is characterized in that a high-crystal silk protein nanofiber aqueous solution is freeze-dried and dissolved in a formic acid solution, then the solution is loaded into a dialysis bag or a semipermeable membrane, and formic acid is replaced by the aqueous solution to obtain the high-strength silk protein nanofiber hydrogel. According to the method provided by the invention, the high-crystal silk protein nanofiber is dissolved in formic acid, on the basis of keeping the original secondary structure and the appearance of the nanofiber, the electrostatic repulsion between fibers is reduced, and a high-concentration silk protein nanofiber formic acid solution is obtained, so that the interaction between silk protein nanofibers is enhanced, then formic acid is removed through the solvent replacement of water and formic acid, the interaction between fibers is fixed by utilizing the gelation of the silk protein nanofiber with the concentration in a water phase, and the mechanical property of the finally obtained hydrogel material is greatly improved.
Description
Technical Field
The invention belongs to the technical field of high-strength biological materials, and particularly relates to a preparation method of high-strength silk protein nanofiber hydrogel.
Background
High strength materials have been the focus of research in the field of materials due to their excellent properties and wide range of applications. High-strength materials existing in nature achieve optimization of performance mainly through multi-stage assembly of micro-nano size and non-covalent interaction. In consideration of the excellent biocompatibility of natural biomaterials, high-strength natural materials such as chitosan and cellulose are dissolved and reconstructed to meet the application requirements of different fields, however, most reconstructed materials lose good mechanical properties and greatly limit the application. How to realize the multilevel assembly of the material in the micro-nano scale in the reconstruction process becomes the key for improving the mechanical property of the material.
The gel material has been a research hotspot of regenerative medicine due to the similarity with human tissue environment, however, compared with other morphological materials, the gel prepared by natural biological materials has lower mechanical property and is difficult to meet the application requirement, and the high-strength hydrogel is always a difficulty in the research of biological materials. Silk, as a typical natural high-strength material, has been prepared into various forms such as fibers, films, hydrogels, scaffolds, microspheres, and the like, and is widely applied in the fields of tissue engineering, flexible electronic devices, drug delivery, and the like. However, similar to the problems faced by other natural materials, the mechanical properties of silk protein hydrogels are too low to meet the requirements of many applications, especially the requirement of cartilage and other tissues with supporting function.
In recent years, researchers improve the mechanical properties of silk protein gel by changing the concentration, chemically crosslinking, adding other components and the like, but the preparation process is relatively complex, the improvement of the mechanical properties is limited, and a new idea and a new method are urgently needed to break through the bottleneck of the prior art. In previous studies, the inventor has developed silk protein nanofibers with abundant beta-sheet structures and successfully applied to the preparation of porous sponges and gels. However, since the surfaces of the silk protein nanofibers are rich in negative charges, the repulsion of charges makes further assembly and action between fibers difficult, and the obtained gel has extremely poor mechanical properties.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a high-strength silk protein nanofiber hydrogel, and the nanofiber hydrogel prepared by the method provided by the present invention has excellent compressive modulus and breaking strength.
The invention provides a preparation method of high-strength silk protein nanofiber hydrogel, which comprises the following steps:
and (3) replacing formic acid in the silk protein nanofiber formic acid solution with the aqueous solution to obtain the high-strength silk protein nanofiber hydrogel.
Preferably, the time for the replacement is 2 to 72 hours.
Preferably, the mass concentration of the silk protein nanofiber formic acid solution is 5-30%.
Preferably, the method of substitution comprises:
putting the silk fibroin nanofiber formic acid solution into a dialysis bag or a semipermeable membrane, and putting the dialysis bag or the semipermeable membrane into an aqueous solution to perform replacement of formic acid and the aqueous solution;
the dialysis bag or the semipermeable membrane silk protein is impermeable, and formic acid and aqueous solution are permeable.
Preferably, the method further comprises, after the replacing:
and soaking the displaced product in the water solution, and repeatedly replacing the water solution until the pH value of the replaced water solution is 6-8.
Preferably, the volume ratio of the aqueous solution to the formic acid in the replacement process is 3: 1 or more.
Preferably, the preparation method of the silk protein nanofiber formic acid solution comprises the following steps:
dissolving the fibroin nanofiber freeze-dried powder in formic acid to obtain a fibroin nanofiber formic acid solution;
the dissolving temperature is 4-80 ℃; the dissolving time is 0.1-24 hours.
Preferably, the aqueous solution comprises:
pure water or an aqueous solution containing ions,
the ions are magnesium ions, calcium ions and/or copper ions.
Preferably, the preparation method of the silk fibroin nanofiber freeze-dried powder comprises the following steps:
freeze-drying the high-crystal silk fibroin nanofiber aqueous solution to obtain silk fibroin nanofiber freeze-dried powder;
the mass concentration of the high-crystalline silk protein nanofiber aqueous solution is 0.01-4%.
Preferably, the preparation method of the high crystal silk protein nanofiber aqueous solution comprises the following steps:
and sequentially carrying out primary concentration and secondary concentration on the silk protein aqueous solution, diluting, and carrying out sealed incubation to obtain the high-crystal silk protein nanofiber aqueous solution.
The high-crystal silk protein nanofiber adopted by the method provided by the invention has a beta-sheet crystal structure and good stability, can still keep the original secondary structure and the original nanofiber appearance after being dissolved in formic acid, optimizes the interaction between the nanofibers through silk protein concentration regulation in a formic acid system, then replaces formic acid and water with each other by utilizing a specific dialysis bag or a semipermeable membrane, and obtains the high-strength silk protein nanofiber hydrogel on the basis of keeping the interaction of the silk protein nanofibers. The invention firstly utilizes a formic acid solvent system to realize the interaction of the silk protein nano fibers, and then replaces formic acid into water through a semipermeable membrane or a dialysis bag to keep the interaction between the fibers, so as to obtain the high-strength silk protein nano fiber hydrogel, the compression modulus and the breaking strength of which respectively reach 5.88MPa and 1.55MPa, and the preparation method is simple and easy to implement at normal temperature and normal pressure.
The invention utilizes formic acid as a solvent to dissolve silk protein nano fibers so as to reduce charge repulsion force and promote the reassembly between the nano fibers to obtain a silk protein film with excellent mechanical property, however, how to transfer a silk protein nano fiber system which is interacted in a formic acid system to an aqueous phase system to prepare high-strength hydrogel lacks a feasible method.
Drawings
FIG. 1 is an atomic force microscope photograph showing the silk fibroin nanofibers prepared in example 1 of the present invention dissolved in water (a) and formic acid (b), respectively;
FIG. 2 is a digital photograph of the silk fibroin nanofiber hydrogel prepared in example 1 of the present invention (a), and its compressed state (b), bent state (c), stretched state (d), before compressed state (e), and after compressed state (f);
FIG. 3 is a microstructure diagram (observed by a cold field emission scanning electron microscope) of the silk fibroin nanofiber hydrogel prepared in example 1 of the present invention;
FIG. 4 is a stress-strain curve of the compression and stretching of silk fibroin nanofiber hydrogel prepared in example 1 of the present invention;
FIG. 5 is an infrared spectrum of a silk fibroin nanofiber hydrogel prepared in example 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of high-strength silk protein nanofiber hydrogel, which comprises the following steps:
and (3) replacing formic acid in the silk protein nanofiber formic acid solution with the aqueous solution to obtain the high-strength silk protein nanofiber hydrogel.
In the invention, the mass concentration of the silk fibroin nanofiber formic acid solution is preferably 5-30%, more preferably 10-25%, most preferably 15-20%, and most preferably 5%, 10%, 15% or 20%.
In the present invention, the preparation method of the silk fibroin nanofiber formic acid solution preferably comprises:
dissolving the silk fibroin nanofiber freeze-dried powder in formic acid to obtain a silk fibroin nanofiber formic acid solution (SNF-FA).
In the invention, the preparation method of the silk fibroin nanofiber freeze-dried powder preferably comprises the following steps:
and (3) freeze-drying the high-crystal silk fibroin nanofiber aqueous solution to obtain the silk fibroin nanofiber freeze-dried powder.
In the invention, the mass concentration of the high-crystalline silk fibroin nanofiber aqueous solution is preferably 0.01-4%, more preferably 0.05-3%, more preferably 0.1-2%, more preferably 0.5-1.5%, and most preferably 1% or 2%.
In the invention, the crystallinity of the high-crystal silk protein nanofiber in the high-crystal silk protein nanofiber aqueous solution is preferably not less than 40%, the diameter is preferably 10-30 nm, more preferably 15-25 nm, and most preferably 20 nm; the length is preferably 200 to 2000nm, more preferably 500 to 1500nm, more preferably 800 to 1200nm, and most preferably 1000 nm.
In the present invention, the preparation method of the high crystalline silk protein nanofiber aqueous solution preferably comprises:
and sequentially carrying out primary concentration and secondary concentration on the silk protein aqueous solution, diluting, and carrying out sealed incubation to obtain the high-crystal silk protein nanofiber aqueous solution.
In the present invention, the method for preparing the silk protein aqueous solution preferably comprises:
degumming silk, drying, dissolving in lithium bromide solution, and dialyzing to obtain silk protein water solution.
In the invention, Na is preferably adopted for degumming2CO3An aqueous solution; the silk, water and Na2CO3The preferable dosage proportion of (4-6) g: (1-3) L: (4-5) g, more preferably (4.5-5.5) g: (1.5-2.5) L: (4.2-4.5) g, most preferably 5 g: 2L: 4.24 g.
In the present invention, the drying is preferably oven drying.
In the invention, the concentration of the lithium bromide solution is preferably 9-10 mol/L, more preferably 9.1-9.5 mol/L, and most preferably 9.2-9.3 mol/L.
In the invention, the usage ratio of the dried product to the lithium bromide solution is preferably (2.5-3.5) g: (8-12) mL, more preferably (2.6-2.8) g: (9-11) mL, most preferably 2.7g:10 mL.
In the present invention, the dialysis time is preferably 2 to 4 days, and more preferably 3 days.
In the invention, the primary concentration is preferably to concentrate the mass concentration of the silk fibroin aqueous solution to 8-12%, more preferably to 9-11%, and most preferably to 10%; the second concentration is preferably to concentrate the silk fibroin aqueous solution after the first concentration to 16-24% by mass, more preferably 18-22% by mass, and most preferably 20% by mass; the dilution is preferably water dilution, and the mass concentration of the silk protein aqueous solution after the secondary concentration is preferably water dilution to 0.01-4%, more preferably 0.05-3%, more preferably 0.1-2%, more preferably 0.5-1.5%, and most preferably 1%.
In the invention, the dissolving temperature is preferably 4-80 ℃, more preferably 10-70 ℃, more preferably 20-60 ℃, more preferably 30-50 ℃, most preferably 40 ℃, and most preferably 4 ℃, 10 ℃, 20 ℃, 40 ℃ or 60 ℃; the dissolving time is preferably 0.1 to 24 hours, more preferably 0.5 to 20 hours, more preferably 1 to 15 hours, more preferably 5 to 12 hours, most preferably 8 to 10 hours, and most preferably 0.5 hour, 1 hour, 4 hours, 12 hours or 24 hours.
In the present invention, the aqueous solution preferably comprises: pure water or an aqueous solution containing ions; the pure water is preferably deionized water; the ions are preferably selected from magnesium ions, calcium ions and/or copper ions.
In the invention, in order to ensure that formic acid can be quickly separated out in the replacement process, an aqueous solution enters the quickly-cured fibroin to form gel, and the volume ratio of the aqueous solution to the formic acid is preferably more than 3: 1, more preferably (3-8): 1, more preferably (4-6): 1, most preferably 5: 1.
in the present invention, in order to ensure sufficient precipitation of formic acid during the replacement process, the replacement time is preferably 2 to 72 hours, more preferably 4 to 70 hours, more preferably 10 to 60 hours, more preferably 20 to 50 hours, more preferably 30 to 40 hours, and most preferably 2 hours, 8 hours, 24 hours, or 48 hours.
In the present invention, the method of substitution preferably comprises:
putting the silk fibroin nanofiber formic acid solution into a dialysis bag or a semipermeable membrane, and putting the dialysis bag or the semipermeable membrane into an aqueous solution to perform replacement of formic acid and the aqueous solution.
In the invention, the dialysis bag or the semipermeable membrane silk protein is not permeable, and formic acid and aqueous solution are permeable; the cut-off molecular weight of the dialysis bag or the semipermeable membrane is preferably 3000-10000, more preferably 5000-8000, and most preferably 6000.
In the invention, formic acid in the dialysis bag or the semipermeable membrane is replaced by aqueous solution, so that the formic acid is precipitated from the dialysis bag or the semipermeable membrane, and water enters to obtain the silk fibroin nanofiber hydrogel.
In the invention, in order to ensure that the formic acid is fully precipitated, the aqueous solution in the replacement process is preferably replaced by a fresh solution for multiple times, namely the fresh aqueous solution is replaced by the fresh aqueous solution after one time of replacement, and the number of replacement of the aqueous solution is preferably more than 2 times, more preferably 2-8 times, more preferably 4-6 times, and most preferably 5 times; the time interval of replacing the aqueous solution is preferably 5 to 15 hours, more preferably 8 to 12 hours, and most preferably 10 hours.
In the present invention, after the replacement is completed, the method preferably further comprises:
and soaking the displaced product in the water solution, and repeatedly replacing the water solution until the pH value of the replaced water solution is 6-8.
In the present invention, it is preferable that the solid silk protein hydrogel after replacement is taken out from the dialysis bag or the semipermeable membrane, and immersed in an aqueous solution to remove residual formic acid; preferably, the water solution is repeatedly changed in the soaking process until the pH value of the changed water solution (the soaked water solution) reaches 6-8, and the final high-strength silk protein nanofiber hydrogel is obtained.
In the present invention, the number of times of repeatedly changing the aqueous solution is preferably 2 or more, more preferably 2 to 8 times, more preferably 3 to 6 times, and most preferably 4 to 5 times.
In the present invention, the repeated water exchange of the aqueous solution is preferably performed such that the pH of the aqueous solution after the exchange (aqueous solution after the soaking) is 6.8 to 7.2, and more preferably 7.
The invention provides the high-strength silk protein nanofiber hydrogel prepared by the method in the technical scheme, and the high-strength silk protein nanofiber hydrogel can be prepared into products with different shapes, such as long strips or stars, through a die or a processing method.
According to the invention, the interaction between the nano-fibers is optimized through regulation and control of silk protein concentration in a formic acid system, then formic acid and water are replaced with each other by utilizing a specific dialysis bag or a semipermeable membrane, and on the basis of keeping the interaction of the silk protein nano-fibers, the high-strength silk protein nano-fiber hydrogel is obtained. The invention firstly utilizes a formic acid solvent system to realize the interaction of the silk protein nano fibers, and then replaces formic acid into water through a semipermeable membrane or a dialysis bag to keep the interaction between the fibers, so as to obtain the high-strength silk protein nano fiber hydrogel, the compression modulus and the breaking strength of which respectively reach 5.88MPa and 1.55MPa, and the preparation method is simple and easy to implement at normal temperature and normal pressure.
Example 1
Freeze-drying the fibroin nanofiber aqueous solution with the mass concentration of 1% to obtain fibroin nanofiber freeze-dried powder; the preparation method of the 1% silk protein nanofiber aqueous solution comprises the following steps:
soaking silk with Na2CO3Aqueous degumming (5g silk: 2L water: 4.24g Na)2CO3) Drying, dissolving in 9.3mol/L lithium bromide solution with bath ratio of (2.7g:10mL), taking out, dialyzing for 3 days to obtain silk protein aqueous solution, sequentially performing primary concentration and secondary concentration on the silk protein aqueous solution until the mass concentration is 20%, diluting with deionized water until the mass concentration is 1%, and sealing and incubating;
dissolving the fibroin nanofiber freeze-dried powder in formic acid at 60 ℃ for 0.5h to obtain a fibroin nanofiber formic acid solution with the mass concentration of 20%;
placing a fibroin nanofiber formic acid solution with the mass concentration of 20% into a dialysis bag (model SP132594, from Yuanye corporation) with the molecular weight cutoff of 3500, then placing the dialysis bag into deionized water with the volume 5 times of that of formic acid, carrying out solvent replacement, replacing the water solution with fresh deionized water outside the dialysis bag for 3 times every 8 hours, and forming solid fibroin nanofiber hydrogel inside the dialysis bag;
and taking out the solid silk protein hydrogel, soaking the solid silk protein hydrogel in deionized water, removing residual formic acid, changing water for 1 time per hour, and measuring the pH value of the water to be 6.8 after changing the water for 3 times to obtain the final hydrogel.
FIG. 1 is an atomic force microscope photograph showing the silk fibroin nanofibers prepared in example 1 of the present invention dissolved in water (a) and formic acid (b), respectively; as can be seen from fig. 1: the silk fibroin in the formic acid solution in the aqueous solution shows the shape of the nanofiber; the length of the nano-fibers in the aqueous solution is distributed in the range of 200-1000 nm, the length of the nano-fibers in the formic acid is reduced to 50-200 nm, and the shorter fiber length provides more opportunities for interaction among the fibers.
Fig. 2 is digital photographs (b-f) of the silk fibroin nanofiber hydrogel (a) prepared in example 1 of the present invention in different states of compression, bending, stretching, etc., and it can be seen that the hydrogel can be processed into different shapes, and the shape remains intact in the compression, bending and stretching states, indicating that it has good processability and mechanical properties.
FIG. 3 is a microstructure diagram of a silk protein nanofiber hydrogel prepared in example 1 of the present invention; as can be seen from fig. 3, the gel maintained a uniform porous structure, and the nanofiber morphology was not apparent, indicating that the fibers were reassembled and tightly bound.
The high strength silk protein nanofiber hydrogel prepared in example 1 was tested for compression modulus and breaking strength:
compression testing was carried out at 25. + -. 0.5 ℃ and a relative humidity of 60. + -. 5% using a universal tester (Instron 5967, sample height: 10 mm; compression speed: 5 mm/min); tensile testing was carried out using a universal tester (Instron3365, sample length: 10 mm; tensile speed: 10 mm/min).
The detection results are shown in fig. 4, and fig. 4 is a stress-strain curve diagram of compression and tension of the silk fibroin nanofiber hydrogel prepared in example 1 of the present invention, and it can be seen from fig. 4 that the silk fibroin nanofiber hydrogel prepared in example 1 of the present invention has excellent mechanical properties, and the compressive modulus and the breaking strength of the silk fibroin nanofiber hydrogel respectively reach 5.88MPa and 1.55 MPa.
FIG. 5 is an infrared spectrum of the silk fibroin nanofiber hydrogel (SNF20) prepared in example 1 of the present invention, wherein the control is the infrared spectrum of the silk fibroin (SNF-FA) in formic acid solution with the same concentration, and the traditional silk fibroin nanofiber hydrogel (SNF 2) prepared by sequentially performing primary concentration and secondary concentration on silk fibroin aqueous solution, and diluting with deionized water to obtain the silk fibroin hydrogel2 wt%, obtained by sealed incubation); as can be seen from FIG. 5, 1168cm in the conventional high-crystalline silk protein nanofiber hydrogel-1And 1064cm-1The absorption peaks at two positions are 1064cm-1Strong 1168cm-1Weak, while 1168cm in formic acid solution and silk protein nanofiber hydrogel prepared by the invention-1And 1064cm-1The absorption peaks at both positions are 1064cm-1Weak 1168cm-1Strong, indicating that good interactions between nanofibers were formed in the formic acid solution and that the interactions were well preserved in the hydrogels prepared in the examples of the invention.
Example 2
Freeze-drying a silk fibroin nanofiber aqueous solution with the mass concentration of 2% (the preparation method is the same as that of example 1, and is different from that of example 1 in that deionized water is used for diluting the silk fibroin nanofiber aqueous solution to the mass concentration of 2%), so as to obtain silk fibroin nanofiber freeze-dried powder;
dissolving the fibroin nanofiber freeze-dried powder in formic acid at 20 ℃ for 4h to obtain a fibroin nanofiber formic acid solution with the mass concentration of 10%;
placing a silk fibroin nanofiber formic acid solution with the mass concentration of 10% into a semipermeable membrane (a source leaf company, model SP131192) with the molecular weight cutoff of 5000, then placing the semipermeable membrane into deionized water with the volume 3 times that of formic acid, carrying out solvent replacement, replacing the external aqueous solution containing precipitated formic acid with fresh deionized water every 4 hours for 4 times, and forming solid silk fibroin nanofiber hydrogel inside a dialysis bag;
and taking out the solid silk protein hydrogel, soaking the solid silk protein hydrogel in deionized water to remove residual formic acid, changing water for 1 time every 2 hours, and measuring the pH value of the water to be 7.2 after changing the water for 4 times to obtain the final hydrogel.
According to the method of the embodiment 1, the mechanical property of the hydrogel prepared in the embodiment 2 of the invention is detected, and the detection result shows that the compressive modulus and the breaking strength of the silk protein nanofiber hydrogel prepared in the embodiment 2 respectively reach 1.71MPa and 0.9 MPa.
Example 3
Freeze-drying the silk fibroin nanofiber aqueous solution with the mass concentration of 1% (the preparation method is the same as that of the example 1) to obtain silk fibroin nanofiber freeze-dried powder;
dissolving the fibroin nanofiber freeze-dried powder in formic acid at 4 ℃ for 24 hours to obtain a fibroin nanofiber formic acid solution with the mass concentration of 15%;
placing 15% silk protein nanofiber formic acid solution in a dialysis bag (model SP131270, from Yuanye corporation) with cut-off molecular weight of 10000, then placing the dialysis bag in water containing magnesium ions (the concentration of the magnesium ions is 100mmol/L) with the volume being 8 times of that of formic acid, carrying out solvent replacement, replacing the external aqueous solution containing precipitated formic acid with fresh water containing magnesium ions every 12 hours for 4 times, and forming solid silk protein nanofiber hydrogel inside the dialysis bag;
and taking out the solid silk protein hydrogel, soaking the solid silk protein hydrogel in deionized water to remove residual formic acid, changing water for 1 time every 4 hours, and measuring the pH value of the water to be 7 after changing the water for 6 times to obtain the final hydrogel.
According to the method in the embodiment 1, the mechanical properties of the product prepared in the embodiment 3 of the invention are detected, and the detection result shows that the compressive modulus and the breaking strength of the silk protein nanofiber hydrogel prepared in the embodiment 3 respectively reach 3.22MPa and 1.3 MPa.
Example 4
Freeze-drying the fibroin nanofiber aqueous solution with the mass concentration of 2% (the preparation method is the same as that of example 2) to obtain fibroin nanofiber freeze-dried powder;
dissolving the fibroin nanofiber freeze-dried powder in formic acid at 40 ℃ for 4h to obtain a fibroin nanofiber formic acid solution with the mass concentration of 5%;
placing a silk fibroin nanofiber formic acid solution with the mass concentration of 5% in a semipermeable membrane (a source leaf company, model SP132594) with the molecular weight cutoff of 3500, then placing the semipermeable membrane into water (the calcium ion concentration is 100mmol/L) containing calcium ions and the volume of the water is 3 times of that of formic acid, carrying out solvent replacement, replacing the external aqueous solution containing precipitated formic acid with fresh water containing calcium ions every half hour for 8 times, and forming solid silk fibroin nanofiber hydrogel inside a dialysis bag;
and taking out the solid silk protein hydrogel, soaking the solid silk protein hydrogel in deionized water to remove residual formic acid, changing water for 1 time every 3 hours, and measuring the pH value of the water to be 7.2 after changing the water for 4 times to obtain the final hydrogel.
According to the method in the embodiment 1, the mechanical properties of the hydrogel prepared in the embodiment 4 of the invention are detected, and the detection result shows that the compressive modulus and the breaking strength of the silk fibroin nanofiber hydrogel prepared in the embodiment 4 respectively reach 1.06MPa and 0.7 MPa.
Example 5
Freeze-drying the fibroin nanofiber aqueous solution with the mass concentration of 2% (the preparation method is the same as that of example 2) to obtain fibroin nanofiber freeze-dried powder;
dissolving the fibroin nanofiber freeze-dried powder in formic acid at 10 ℃ for 12h to obtain a fibroin nanofiber formic acid solution with the mass concentration of 15%;
placing 15% silk fibroin nanofiber formic acid solution in a semipermeable membrane (model SP131192, from Yuanye corporation) with molecular weight cutoff of 5000, then placing the semipermeable membrane in water (the concentration of copper ions is 100mmol/L) containing copper ions and the volume of which is 8 times of that of formic acid, carrying out solvent replacement, replacing the external aqueous solution containing precipitated formic acid with fresh water containing copper ions every 3 hours for 8 times, and forming solid silk fibroin nanofiber hydrogel inside a dialysis bag;
and taking out the solid silk protein hydrogel, soaking the solid silk protein hydrogel in deionized water to remove residual formic acid, changing water for 1 time every 2 hours, and measuring the pH value of the water to be 7 after changing the water for 6 times to obtain the final hydrogel.
According to the method in the embodiment 1, the mechanical properties of the hydrogel prepared in the embodiment 5 of the invention are detected, and the detection result shows that the compressive modulus and the breaking strength of the silk protein nanofiber hydrogel prepared in the embodiment 5 respectively reach 3.86MPa and 1.5 MPa.
The method provided by the invention reduces the charge repulsion force through formic acid, optimizes the non-covalent action, and then realizes the replacement of formic acid and water through a dialysis bag or a semipermeable membrane, so that the method for obtaining the high-strength hydrogel provides experimental basis for the mechanical regulation of other materials, and can be widely applied to the field of high-strength gel preparation.
The invention regulates and controls the interaction between silk protein nanofibers by the concentration of the silk protein nanofibers dissolved in formic acid, and then replaces the formic acid with water through a dialysis bag or a semipermeable membrane to obtain the high-strength silk protein hydrogel material. The method for obtaining the hydrogel on the basis of keeping the interaction of the nano fibers by reducing the charge repulsion through formic acid, optimizing the interaction of the nano fibers and then replacing the formic acid with water can be widely applied to the field of high-strength material preparation.
While the invention has been described and illustrated with reference to specific embodiments thereof, such description and illustration are not intended to limit the invention. It will be clearly understood by those skilled in the art that various changes in form and details may be made therein without departing from the true spirit and scope of the invention as defined by the appended claims, to adapt a particular situation, material, composition of matter, substance, method or process to the objective, spirit and scope of this application. All such modifications are intended to be within the scope of the claims appended hereto. Although the methods disclosed herein have been described with reference to particular operations performed in a particular order, it should be understood that these operations may be combined, sub-divided, or reordered to form equivalent methods without departing from the teachings of the present disclosure. Accordingly, unless specifically indicated herein, the order and grouping of the operations is not a limitation of the present application.
Claims (10)
1. A preparation method of high-strength silk protein nanofiber hydrogel comprises the following steps:
and (3) replacing formic acid in the silk protein nanofiber formic acid solution with the aqueous solution to obtain the high-strength silk protein nanofiber hydrogel.
2. The method according to claim 1, wherein the time for the replacement is 2 to 72 hours.
3. The method according to claim 1, wherein the mass concentration of the silk protein nanofiber formic acid solution is 5-30%.
4. The method of claim 1, wherein the permuting comprises:
putting the silk fibroin nanofiber formic acid solution into a dialysis bag or a semipermeable membrane, and putting the dialysis bag or the semipermeable membrane into an aqueous solution to perform replacement of formic acid and the aqueous solution;
the dialysis bag or the semipermeable membrane silk protein is impermeable, and formic acid and aqueous solution are permeable.
5. The method of claim 1, wherein the permuting further comprises:
and soaking the displaced product in the water solution, and repeatedly replacing the water solution until the pH value of the replaced water solution is 6-8.
6. The method of claim 1, wherein the volume ratio of aqueous solution to formic acid in the displacement process is in the range of 3: 1 or more.
7. The method according to claim 1, wherein the preparation method of the silk fibroin nanofiber formic acid solution comprises:
dissolving the fibroin nanofiber freeze-dried powder in formic acid to obtain a fibroin nanofiber formic acid solution;
the dissolving temperature is 4-80 ℃; the dissolving time is 0.1-24 hours.
8. The method of claim 1, wherein the aqueous solution comprises:
pure water or an aqueous solution containing ions,
the ions are magnesium ions, calcium ions and/or copper ions.
9. The method of claim 7, wherein the preparation method of the silk fibroin nanofiber freeze-dried powder comprises:
freeze-drying the high-crystal silk fibroin nanofiber aqueous solution to obtain silk fibroin nanofiber freeze-dried powder;
the mass concentration of the high-crystalline silk protein nanofiber aqueous solution is 0.01-4%.
10. The method of claim 9, wherein the method for preparing the aqueous solution of high crystalline silk protein nanofibers comprises:
and sequentially carrying out primary concentration and secondary concentration on the silk protein aqueous solution, diluting, and carrying out sealed incubation to obtain the high-crystal silk protein nanofiber aqueous solution.
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| PCT/CN2022/086039 WO2023137880A1 (en) | 2022-01-18 | 2022-04-11 | Preparation method for high-strength silk protein nanofiber hydrogel |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015144056A1 (en) * | 2014-03-27 | 2015-10-01 | 苏州丝美特生物技术有限公司 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| CN106310380A (en) * | 2016-08-19 | 2017-01-11 | 苏州大学 | Nano-fibrosis silk fibroin gel and preparation method thereof |
| CN109096501A (en) * | 2018-07-16 | 2018-12-28 | 武汉纺织大学 | A kind of fibroin three-dimensional porous rack and preparation method thereof |
| CN112876711A (en) * | 2021-01-20 | 2021-06-01 | 苏州大学 | High-strength silk protein nanofiber membrane and preparation method thereof |
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| JP2015093857A (en) * | 2013-11-13 | 2015-05-18 | 国立大学法人信州大学 | Water-insoluble silk protein |
| CN106139246A (en) * | 2016-08-19 | 2016-11-23 | 苏州大学 | A kind of regenerated silk azelon support and preparation method thereof |
| CN107118384A (en) * | 2017-05-02 | 2017-09-01 | 丝纳特（苏州）生物科技有限公司 | A kind of silk-fibroin sponge and preparation method thereof |
| CN107080860B (en) * | 2017-05-02 | 2020-08-14 | 丝纳特（苏州）生物科技有限公司 | Silk protein sponge and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015144056A1 (en) * | 2014-03-27 | 2015-10-01 | 苏州丝美特生物技术有限公司 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| CN106310380A (en) * | 2016-08-19 | 2017-01-11 | 苏州大学 | Nano-fibrosis silk fibroin gel and preparation method thereof |
| CN109096501A (en) * | 2018-07-16 | 2018-12-28 | 武汉纺织大学 | A kind of fibroin three-dimensional porous rack and preparation method thereof |
| CN112876711A (en) * | 2021-01-20 | 2021-06-01 | 苏州大学 | High-strength silk protein nanofiber membrane and preparation method thereof |
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