CN115260300A - (methyl) acryloyl modified silk protein and preparation method and application thereof - Google Patents

(methyl) acryloyl modified silk protein and preparation method and application thereof Download PDF

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CN115260300A
CN115260300A CN202211040714.6A CN202211040714A CN115260300A CN 115260300 A CN115260300 A CN 115260300A CN 202211040714 A CN202211040714 A CN 202211040714A CN 115260300 A CN115260300 A CN 115260300A
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silk protein
meth
carboxylated
acryloyl
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郭成辰
王家奇
孙子扬
郑小睿
尚鸿鹏
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Westlake University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • 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
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • 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
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • G—PHYSICS
    • G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
    • G03F—PHOTOMECHANICAL PRODUCTION OF TEXTURED OR PATTERNED SURFACES, e.g. FOR PRINTING, FOR PROCESSING OF SEMICONDUCTOR DEVICES; MATERIALS THEREFOR; ORIGINALS THEREFOR; APPARATUS SPECIALLY ADAPTED THEREFOR
    • G03F7/00—Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor
    • G03F7/004—Photosensitive materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof

Abstract

The application relates to a (methyl) acryloyl modified silk protein and a preparation method and application thereof. The (meth) acryloyl-modified silk protein is prepared by the following method: (1) Reacting silk protein with dicarboxylic anhydride to obtain carboxylated silk protein; and (2) reacting the carboxylated silk protein with a compound containing an amino group and a (meth) acryloyl group to prepare the (meth) acryloyl group modified silk protein. The preparation method has the characteristics of preparation condition temperature and low raw material toxicity. The prepared (methyl) acryloyl modified silk protein has good solubility in water, can be further prepared into aqueous phase photoresist or photocrosslinking hydrogel, and the hydrogel has application prospects in tissue repair materials.

Description

(methyl) acryloyl modified silk protein and preparation method and application thereof
Technical Field
The invention relates to the field of novel protein-based natural high polymer materials, in particular to (methyl) acryloyl modified silk protein and a preparation method and application thereof.
Background
Fibroin derived from silkworm cocoons has excellent mechanical properties, biocompatibility and biodegradability, and has recently received wide attention in the biomedical field, such as biosensors, drug carriers, surgical implant materials, and the like. Natural silk is composed of silk fibroin and sericin, wherein the silk fibroin is wrapped by sericin as an inner core. After degumming, dissolving, purifying and other treatment methods, single regenerated silk fibroin (silk protein for short) can be obtained. Through further processing, materials of various forms, including hydrogels, films, sponges, and the like, may be prepared.
Although natural silk proteins have excellent biocompatibility and biodegradability and are widely used in various fields, their functionality, particularly, in terms of stimulus responsiveness and bio-responsiveness, etc., is limited. Grafting a group with specific functionality on a fibroin molecular chain by a chemical modification method is an effective way for expanding the application of fibroin. Wherein, the silk protein is modified by methacryloyl group to obtain the silk protein-based functional material with photoresponse. The material can be used as a photoresist or as a photo-crosslinking bio-ink. To date, researchers have attempted to convert isocyanate ethyl methacrylate into methacryloyl groups by reacting it with hydroxyl groups in serine and tyrosine residues of silk proteins (Vamsi k. Yadavali et al. Adv. Mater.2013,25, 6207-6212), but the reaction conditions are harsh, require large amounts of organic solvents, and the raw material, isocyanate ethyl methacrylate, is expensive and does not meet the green chemistry principle. Further, the grafting ratio of methacryloyl groups cannot be secured. In addition, some researchers use glycidyl methacrylate to react with amino groups on the side chain of silk protein, but the reaction is limited by the low content of amino groups on the side chain in natural silk protein, the proportion of modified methacryloyl groups is low, and the photo-crosslinking conditions are harsh.
Disclosure of Invention
Technical purpose
It is an object of the present invention to provide a method for preparing a (meth) acryloyl-modified silk protein.
It is another object of the present invention to provide a (meth) acryloyl-modified silk protein prepared by the method.
Still another object of the present invention is to provide use of the (meth) acryloyl-modified silk protein in preparation of an aqueous phase photoresist or a photo-crosslinked hydrogel.
Technical scheme
In one aspect, the present invention provides a method of preparing a (meth) acryloyl-modified silk protein, the method comprising the steps of:
(2) Reacting silk protein with dicarboxylic anhydride to produce carboxylated silk protein; and
(2) Reacting the carboxylated silk protein with a compound containing an amino group and a (meth) acryloyl group to prepare the (meth) acryloyl group modified silk protein.
In a specific embodiment, the step (1) comprises:
reacting the fibroin with dicarboxylic anhydride in the presence of lithium chloride to obtain carboxylated fibroin;
in some embodiments, step (1) may further comprise purification and drying steps. For example, the solution obtained by the reaction in step (1) may be purified by dialysis (e.g., dialysis in pure water) and dried by lyophilization.
In some embodiments, the silk protein of step (1) is prepared by:
1': adding silkworm cocoon into sodium carbonate water solution, heating until the solution is boiled and keeping for 30-120 minutes, rinsing the silk after degumming in water for many times, and then drying at room temperature;
2': and adding the silk after degumming into an aqueous solution of lithium bromide, heating, and dialyzing the solution after the silk is dissolved to obtain a silk protein solution.
In some embodiments, in step (1), the dicarboxylic anhydride may be succinic anhydride, glutaric anhydride, phthalic anhydride.
In some embodiments, in step (1), the silk protein is reacted with the dicarboxylic acid anhydride in a solvent that is dimethyl sulfoxide, dimethylformamide, dimethylacetamide, or methylpyrrolidone.
In some embodiments, in step (1), the mass ratio of the silk protein to the dicarboxylic anhydride is 1.1 to 1, preferably 1:1 to 1:5, such as 1.1, 1:2, 1:3, 1:5, 1.
In some embodiments, in step (1), the reaction temperature of the silk protein with the dicarboxylic acid anhydride is 20 to 60 ℃; the reaction time of the silk protein with the dicarboxylic acid anhydride is 5 minutes to 72 hours.
In some embodiments, the carboxylated silk protein obtained in step (1) has a degree of carboxylation of 0.1 to 12%.
In a specific embodiment, the step (2) comprises:
(2-1) dissolving the carboxylated silk protein, mixing the dissolved carboxylated silk protein with N-hydroxysuccinimide and carbonyldiimine (for example, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine or dicyclohexylcarbodiimide), and reacting the mixture to obtain activated carboxylated silk protein;
(2-2) mixing the activated carboxylated silk protein obtained in the above step with a compound containing an amino group and a (meth) acryloyl group for reaction.
In some embodiments, step (2) may further comprise purification and drying steps. For example, the product obtained in step (2-2) can be purified by dialysis to obtain an aqueous (meth) acryloyl-modified silk protein solution, and then the solution is dried to obtain a (meth) acryloyl-modified silk protein. In some embodiments, dialysis can be performed using pure water or a buffer solution, such as pure water, TBS buffer, PBS buffer, preferably PBS buffer.
In some embodiments, in step (2-1), the mass ratio of carboxylated silk protein to N-hydroxysuccinimide may be from 1.
In some embodiments, in step (2-1), the mass ratio of carboxylated silk fibroin to 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine can be 1:1 to 1:5, preferably 1:2 to 1:3, such as 1:1, 1:2, 1:3, 1:5.
In some embodiments, in step (2-1), the time for the mixed reaction of the carboxylated silk protein with N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide may be 0.1 to 5 hours, preferably 0.5 to 2.5 hours, such as 0.5, 1.5, 2.5 hours.
In some embodiments, in step (2), the compound containing an amino group and a (meth) acryloyl group may be represented by the following general formula:
Figure BDA0003820195690000031
in the above formula, R represents methyl or H, X represents O or N, and N is an integer of 0 to 3.
In particular, the compound containing an amino group and a (meth) acryloyl group may be aminoethyl (meth) acrylate, N- (2-aminoethyl) (meth) acrylamide, N- (3-aminopropyl) (meth) acrylamide or a salt thereof, in particular a hydrochloride salt, preferably aminoethyl (meth) acrylate hydrochloride salt.
In some embodiments, in step (2), the mass ratio of carboxylated silk protein to the compound containing an amino group and a (meth) acryloyl group may be 1.
In some embodiments, in step (2), the time for mixing and reacting the carboxylated silk protein and the compound containing an amino group and a (meth) acryloyl group may be 1 hour to 24 hours, preferably 3 hours to 15 hours, such as 1 hour, 3 hours, 10 hours, 12 hours, 24 hours.
In still another aspect, the present invention provides a (meth) acryloyl-modified silk protein prepared by the above method.
In particular, when the compound containing an amino group and a (meth) acryloyl group is aminoethyl methacrylate hydrochloride, the methacryloyl group-modified silk protein contains a structure of the following formula in its side chain
Figure BDA0003820195690000041
In particular, when the compound containing an amino group and a (meth) acryloyl group is N- (3-aminopropyl) methacrylic acid hydrochloride, the methacryloyl group-modified silk protein contains a structure of the following formula in its side chain
Figure BDA0003820195690000042
In yet another aspect, the present invention provides a silk protein composition comprising the above (meth) acryloyl-modified silk protein according to the present invention, a polymerization initiator, and a solvent.
In particular embodiments, the polymerization initiator may be a photoinitiator, a thermal initiator, or the like. The specific type of the photoinitiator and the thermal initiator is not particularly limited, and may be appropriately selected depending on the polymerization conditions and the like. The polymerization initiator is preferably a photoinitiator, for example LAP.
In a particular embodiment, the solvent is an aqueous solvent, preferably water.
In addition, the silk protein composition of the present application may further contain other vinyl monomers (including monovinyl monomers and polyvinyl monomers as a crosslinking monomer), additives (such as fillers, colorants, fragrances, preservatives, thickeners, diluents, etc.) as needed for product performance, etc., but is not limited thereto.
In some embodiments, the silk protein composition is used as a photoresist comprising the above (meth) acryloyl-modified silk protein according to the present invention, a photoinitiator, and a solvent.
The present invention provides the use of the above-described (meth) acryloyl-modified silk protein according to the present invention or the above-described silk protein composition according to the present invention for preparing a photoresist.
The description of the photoinitiator and the solvent is the same as that described above.
The photoresist may also contain other components for photoresist such as other vinyl monomers (including monovinyl monomers and polyvinyl monomers as crosslinking monomers), additives (e.g., fillers, colorants, preservatives, thickeners, diluents, etc.) as needed for product performance, etc., but is not limited thereto.
In some embodiments, the photoresist is an aqueous photoresist, i.e., the solvent is an aqueous solvent, preferably water.
The formulation of the photoresist is not particularly limited as long as the components can be uniformly mixed. For example, the photoresist can be prepared as follows: preparing the (methyl) acryloyl group modified silk protein into aqueous solution, and dissolving a photoinitiator LAP into the aqueous solution to obtain the silk protein. In one embodiment, the (meth) acryloyl modified silk protein according to the present invention is prepared into a 10% aqueous solution, and then 1% photoinitiator LAP is dissolved.
The method of applying the photoresist may employ a method known in the art. For example, 200 microliters of the previously prepared photoresist can be spin-coated onto a silicon wafer at a speed of 3000 rpm. After the silicon wafer was masked with a mask having a pattern, it was irradiated with a 405nm laser for 30 seconds. And after the irradiation is finished, the silicon wafer is put into water for development for 15 minutes, and the exposure area is left on the silicon wafer to form a pattern.
In another aspect, the silk protein composition is used as a gel composition for preparing a gel. That is, the present invention provides the use of a (meth) acryloyl-modified silk protein according to the present invention or the above-described silk protein composition according to the present invention for preparing a gel.
There is no particular limitation on the method for preparing the gel, as long as a gel that meets the requirements can be formed. For example, the components of the silk fibroin composition can be mixed together and then polymerization can be initiated to provide a gel, although the invention is not limited thereto.
In some embodiments, the gel is a hydrogel, i.e., the solvent is an aqueous solvent, preferably water.
In some embodiments, the gel may be formed as follows: dissolving the methacryloyl modified silk protein in a solvent to prepare an aqueous solution, dissolving an initiator, and then initiating polymerization to obtain the methacryloyl modified silk protein.
In some embodiments, using water as the solvent and a photoinitiator as the initiator, a gel is formed as follows: the methacryloyl modified silk protein is prepared into an aqueous solution, then a photoinitiator is dissolved, and then an LED light source is used for irradiation, so that the methacryloyl modified silk protein is obtained.
In some embodiments, the hydrogel can be prepared according to the following method: the (meth) acryloyl modified silk protein according to the invention is prepared into a 10% aqueous solution, 1% photoinitiator LAP is dissolved, and the solution loses fluidity and turns into gel after being irradiated for 15 seconds in a glass bottle by using a 405nm LED light source.
In another aspect, the present invention provides a gel comprising the polymerization product of the above (meth) acryloyl-modified silk protein according to the present invention. In particular, the gel is a hydrogel.
Hydrogel materials have been widely used in tissue repair materials, particularly cartilage tissue. In a further aspect, therefore, the present invention provides the use of a hydrogel as described above in the preparation of a tissue repair material.
In a specific embodiment, the tissue is cartilage tissue.
Advantageous effects
(1) Silk protein required by the method is directly extracted from silkworm cocoons, and the silk protein is wide in source, cheap and easy to obtain; the synthesis method of the carboxylated silk protein is simple and convenient, and the raw materials required by synthesis are wide in source, cheap and easy to obtain.
(2) The N-hydroxysuccinimide, 1-ethyl- (3-dimethylaminopropyl) carbodiimide, aminoethyl methacrylate hydrochloride, N- (3-aminopropyl) methacrylic acid hydrochloride and the like required by the method are common industrial raw materials and have low price.
(3) The raw materials required by the method of the invention have low toxicity and accord with the principle of green chemistry.
(4) The method disclosed by the invention is mild in preparation step conditions, simple to operate and very suitable for batch production.
(5) The preparation steps of the method can quantitatively and controllably modify the methacryloyl onto the silk protein, and the method has a wider modification range.
(6) The methacryl modified silk protein obtained by the method has good solubility in water, and can be further prepared into aqueous phase photoresist or photocrosslinking hydrogel.
Drawings
FIG. 1 shows a powder photograph of methacryloyl-modified silk protein prepared in example 1 of the present invention.
FIG. 2 is a nuclear magnetic diagram of hydrogen spectra of methacryloyl-modified silk proteins prepared in example 1 of the present invention in deuterated dimethyl sulfoxide.
FIG. 3 is a graph showing an infrared absorption spectrum of a methacryloyl-modified silk protein prepared in example 1 of the present invention.
FIG. 4 is a nuclear magnetic diagram of hydrogen spectrum of methacryloyl group-modified silk protein prepared in example 5 of the present invention in deuterated dimethyl sulfoxide.
FIG. 5 is a graph showing an infrared absorption spectrum of a methacryloyl-modified silk protein prepared in example 5 of the present invention.
FIG. 6 is a graph showing the grafting yield of methacryloyl-modified silk proteins prepared in examples 1 to 7 of the present invention as a function of the charge ratio.
FIG. 7 is a photograph of a methacryloyl-modified silk protein prepared in example 1 of the present invention, which was subjected to photolithography patterning. The left image is an image after development photographed by an optical microscope, the middle image is an image photographed by a scanning electron microscope, and the right image is an enlarged view of a partial region of the middle image.
FIG. 8 is a photograph of a hydrogel formed by photocrosslinking methacryloyl-modified silk protein prepared in example 1 of the present invention.
FIG. 9 is a graph showing the rheological properties of hydrogel formed by photocrosslinking of methacryloyl-modified silk protein prepared in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention are described in detail below by means of specific embodiments, which, however, are not intended to limit the scope of the present invention.
Term(s) for
In the present invention, the silk protein refers to silk protein-based materials, such as natural silk protein, recombinant silk protein, regenerated silk protein having different molecular weights, and the like.
In the present invention, the carboxylated silk protein refers to carboxylated silk protein modified with acid anhydride.
In the present invention, the (meth) acryloyl group includes acryloyl groups and methacryloyl groups.
Materials and instruments
N-hydroxysuccinimide (140 mg) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide were purchased from Chinesia chemical industry development Co., ltd, and aminoethyl methacrylate hydrochloride was purchased from Beijing Yinaoka science Co., ltd.
Preparation example 1: preparation of carboxylated silk proteins
(1) 1g of silk protein was dissolved in a dimethyl sulfoxide solution (50 mL) having a lithium chloride concentration of 1mol/L, and 10g of succinic anhydride was added thereto, and the mixture was heated to 50 ℃ to react for 6 hours.
(2) Dialyzing the reacted solution to obtain a carboxylated silk protein aqueous solution. And (3) freeze-drying the solution to obtain the carboxylated silk protein dry powder.
Example 1
(1) 100mg of the carboxylated silk protein obtained in preparation example 1 was dissolved in 1X MES buffer solution (5 mL) at room temperature, N-hydroxysuccinimide (140 mg) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide (200 mg) were added, and after 0.5 hour of reaction, aminoethyl methacrylate hydrochloride (5 mg) was added and the reaction was continued for 3 hours.
(2) The reacted solution was dialyzed against 0.1 × pbs buffer solution to obtain a methacryl-modified silk protein buffer saline solution. And (3) carrying out freeze drying on the solution to obtain the methacryloyl modified silk protein dry powder.
Example 2
Prepared in the same manner as in example 1, except that aminoethyl methacrylate hydrochloride (10 mg) was added after the reaction for 0.5h, and the reaction was continued for 3h.
Example 3
Prepared in the same manner as in example 1, except that aminoethyl methacrylate hydrochloride (20 mg) was added after the reaction for 0.5h, and the reaction was continued for 3h.
Example 4
Prepared in the same manner as in example 1, except that aminoethyl methacrylate hydrochloride (50 mg) was added after the reaction for 0.5h, and the reaction was continued for 3h.
Example 5
Prepared in the same manner as in example 1, except that after 0.5h of reaction, aminoethyl methacrylate hydrochloride (1000 mg) was added and the reaction was continued for 3h.
Example 6
Prepared in the same manner as in example 1, except that after 1.5h of reaction, aminoethyl methacrylate hydrochloride (10 mg) was added and the reaction was continued for 12 h.
Example 7
Was prepared in the same manner as in example 6 except that N-hydroxysuccinimide (300 mg) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide (400 mg) were added.
Comparative example 1
40g of silkworm cocoon was boiled in 1L of 0.05M sodium carbonate solution at 100 ℃ for 30 minutes, and then washed several times with distilled water, followed by drying at room temperature, and 20g was dissolved in 100mL of 9.3M lithium bromide solution. Immediately after dissolution, the ethyl methacrylate solution was added to the mixture, which was then washed in a cold ethanol/acetone mixture and centrifuged, followed by lyophilization.
Comparative example 2
40g of silkworm cocoon was boiled in 1L of 0.05M sodium carbonate solution at 100 ℃ for 30 minutes, and then washed several times with distilled water, followed by drying at room temperature, and 20g was dissolved in 100mL of 9.3M lithium bromide solution. Immediately after dissolution, the glycidyl methacrylate solution was added to the mixture and stirred at 60 ℃ for 3h. The resulting solution was then filtered and dialyzed for 4 days and lyophilized.
Test example 1:
calculation of modification ratio of methacryloyl group: the peaks at 6.06 and 5.69ppm in the nuclear magnetic hydrogen spectrum correspond to two hydrogen atoms of a carbon-carbon double bond on a methacryloyl group, the peak at 1.87ppm corresponds to three hydrogen atoms of a methyl group on a methacryloyl group, and the peak at 1.26ppm corresponds to three hydrogen atoms of a methyl group on a serine alanine. The modification ratio of methacryloyl groups can be calculated by comparing the peak areas.
The yield (%) of the methacryl-modified silk protein prepared in each example was calculated by a weighing method.
The preparation conditions of examples and comparative examples and the modification rates and yields of the modifying groups to give modified products are shown in Table 1 below.
TABLE 1
Figure BDA0003820195690000091
As shown in Table 1 above, the modification rate of methacryloyl group can be increased by increasing the reaction time or increasing the mass ratio of the carboxylated silk protein to the methacryloyl derivative, and the modification rate is preferably controlled to be 2% to 10%.
Test example 2: determination of Hydrogen Spectrum data and Infrared absorption Spectrum data of methacryloyl-modified Silk proteins
The results of measuring hydrogen spectrum data and infrared absorption spectrum data of the methacryloyl-modified silk proteins obtained in example 1 and example 5 are shown in FIGS. 2 to 5, respectively.
Test example 3: relation between charge mass ratio of carboxylated silk protein and methacryloyl derivative and final methacryloyl grafting rate
FIG. 6 is a graph showing the trend of the charge mass ratio of carboxylated silk protein to methacryloyl derivative and the final methacryloyl graft ratio in examples 1 to 5. The abscissa represents the molar ratio of methacryloyl groups to carboxyl groups charged during the reaction, and the ordinate represents the molar ratio of methacryloyl groups to the total number of amino acids in the silk protein modified with methacryloyl groups obtained after the reaction.
As can be seen from FIG. 6, the final methacryloyl group grafting rate increases with increasing molar ratio of methacryloyl groups to carboxyl groups fed during the reaction, but when the molar ratio fed reaches 120%, the increase in grafting rate becomes more gradual with increasing molar ratio of methacryloyl groups to carboxyl groups fed.
Test example 4: preparation of aqueous phase photoresist
The methacryl-modified silk protein obtained in example 2 was prepared as a 10% aqueous solution, and then 1% photoinitiator LAP was dissolved, and 200 μ l was spin-coated on a silicon wafer at 3000 rpm. After the silicon wafer was masked with a mask having a pattern, it was irradiated with a 405nm laser for 30 seconds. After the irradiation was completed, the silicon wafer was put into water and developed for 15 minutes, and the finished product was as shown in FIG. 7. The exposed regions were left on the silicon wafer to form a pattern, which was photographed using an optical and scanning electron microscope. The formed pattern has sharp edges and resolution less than 30 microns.
Test example 5: preparation of photo-crosslinked hydrogels
The methacryl-modified silk protein obtained in example 2 was prepared as a 10% aqueous solution, and then 1% photoinitiator LAP was dissolved and irradiated in a glass bottle for 15 seconds using a 405nm LED light source. As a result, as shown in FIG. 8, it was observed that the solution lost fluidity and was converted into a gel, which was further formed as a hydrogel due to the formation of a network structure by the methacryloyl group crosslinking.
Test example 6: hydrogel rheological Property testing
The hydrogel obtained in test example 5 was subjected to rheological property test using a rotational rheometer, and the loss of viscoelasticity of the gel was measured by horizontal rotation after slightly pressing the gel, with the results shown in fig. 9. The curves marked by the triangular patterns in fig. 9 represent the storage modulus of the gel, with values above 1000 indicating that the gel can store more elastic deformation energy. The curve marked by the square pattern represents the loss modulus of the gel, with a value of about 100 indicating a lower energy loss during elastic deformation of the gel. The curve marked by the circle pattern represents the angle of rotation during the rotational rheometer test.
Rheological property tests prove that the hydrogel product can obtain hydrogel with better mechanical property at the concentration of 10%. The silk protein obtained in comparative example 1 could not have a high solubility in pure water. Compared with the methacryl modified silk protein prepared in the comparative examples 1-2, the methacryl modified silk protein of the present application has obvious advantages in preparing hydrogel compared with the methacryl modified silk protein prepared in the comparative examples 1-2.
In summary, the present application develops a novel method for preparing methacryl-modified silk protein. Specifically, firstly, natural silk protein is carboxylated, and functional groups (such as hydroxyl and amino) on the side chain of the silk protein molecular chain are reacted with dicarboxylic anhydride to obtain silk protein with the degree of side chain carboxylation of about 12%. Higher degree of carboxylation may allow the carboxylated silk protein to possess higher reactivity. Secondly, the compound containing both amino and methacryloyl can be covalently modified on the silk protein in a high proportion by utilizing biological coupling reaction such as amidation reaction and the like, and the methacryloyl modified silk protein is obtained. The method is a full-aqueous phase method, and has the advantages of adjustable modification degree, simple and convenient operation, environmental protection and the like. The methacryl modified silk protein obtained by the invention has good solubility in water, can reach the concentration of more than 10 percent, and can be further prepared into aqueous phase photoresist or photocrosslinking hydrogel.

Claims (10)

1. A method of preparing a (meth) acryloyl modified silk protein, the method comprising the steps of:
(1) Reacting silk protein with dicarboxylic anhydride to obtain carboxylated silk protein; and
(2) Reacting the carboxylated silk protein with a compound containing an amino group and a (meth) acryloyl group to prepare the (meth) acryloyl group modified silk protein.
2. The method of claim 1, wherein the step (1) comprises: reacting the fibroin with dicarboxylic anhydride in the presence of lithium chloride to obtain carboxylated fibroin; and/or
The carboxylation degree of the carboxylated silk protein obtained in the step (1) is 0.1-12%; and/or
In the step (2), the compound containing an amino group and a (meth) acryloyl group is represented by the following general formula:
Figure FDA0003820195680000011
in the above formula, R represents methyl or H, X represents O or N, N is an integer of 0 to 3,
in particular, the compound containing an amino group and a (meth) acryloyl group is aminoethyl methacrylate, N- (2-aminoethyl) methacrylamide, N- (3-aminopropyl) methacrylamide or their salts, in particular hydrochloride salts, preferably aminoethyl methacrylate hydrochloride;
and/or
In step (2), the mass ratio of the carboxylated silk protein to the compound containing an amino group and a (meth) acryloyl group is 1; and the time for reacting the carboxylated silk protein with the compound containing an amino group and a (meth) acryloyl group is 1 to 24 hours.
3. The method of claim 1, wherein the step (2) comprises:
(2-1) dissolving the carboxylated silk protein, mixing the dissolved carboxylated silk protein with N-hydroxysuccinimide and carbonyldiimine (for example, 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine or dicyclohexylcarbodiimide), and reacting the mixture to obtain activated carboxylated silk protein;
(2-2) mixing the activated carboxylated silk protein obtained in the step (2-1) with a compound containing an amino group and a (meth) acryloyl group for reaction to obtain a (meth) acryloyl group modified silk protein.
4. The method of claim 3, wherein,
in the step (2-1), the step (c),
the mass ratio of the carboxylated silk protein to the N-hydroxysuccinimide is 1.5 to 1:4;
the mass ratio of the carboxylated silk protein to the carbonyldiimine is 1:1 to 1:5;
the time for mixing and reacting the carboxylated silk protein with the N-hydroxysuccinimide and the carbonyldiimine is 0.1 to 5 hours.
5. A (meth) acryloyl modified silk protein prepared by the method of any one of claims 1-4.
6. A silk protein composition comprising the (meth) acryloyl-modified silk protein according to claim 5, a polymerization initiator, and a solvent;
in particular, the silk protein composition is a photoresist, preferably an aqueous phase photoresist, or the silk protein composition is a gel composition.
7. The silk protein composition of claim 6, wherein the polymerization initiator is a photoinitiator and the solvent is water.
8. Use of a (meth) acryloyl-modified silk protein according to claim 5 or a silk protein composition according to claim 6 or 7 for the preparation of a photoresist, in particular an aqueous phase photoresist, a gel, in particular a hydrogel.
9. A gel, in particular a hydrogel, which is a polymerization product of the (meth) acryloyl-modified silk protein according to claim 5.
10. Use of a gel according to claim 9 for the preparation of a tissue repair material, in particular, the tissue is cartilage tissue.
CN202211040714.6A 2022-08-29 2022-08-29 (methyl) acryloyl modified silk protein and preparation method and application thereof Pending CN115260300A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116041728A (en) * 2023-01-16 2023-05-02 西湖大学 Catechol modified silk protein and preparation method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116041728A (en) * 2023-01-16 2023-05-02 西湖大学 Catechol modified silk protein and preparation method and application thereof

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