CN114618010A - Multifunctional keratin-based hydrogel and preparation method thereof - Google Patents
Multifunctional keratin-based hydrogel and preparation method thereof Download PDFInfo
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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- 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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
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- 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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
Abstract
The invention relates to an injectable, self-repairing and self-adaptive keratin-based hydrogel and a preparation method thereof, belonging to the field of biomass materials. According to the invention, keratin is prepared by a reduction method, then keratin is used as a base material, and a keratin solution is treated under an alkaline condition of pH 10-13, so that the keratin-based hydrogel with dynamic covalent crosslinking is finally obtained. The keratin hydrogel disclosed by the invention is simple in preparation method and short in gelling time, has the functions of injectability, self-repairing and self-adaption, and has wide application value in the aspects of biomedical materials, tissue engineering, drug delivery and the like.
Description
Technical Field
The invention belongs to the field of biomass materials, and particularly relates to an injectable, self-repairing and self-adaptive keratin-based hydrogel and a preparation method thereof.
Background
With the development of biomaterials, intelligent wound healing materials have become one of the materials of interest in the biomedical field. Compared with traditional Materials, smart Materials can adapt to different requirements and environmental changes [ Lei Z Y, Wang, Q K, Sun S T, et al. a bipolar minor hydrogel as a self-reliable, mechanical adaptive skin for high throughput sensing. advanced Materials,2017,29(22): 1700321.1-6; zhang L Z, Liu Z H, Wu X L, et al.a high adapted self-healing hydrogel with unprecedented mechanical properties, advanced Materials,2019,31(23):1901402.1-8], Yongsan Li et al [ Li Y, Wang X, Fu Y, et al.self-adapting hydrogel into self-healing. acs Applied Materials & Interfaces,2018,10: 26046. self-healing hydrogel ] shows the self-adaptation properties of chitosan-based self-healing hydrogels, the unique mobility of which comes from its dynamic schiff base network, during liver laceration, this self-adapting hydrogel shows a great promising potential over the conventional drug-delivery hydrogels, which has a great medical advantage as a new material for application. Although hydrogels with properties intermediate between solid and liquid properties have the potential to be used as smart dressings that can help alleviate pain caused by overheating of a patient's wound surface, most conventional hydrogel dressings still require preparation and shaping prior to use [ Zhao W, Sharon G.A self-healing hydrogel as an inorganic absorbent carrier for cellular pharmaceuticals, biomaterials,2018,185: 86-96; li L, Wang N, Xun J, et al.Biogradable and injectable in situ cross-linking reagents for porous additive prediction. biomaterials,2014,35(12) 3903-; khan M, Koivisto J, Hukka T, et al, composite hydrogels using biological additive approach with structure relationship and self-healing ability as future Applied Materials & Interfaces,2018,10(14):11950 and 11960 ]. Therefore, the preparation of injectable, self-adaptive hydrogels with rapid self-healing capacity is very important for the development of advanced wound repair materials that can be used to treat irregular wounds.
Disclosure of Invention
It has long been known how to extract keratin from epidermal structures such as animal hair, nails or feathers, and to apply it to the textile and cosmetic fields. Keratin can significantly promote wound healing because of its many key features required for wound dressings, such as the ability to form a gel upon absorption of wound exudate, good water absorption, optimal water vapor transmission rate, non-toxicity and biodegradability, promotion of hemostasis, promotion of fibroblast proliferation and migration, and collagen production, among others. Meanwhile, keratin-based photosensitive bio-inks can be printed in three dimensions (3D) to form complex scaffolds for the treatment of topical skin burns, experiments have shown that the printed hydrogels have improved in vivo healing parameters on a porcine thermal burn model [ Navarro J, Clohessy RM, Holder RC. in vivo evaluation of three-dimensional printed, key-based hydrogels in a porous thermal burn model. tissue Engineering Part A, 202026 (5-6):265-278 ]. Therefore, the invention selects keratin as a raw material to prepare the injectable, self-repairing and self-adaptive hydrogel.
However, the traditional keratin hydrogel has single function and does not have the functions of shear thinning, self-repairing and the like. Part of the disulfide bonds of keratin can be reduced to free thiol groups, and the resulting keratin has a larger average molecular weight. The disulfide bonds and thiol groups contained in the keratin subjected to reduction treatment can also be used for constructing a disulfide bond-thiol exchange chemical reaction in which the thiol groups are oxidized to form disulfide bonds; however, the reaction proceeds very slowly under physiological conditions (pH 7.4) because it involves deprotonation of the thiol group and thiolate ion (RS)-) Formation of thiolate ion (RS)-) And reacting with oxygen molecule to generate reactive free radical substance [ Daniel Bermejo-Velasco D, Azemar A, Oommen O P, et al. modulated thio pKa protein formulations at physiological pH ] An elastomer strain to design strain crosslinked hydrophilic acids hydrogels, biomacromolecules,2019,20(3): 1412. 1420 ]. Basic conditions (pH) due to the pKa of thiols of about 8-10>8.5) the reaction can be driven forward. Another strategy for disulfide bond formation is a disulfide exchange reaction, which still involves deprotonation of the sulfhydryl group, and the resulting product continues to undergo nucleophilic substitution reactions with other disulfide bonds having labile sulfhydryl groups. The dynamic covalent crosslinking hydrogel not only has unique physicochemical properties capable of simulating dynamic extracellular matrix, but also has the functions of shear thinning, self-repairing and even self-adapting and the like. The reversibility of the hydrogel can be achieved by: reversible physical interactions (e.g., hydrogen bonding interactions, hydrophobic self-assembly, and host-guest interactions), or the establishment of dynamic covalent bonds, including imine, borate, disulfide, and Diels-Alder reactions [ Teng L, Chen Y, Jia Y G, et al.Supramolecular and dynamic covalent hydrogel scaffolds:from gelation chemistry to enhanced cell retention and cartilage regeneration[J]J. mater. chem.b,2019,7(43) ]. Therefore, the invention develops the hydrogel with dynamic covalent crosslinking by using the sulfydryl-disulfide bond exchange (the breaking and reforming of reversible bonds) of keratin through two alkali treatments, so that the hydrogel has the functions of shear thinning, self-repairing, even self-adapting (automatically adapting to continuously changing environment) and the like.
The purpose of the application is to provide a preparation method of the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
It is another object of the present invention to provide a keratin-based hydrogel that is injectable, self-repairing and self-adaptive.
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation, which is provided by the invention, comprises two alkali treatment steps:
(1) treating common biological materials serving as raw materials by using an alkali solution to prepare keratin;
(2) preparing hydrogel with injectable, self-repairing and self-adapting functions by taking the keratin prepared in the step (1) as a raw material through secondary alkali treatment;
the common biomaterial is any one selected from among human hair, wool, nails, horns, and bird feathers, which are subjected to degreasing and cleaning processes.
Preferably, the step (1) comprises the following operation procedures: weighing 1 part by weight of raw materials, and shearing the raw materials to about 1 cm; placing the raw material in an alkali solution with the pH value of 10-13, and stirring at a low speed for 4-12 hours at the temperature of 30-55 ℃; filtering or centrifuging to obtain keratin mixed solution, dialyzing the keratin mixed solution for 48 hours by a dialysis device with the molecular weight cutoff of 3000Da, and freeze-drying to obtain the keratin.
Preferably, the step (2) comprises the following operation procedures: dissolving the keratin in deionized water at a mass concentration of 5-30%; adjusting the pH value of the keratin solution to 9-11 by adopting an alkaline reagent, and then treating for 0.2-3.0 hours at 20-40 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Preferably, the alkali solution in the step (1) contains 0.01 to 0.5mol/L of a reducing agent, and the reducing agent is any one of L-cysteine, sodium sulfide, thioglycolic acid, mercaptoethanol or sodium bisulfite.
Preferably, the alkaline solution in the step (1) further contains 0.02 to 0.5mol/L sodium dodecyl sulfate and 0.1 to 2.0mol/L urea.
Preferably, the base is any one of sodium hydroxide, potassium hydroxide and sodium bicarbonate.
Drawings
FIG. 1 is a schematic representation of the thiol-disulfide exchange reaction of keratin under more basic conditions;
FIG. 2 is a view showing the flow conditions of the samples in the example of the present invention and the comparative example under the same inclination angle;
FIG. 3 is a graph of the syringeability and self-healing properties of hydrogels according to embodiments of the present invention;
FIG. 4 is a graph of the reciprocal healing properties between different hydrogels in an example of the present invention;
FIG. 5 is a graph of the dynamic motion of a hydrogel of the invention driven by gravity over time;
FIG. 6 shows the growth of L929 cells cultured with the hydrogel leaching solution of the present invention for 24 hours;
figure 7 is a graph of the adhesion of the hydrogel of example 3 to porcine skin tissue before and after distortion.
Detailed Description
The invention is further illustrated by the following examples. It should be noted that the following examples are not to be construed as limiting the scope of the present invention, and that the skilled person would be able to make insubstantial modifications and adaptations of the invention in light of the above teachings.
Example 1
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.01mol/L L-cysteine solution is prepared, and the pH value of the solution is adjusted to 10 by using sodium hydroxide solution. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted in an oven at 55 ℃ for 4 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 50mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 9 by using a sodium hydroxide solution, and standing for 3 hours at 40 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 2
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.5mol/L thioglycolic acid solution is prepared, and the pH value of the solution is adjusted to 11 by using sodium hydroxide. Weighing 2.5g clean ox horn, crushing to 1cm, placing in the above solution, and extracting at 45 deg.C for 5 hr. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 100mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 9.5 by using a sodium hydroxide solution, and standing for 2 hours at 20 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 3
The preparation method of the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions comprises the following steps:
(1) 50mL of 0.1mol/L sodium sulfide solution is prepared, and the pH value of the solution is adjusted to 12 by using sodium hydroxide. 2.5g of defatted feathers were weighed, cut to 1cm and placed in the above solution, and extracted at 40 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 150mg of keratin powder, dissolving the keratin powder in 1mL of deionized water, adjusting the pH value of the solution to 10 by using a sodium hydroxide solution, and standing the solution for 1 hour at 30 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 4
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.2mol/L mercaptoethanol solution is prepared, and the pH value of the solution is adjusted to 13 by using sodium hydroxide. 2.5g of defatted nails were weighed, cut to 1cm and placed in the above solution, and extracted at 35 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing keratin powder 200mg, dissolving in 1mL deionized water, adjusting the pH value of the solution to 10.5 by using sodium hydroxide solution, and standing for 0.5 hour at 25 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 5
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.05mol/L sodium bisulfite solution is prepared, and the pH value of the solution is adjusted to 10.5 by using sodium hydroxide. 2.5g of defatted nails were weighed, cut to 1cm and placed in the above solution, and extracted at 30 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with molecular weight cutoff of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 300mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 11 by using a sodium hydroxide solution, and standing for 0.2 hour at 35 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 6
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.1mol/L L-cysteine solution containing 0.01mol/L sodium lauryl sulfate was prepared, and the pH of the solution was adjusted to 13 using potassium hydroxide. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 30 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 300mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 11 by using a potassium hydroxide solution, and standing for 0.2 hour at 20 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 7
The preparation method of the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions comprises the following steps:
(1) 50mL of 0.2mol/L thioglycolic acid solution containing 0.1mol/L sodium lauryl sulfate was prepared, and the pH of the solution was adjusted to 12 using potassium hydroxide. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 35 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 250mg of keratin powder, dissolving the keratin powder in 1mL of deionized water, adjusting the pH value of the solution to 10.5 by using a potassium hydroxide solution, and standing the solution at 25 ℃ for 0.5 hour to obtain the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation.
Example 8
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.4mol/L sodium sulfide solution containing 0.2mol/L sodium dodecyl sulfate was prepared, and the pH of the solution was adjusted to 11 with potassium hydroxide. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 40 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing keratin powder 200mg, dissolving in 1mL deionized water, adjusting the pH value of the solution to 10 by using a potassium hydroxide solution, and standing for 1 hour at 30 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 9
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.3mol/L thioglycolic acid solution containing 0.3mol/L sodium dodecyl sulfate was prepared, and the pH of the solution was adjusted to 10.5 with potassium hydroxide. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 45 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 100mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 9.5 by using a potassium hydroxide solution, and standing for 2 hours at 35 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 10
The preparation method of the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions comprises the following steps:
(1) 50mL of 0.5mol/L sodium bisulfite solution containing 0.5mol/L sodium lauryl sulfate was prepared, and the pH of the solution was adjusted to 10 with potassium hydroxide. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 55 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 50mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 9 by using a potassium hydroxide solution, and standing for 3 hours at 40 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 11
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.5mol/L L-cysteine solution containing 0.1mol/L urea was prepared, and the pH of the solution was adjusted to 12 using sodium bicarbonate. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution and extracted at 40 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 250mg of keratin powder, dissolving the keratin powder in 1mL of deionized water, adjusting the pH value of the solution to 10 by using sodium bicarbonate solution, and standing the solution for 2 hours at the temperature of 30 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 12
The preparation method of the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions comprises the following steps:
(1) 50mL of 0.4mol/L thioglycolic acid solution containing 0.2mol/L urea is prepared, and the pH value of the solution is adjusted to 10 by using sodium bicarbonate. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 30 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 300mg of keratin powder, dissolving the keratin powder in 1mL of deionized water, adjusting the pH value of the solution to 9.5 by using a sodium bicarbonate solution, and standing the solution for 1 hour at 35 ℃ to obtain the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation.
Example 13
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.01mol/L sodium sulfide solution containing 0.5mol/L urea is prepared, and the pH value of the solution is adjusted to 11 by using sodium bicarbonate. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 55 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 50mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 11 by using a sodium bicarbonate solution, and standing for 3 hours at 25 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 14
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.3mol/L mercaptoethanol solution containing 1mol/L urea is prepared, and the pH value of the solution is adjusted to 13 by using sodium bicarbonate. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 45 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with molecular weight cutoff of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing keratin powder 200mg, dissolving in 1mL deionized water, adjusting the pH value of the solution to 9.5 by using sodium bicarbonate solution, and standing for 0.2 hour at 40 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Example 15
The preparation method of the keratin-based hydrogel with the functions of injectability, self-repair and self-adaptation comprises the following steps:
(1) 50mL of 0.2mol/L sodium bisulfite solution containing 2mol/L urea was prepared, and the pH of the solution was adjusted to 11.5 with sodium bicarbonate. 2.5g of defatted wool was weighed, cut to 1cm and placed in the above solution, and extracted at 35 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Weighing 100mg of keratin powder, dissolving in 1mL of deionized water, adjusting the pH value of the solution to 10.5 by using a sodium bicarbonate solution, and standing for 0.5 hour at 20 ℃ to obtain the keratin-based hydrogel with injectable, self-repairing and self-adaptive functions.
Comparative example 1
(1) 50mL of 0.01mol/L L-cysteine solution was prepared, and the pH of the solution was adjusted to 10 using sodium hydroxide solution. 2.5g of degreased wool is weighed, cut to 1cm and placed in the above solution, and then placed in an oven at 55 ℃ for extraction for 4 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Keratin powder (50 mg) was dissolved in 1mL of deionized water and allowed to stand at 40 ℃ for 3 hours to give comparative sample 1.
Comparative example 2
(1) 50mL of 0.5mol/L thioglycolic acid solution is prepared, and the pH value of the solution is adjusted to 11 by using sodium hydroxide. 2.5g of clean ox horn was weighed, crushed to 1cm and placed in the above solution and extracted at 45 ℃ for 5 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) 100mg of keratin powder was weighed out and dissolved in 1mL of deionized water, and allowed to stand at 20 ℃ for 2 hours to obtain comparative sample 2.
Comparative example 3
(1) 50mL of 0.1mol/L sodium sulfide solution is prepared, and the pH value of the solution is adjusted to 12 by using sodium hydroxide. 2.5g of defatted feathers were weighed, cut to 1cm and placed in the above solution, and extracted at 40 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) 100mg of keratin powder was weighed out and dissolved in 1mL of deionized water, and allowed to stand at 30 ℃ for 1 hour to obtain comparative sample 3.
Comparative example 4
(1) 50mL of 0.2mol/L mercaptoethanol solution is prepared, and the pH value of the solution is adjusted to 13 by using sodium hydroxide. 2.5g of defatted nails were weighed, cut to 1cm and placed in the above solution, and extracted at 35 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) Keratin powder 200mg was weighed out and dissolved in 1mL of deionized water, the pH of the solution was adjusted to 10.5 using sodium hydroxide solution, and the solution was allowed to stand at 25 ℃ for 0.5 hour to obtain comparative sample 4.
Comparative example 5
(1) 50mL of 0.05mol/L sodium bisulfite solution is prepared, and the pH value of the solution is adjusted to 10.5 by using sodium hydroxide. 2.5g of defatted nails were weighed, cut to 1cm and placed in the above solution, and extracted at 30 ℃ for 15 hours. Immediately filtering, putting the filtrate into a dialysis bag with cut-off molecular weight of 3000Da for dialysis, and freeze-drying after 2 days of dialysis to obtain keratin powder.
(2) 300mg of keratin powder was weighed out and dissolved in 1mL of deionized water, and allowed to stand at 35 ℃ for 0.2 hour to obtain comparative sample 5.
And (3) performance testing:
free flow: 0.5mL of the hydrogel obtained in example and comparative example was taken under the experimental conditions and put into a transparent glass sample bottle, and the state of the sample was observed with tilting the same angle. As shown in fig. 2, the sample obtained by the two-stage alkali treatment was in a more viscous gel state, whereas the sample obtained in the comparative example was in an arbitrarily flowing solution state.
Injection experiment: the hydrogel in the examples was loaded into a 5mL syringe and the hydrogel was manually extruded onto the die. The injectability of the hydrogel was evaluated by dropping the hydrogel onto a mold. The hydrogel was smoothly passed through a 200 μm-sized needle by pressing the hydrogel with hand while pressing and the hydrogel extruded onto the mold had a fixed shape (see fig. 3), the hydrogel had a rougher surface at the initial stage of injection, but the rough surface then became very smooth, indicating that the hydrogel obtained had good injectability and self-healing properties.
Self-healing properties: referring to fig. 4, the hydrogel was injected into the disc by a syringe, the original state of the hydrogel was observed and recorded, and photographs were taken at different time intervals to record the appearance thereof. In addition, by placing the hydrogels of example 2 and example 3 in the same disc (at a certain distance apart), respectively, the original states of the two hydrogels were observed and recorded, and photographs were taken at different time intervals for evaluating the self-healing properties of the hydrogels. Figure 4 shows that the hydrogels of example 2 and example 3 moved over time and eventually the two hydrogels rejoined together, further demonstrating the self-healing properties of the keratin-based hydrogels.
Self-adaptability: referring to fig. 5, two layers of spherical glass beads were placed in the sample vial to simulate different morphologies of the internal tissues of animals. The prepared hydrogel was added to the sample vial and photographs were taken at different time intervals to record the morphological changes of the hydrogel in the sample vial to verify the adaptivity of the hydrogel. The hydrogel obtained in the examples gradually covered the beads of the bottom layer due to gravity and surface tension, indicating that the hydrogel obtained in the examples had good adaptivity. Therefore, the self-adaptive hydrogel can be better attached to the wound by changing the shape, and the aims of better treatment effect and drug delivery are fulfilled.
Cytotoxicity: referring to FIG. 6, the indirect contact-leach liquor method was used to evaluate the cytotoxicity of the hydrogels of examples 1, 2 and 3, and the test was performed using mouse fibroblasts (L929) and the test standard was ISO 10993. At 24h, the cell morphology of the control group and the sample group is mostly spindle-shaped or star-shaped structure, the growth state is similar, and the cell growth state is good. The observation result shows that the hydrogel leaching liquor has no obvious cytotoxicity.
Skin adhesiveness: referring to fig. 7, a twist experiment was performed to study the adhesion of the hydrogel to the pigskin. Briefly, a hydrogel was formed in situ on the surface of the pigskin by syringe. Then, a torsional stress was applied to the pigskin to simulate the adhesive flexibility of the test hydrogel on the skin of the animal. As can be seen in FIG. 7, the hydrogel adhered well to the skin under the action of external force, indicating the potential ability of the hydrogel to serve as a wound dressing.
In conclusion, the keratin-based hydrogel obtained by the two alkali treatment steps obtains dynamic covalent crosslinking through a sulfhydryl-disulfide bond exchange reaction, so that the obtained keratin-based hydrogel has excellent injectable, self-repairing and self-adaptive functions and better biocompatibility. The preparation method of the keratin hydrogel provided by the invention is simple and has short gelling time, can effectively overcome the technical obstacles of the hydrogel for biomedical materials, tissue engineering and drug delivery, and endows the keratin hydrogel with wide application value.
Claims (7)
1. A method for preparing an injectable, self-repairing and self-adapting keratin-based hydrogel, characterized in that it comprises two alkali treatment steps:
(1) treating common biological materials serving as raw materials by using an alkali solution to prepare keratin;
(2) preparing hydrogel with injectable, self-repairing and self-adapting functions by taking the keratin prepared in the step (1) as a raw material and performing second alkali treatment;
the common biomaterial is any one selected from among human hair, wool, nails, horns, and bird feathers, which are subjected to degreasing and cleaning processes.
2. The method for preparing the injectable, self-repairing and self-adaptive keratin-based hydrogel according to claim 1, wherein the step (1) comprises the following procedures: weighing 1 part by weight of raw materials, and shearing the raw materials to about 1 cm; placing the raw material in an alkali solution with the pH value of 10-13, and stirring at a low speed for 4-12 hours at the temperature of 30-55 ℃; filtering or centrifuging to obtain keratin mixed solution, dialyzing the keratin mixed solution for 48 hours by a dialysis device with the molecular weight cutoff of 3000Da, and freeze-drying to obtain the keratin.
3. The process for the preparation of keratin-based hydrogels with injectable, self-repairing and adaptive functions as claimed in claim 2, wherein the step (2) comprises the following procedures: dissolving the keratin in deionized water at a mass concentration of 5-30%; adjusting the pH value of the keratin solution to 9-11 by adopting an alkaline reagent, and then treating the keratin solution at the temperature of 20-40 ℃ for 0.2-3.0 hours to obtain the keratin-based hydrogel with injectable, self-repairing and self-adapting functions.
4. The method for preparing the keratin-based hydrogel with injectable, self-repairing and self-adapting functions according to claim 3, wherein the alkaline solution in the step (1) contains 0.01 to 0.5mol/L of a reducing agent, and the reducing agent is any one of L-cysteine, sodium sulfide, thioglycolic acid, mercaptoethanol or sodium bisulfite.
5. The method for preparing the injectable, self-repairing and self-adaptive keratin-based hydrogel according to claim 4, wherein the alkaline solution of step (1) further comprises 0.02 to 0.5mol/L sodium dodecyl sulfate and 0.1 to 2.0mol/L urea.
6. The method for preparing the injectable, self-repairing and self-adaptive keratin-based hydrogel according to any one of claims 1 to 5, wherein the base is any one of sodium hydroxide, potassium hydroxide and sodium bicarbonate.
7. A keratin-based hydrogel that is injectable, self-repairing and self-adapting, wherein the keratin-based hydrogel is prepared by the method of claims 1-6.
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| CN116041727A (en) * | 2023-01-30 | 2023-05-02 | 国科温州研究院(温州生物材料与工程研究所) | Synthesis preparation method and application of carbon-carbon double bond modified keratin injectable hydrogel |
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