CN113069586A - Preparation method of double-layer wound dressing containing wax-coated silk fibroin fabric layer and regenerated silk fibroin silver-loaded medicine layer - Google Patents
Preparation method of double-layer wound dressing containing wax-coated silk fibroin fabric layer and regenerated silk fibroin silver-loaded medicine layer Download PDFInfo
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
- A61L2300/104—Silver, e.g. silver sulfadiazine
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
Abstract
The invention relates to the field of biomedical materials, and discloses a preparation method of a double-layer wound dressing containing a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded drug layer, wherein carnauba wax is dissolved in a morpholine solution, and a silk fibroin fabric is soaked in the wax solution to obtain the wax-coated silk fibroin fabric; and uniformly mixing the silver nitrate aqueous solution and the silk fibroin aqueous solution, and exposing the mixture to ultraviolet light to form the silk fibroin-nano silver solution. And finally, arranging the solution on the wax-coated silk fibroin fabric, freeze-drying, sublimating ice into water vapor, and discharging to obtain the double-layer wound dressing containing the wax-coated silk fibroin fabric layer and the regenerated silk fibroin silver-loaded medicine layer.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a preparation method of a double-layer wound dressing containing a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer.
Background
Silk Fibroin (SF) is a natural fiber protein derived from silk, the main components of which are glycine and alanine, specific nonpolar amino acid sequences. Silk fibroin is widely used in biomedical applications due to its unique physical, mechanical and biological properties, including strength, toughness, elasticity, lightweight, biocompatibility, biodegradability, minimal inflammatory response, wound healing promoting ability and simple chemical modification to meet the application goals. In recent years, it has become a new advanced tissue engineering biomaterial. Since silk fibroin has the ability to promote the adhesion and proliferation of various cells (including keratinocytes and fibroblasts), it has become a potential biomaterial and can be used to manufacture wound dressings of various formulations and styles.
Nano silver is a metal nanoparticle with broad-spectrum antibacterial property and high efficiency, and is often used for treating wound infection due to its advantages of easy preparation and low cost. The nano silver has unique small size effect and oxidation effect, and can easily enter pathogens to play a role in sterilization even at low concentration. The antibacterial principle of nano silver can be briefly summarized as follows: the membrane structure is damaged by the damage of the biological membrane caused by the adhesion of microorganisms, and then the microorganisms enter the interior of the microorganisms to generate active oxygen and free radicals by utilizing the oxidability of the microorganisms to inactivate biological macromolecules such as protein, nucleic acid and the like, so that pathogenic microorganisms can be directly killed, and the microorganisms can be prevented from continuously proliferating. The characteristics of the nano silver determine that the nano silver has efficient killing and inhibiting effects on most pathogenic microorganisms, does not generate drug-resistant strains, and can be used as an antibacterial agent to be applied to wound dressings.
Disclosure of Invention
The invention provides a preparation method of a double-layer wound dressing containing a wax-coated fibroin fabric layer and a regenerated fibroin silver-loaded medicine layer, and the preparation method comprises the steps of incorporating a fibroin non-adhesive layer (the wax-coated fibroin fabric layer) into the wound dressing, so that the wound dressing has non-stickiness, is beneficial to preventing the dehydration of a wound surface and easy to remove, and does not worry about secondary wound caused by residue on the surface of the wound after use; meanwhile, the drug-loaded layer combined with the non-adhesive layer is also a substrate formed by silk fibroin, has good biocompatibility and excellent mechanical property, and the loaded drug silver can resist inflammation and bacteria, so that the wound healing is accelerated under the double guarantee. The double-layer material has good complementary synergistic effect, and has good treatment effect on wound healing and large-area wound infection.
The specific technical scheme of the invention is as follows:
a preparation method of a double-layer wound dressing containing a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer comprises the following steps:
step 1: preparing a wax-coated silk fibroin fabric: dissolving carnauba wax in morpholine solution, stretching silk fibroin fabric at room temperature, soaking in the obtained wax solution for 10-30min, and air-drying overnight to obtain wax-coated silk fibroin fabric.
Wax is a naturally edible coating material, carnauba wax, recovered from the bottom of the leaves of the carnauba tree. It is mainly used to retard water loss and increase gloss. The fibroin non-adhesive layer (the wax-coated fibroin fabric layer) is incorporated into the wound dressing, so that the wound dressing has non-stickiness, is favorable for preventing the dehydration of a wound surface, is easy to remove, and does not need to worry about secondary wound caused by residue on the surface of the wound after being used.
Step 2: preparation of silk fibroin aqueous solution: taking the silkworm cocoons, shearing the silkworm cocoons into sheets, putting the sheet-shaped silkworm cocoons into a container, adding water, washing by ultrasonic oscillation, taking out the silkworm cocoons and washing the silkworm cocoons after the washing is finished, and repeating the operation for a plurality of times; drying the silkworm cocoon; preparing a sodium carbonate solution, heating to boil, adding dried silkworm cocoons, and degumming under a stirring condition; after degumming, taking out the silk fibroin, washing with water to remove the attached sericin, and completely drying after washing; preparing a lithium bromide solution, mixing the lithium bromide solution with dried silk fibroin, and heating for dissolving; pouring the obtained silk fibroin solution into a dialysis bag for dialysis for 40-50h to remove lithium bromide, continuously stirring the solution in the dialysis bag, and replacing dialysate; and (4) after dialysis, freezing and centrifuging the obtained crude silk fibroin aqueous solution, removing precipitates, taking supernatant to obtain refined silk fibroin aqueous solution, and refrigerating for later use.
And step 3: preparing a silk fibroin-nano silver mixed solution: preparing silver nitrate mother liquor, mixing the silver nitrate mother liquor with the refined silk fibroin aqueous solution, uniformly stirring, adding water for dilution, and irradiating the obtained mixed solution under ultraviolet light to generate nano silver particles to obtain the silk fibroin-nano silver mixed solution.
The mixed solution generated in the step is a uniform dispersion system, silk fibroin is used as a dispersing agent and a stabilizing agent, and silver ions are prepared into nano silver particles through ultraviolet irradiation so as to facilitate the treatment of the next step, such as reverse molding, qualitative treatment, freeze drying and the like.
And 4, step 4: preparing a double-layer wound dressing: and (3) casting the silk fibroin-nano silver mixed solution obtained in the step (3) on a mould on a stretched wax-coated silk fibroin fabric, placing the mould into a mould for pre-freezing at the temperature of minus 25 to minus 15 ℃, freezing at the temperature of minus 85 to minus 75 ℃ overnight after 0.5 to 1.5 hours, immediately freezing and drying after freezing and molding to obtain a double-layer wound dressing, placing the double-layer wound dressing into a methanol solution for treatment, washing the residual methanol with water, and then drying and storing at the temperature of 55 to 65 ℃.
By utilizing the freeze-drying technology, a double-layer material structure can be formed under the environment-friendly and pollution-free condition, the degradation rate of the material can be greatly delayed by using methanol solution for treatment, so that the porous structure of the material is richer, and the absorption of wound exudate, accommodation of cells and tissue adsorption growth are facilitated.
Firstly, dissolving carnauba wax in a morpholine solution, stretching a silk fibroin fabric at room temperature, soaking the silk fibroin fabric in the wax solution, and airing to obtain a wax-coated silk fibroin fabric; then, fully and uniformly mixing the silver nitrate aqueous solution and the silk fibroin aqueous solution to obtain a uniform system, adsorbing silver ions on silk fibroin molecules or dissociating the silk fibroin molecules in the mixed solution, exposing the silk fibroin molecules to ultraviolet light, and reducing and stacking the silver ions to form nano silver particles when the silk fibroin serves as a stabilizer, a dispersing agent and a protective agent; at the moment, most of nano-silver particles are deposited and adsorbed on silk fibroin molecules to form a silk fibroin-nano silver uniform dispersion solution system. And finally, casting the mixed solution on a wax-coated silk fibroin fabric stretched on a polytetrafluoroethylene mold, fully freezing at ultralow temperature, forming a porous framework structure by silk fibroin molecules in the system, and solidifying the solvent water into solid ice to support the stability of the framework structure. And then freeze-drying, wherein the ice sublimates the water vapor to be discharged out of the system in the freeze-drying process, and the wound dressing with the double-layer structure can be obtained.
The drug-loaded layer combined with the non-adhesive layer is also a substrate formed by silk fibroin, has good biocompatibility and excellent mechanical property, and the loaded drug silver can resist inflammation and bacteria, so that the wound healing is accelerated under the double guarantee. The double-layer material has good complementary synergistic effect, and has good treatment effect on wound healing and large-area wound infection.
Preferably, in step 1, the concentration of the morpholine solution is 0.025-0.1% w/v.
Preferably, in the step 2, 13-17 silkworm cocoons are taken, each silkworm cocoon is cut into 4-8 pieces, and 200ml of water is added; the ultrasonic oscillation power is 80-120Hz, and the washing is carried out for 3-7 min.
Preferably, in the step 2, the drying temperature of the silkworm cocoons is 55-65 ℃; the concentration of the sodium carbonate solution is 0.01-0.03M; degumming for 15-60 min; the concentration of the lithium bromide solution is 9.0-9.5M, the heating and dissolving temperature is 55-65 ℃, and the dissolving time is 4-6 h.
Preferably, in step 2, the length of the dialysis bag is 13-17 cm; the freezing and centrifuging conditions are 8000-10000rpm, 1-5 ℃, 15-25min, and the steps are repeated twice.
Preferably, the dialysis bag is pretreated before use: preparing an EDTA-disodium solution with a concentration of 1mM, and adjusting the pH =7.8-8.2 with a 0.1M sodium hydroxide solution; preparing 2% w/v sodium bicarbonate solution; uniformly mixing the two solutions according to the volume ratio of 1:1, heating to boil, putting the dialysis bag into the solution, boiling for 5-15min, taking out, and then thoroughly cleaning with ultrapure water; after the cleaning, the mixture is put into the EDTA-disodium solution to be boiled for 5-15min, taken out and put into ultrapure water to be soaked for standby.
Preferably, in the step 3, the concentration of the silk fibroin after being diluted by water is 3-6% g/ml; the concentration of silver nitrate is 0.02-0.08% g/ml; the wavelength of ultraviolet light is 365nm, and the irradiation time is 4-6 h.
Preferably, in step 4, the concentration of the methanol solution is 85-95% v/v, and the treatment time is 20-40 min.
The methanol solution treatment induces the generation of additional beta-sheet in the silk fibroin, and can improve the mechanical property of the hydrogel.
Compared with the prior art, the invention has the following technical effects: the silk fibroin non-adhesive layer (wax-coated silk fibroin fabric layer) is incorporated into the wound dressing, so that the wound dressing has non-stickiness, and secondary wound caused by residue on the surface of a wound is avoided after the wound dressing is used; meanwhile, the drug-loaded layer combined with the non-adhesive layer is also a substrate formed by silk fibroin, has good biocompatibility and excellent mechanical property, and the loaded drug silver can resist inflammation and bacteria, so that the wound healing is accelerated under the double guarantee. The double-layer material has good complementary synergistic effect, and has good treatment effect on wound healing and large-area wound infection.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
1) Preparing a wax-coated silk fibroin fabric: carnauba wax was dissolved in 0.025% w/v morpholine solution. Stretching the silk fibroin fabric at room temperature, soaking in a wax solution for 20min, and then airing overnight to obtain the wax-coated silk fibroin fabric.
2) Preparation of silk fibroin aqueous solution: taking out 13 silkworm cocoons, cutting each silkworm cocoon into 4 pieces, placing into a beaker, adding 200ml of ultrapure water into the beaker, placing into an ultrasonic cleaning instrument, performing vibration washing for 5min at 100Hz, taking out the silkworm cocoons after the vibration washing is finished, and washing with ultrapure water to remove insoluble substances on the silkworm cocoons. After repeating the above operation 3 times, the mixture was dried in a constant temperature oven at 60 ℃ and weighed to 4g after the drying. Preparing 0.02M sodium carbonate solution with 2L large-size beaker, heating to boil, adding dried Bombyx Bombycis, degumming for 15min while stirring with glass rod to fully degum. After degumming, taking out the silk fibroin, washing with ultrapure water to remove the attached sericin, and after washing, putting the silk fibroin into a 60 ℃ constant temperature drying oven for thorough drying, wherein the weight of the silk fibroin is 3.0 g. Preparing 13ml of 9.3M lithium bromide solution, mixing with dry silk fibroin, and dissolving in a 60 ℃ constant-temperature water bath kettle for 4 h. Cutting a dialysis bag 13cm long, pouring the dissolved silk fibroin solution into the bag, placing the bag in 2L of ultrapure water, performing dialysis for 48h to remove lithium bromide, continuously stirring with a magnetic stirrer, and replacing dialysate (ultrapure water) at 1, 4, 8, 12, 24, 36, and 40 h. After dialysis was completed, 35ml of crude silk fibroin aqueous solution was obtained, which was subjected to refrigerated centrifugation at 9000rpm at 4 ℃ for 20min, and repeated twice. Removing precipitate, collecting supernatant to obtain 30ml refined silk fibroin aqueous solution, and storing in refrigerator at 4 deg.C for use.
3) Preparing a silk fibroin-nano silver mixed solution: taking the refined silk fibroin aqueous solution prepared in the step 2), dissolving silver nitrate powder into ultrapure water to form silver nitrate mother liquor with a known concentration, mixing the silver nitrate mother liquor with the silk fibroin aqueous solution, and slightly stirring to fully and uniformly mix the silver nitrate mother liquor and the silk fibroin aqueous solution; ultra pure water was added for titration to give a final concentration of 3% for silk fibroin and 0.02% for silver nitrate. And (3) placing the mixed solution below an ultraviolet irradiation instrument for irradiation for 4 hours, and generating nano silver particles after the irradiation is finished.
4) Preparing a double-layer wound dressing of a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer: and (3) casting the mixed solution obtained in the step (3) on a mould on the stretched wax-coated silk fibroin fabric, placing the mould into a freezer at the temperature of-20 ℃ for pre-freezing, placing the mould into a freezer at the temperature of-80 ℃ for freezing overnight after one hour, and immediately placing the mould into a freeze drying box for freeze drying after freezing mould making and molding. After freeze-drying, obtaining a double-layer wound dressing of the wax-coated silk fibroin fabric layer and the regenerated silk fibroin silver-loaded medicine layer, and putting the double-layer wound dressing into a methanol solution with the concentration of 90% v/v for treatment for 30 min; after the excess methanol is washed by ultrapure water, the mixture is put into a constant-temperature drying oven at 60 ℃ for drying and preservation for later use.
The dressing obtained by the method has the tensile modulus of 200.7MPa and the elongation of 29.5 percent. After 12h of adhesion to partial and full layer of wound, the number of cell adhesion was 5X 104And 7X 104Taking the area of partial and whole layer of wound as 2.5cm2The dressing was attached to the wound and the wound area was reduced to 2.1 and 1.9cm on day 32Day 7, scaling to 1.6 and 1.3cm2On day 14, only 0.4 and 0.2cm of wound area remained2。
Example 2
1) Preparing a wax-coated silk fibroin fabric: carnauba wax was dissolved in 0.05% w/v morpholine solution. Stretching the silk fibroin fabric at room temperature, soaking in a wax solution for 20min, and then airing overnight to obtain the wax-coated silk fibroin fabric.
2) Preparation of silk fibroin aqueous solution: taking out 15 silkworm cocoons, cutting each silkworm cocoon into 6 pieces, placing into a beaker, adding 200ml of ultrapure water into the beaker, placing into an ultrasonic cleaning instrument, performing vibration washing for 5min at 100Hz, taking out the silkworm cocoons after the vibration washing is finished, and washing with ultrapure water to remove insoluble substances on the silkworm cocoons. After repeating the above operation 3 times, the mixture was dried in a constant temperature oven at 60 ℃ and the weight of the dried mixture was 5 g. Preparing 0.02M sodium carbonate solution with 2L large-size beaker, heating to boil, adding dried Bombyx Bombycis, degumming for 30min while stirring with glass rod to fully degum. After degumming, taking out the silk fibroin, washing with ultrapure water to remove the attached sericin, and after washing, putting the silk fibroin into a 60 ℃ constant temperature drying oven to be dried completely, wherein the weight of the silk fibroin is 3.5 g. Preparing 15ml of 9.3M lithium bromide solution, mixing with dry silk fibroin, and dissolving in a 60 ℃ constant-temperature water bath kettle for 5 hours. Cutting a dialysis bag with a length of 15cm, pouring the dissolved silk fibroin solution into the dialysis bag, placing the dialysis bag in 2L of ultrapure water for 48h to remove lithium bromide, continuously stirring the dialysis bag by a magnetic stirrer, and replacing the dialysate (ultrapure water) at 1, 4, 8, 12, 24, 36 and 40 h. After dialysis was completed, 40ml of crude silk fibroin aqueous solution was obtained, which was subjected to refrigerated centrifugation at 9000rpm at 4 ℃ for 20min, and repeated twice. Removing precipitate, collecting supernatant to obtain 35ml refined silk fibroin aqueous solution, and storing in refrigerator at 4 deg.C for use.
3) Preparing a silk fibroin-nano silver mixed solution: taking the refined silk fibroin aqueous solution prepared in the step 2), dissolving silver nitrate powder into ultrapure water to form silver nitrate mother liquor with a known concentration, mixing the silver nitrate mother liquor with the silk fibroin aqueous solution, and slightly stirring to fully and uniformly mix the silver nitrate mother liquor and the silk fibroin aqueous solution; ultra pure water was added for titration to give a final concentration of 5% for silk fibroin and 0.04% for silver nitrate. And (3) placing the mixed solution below an ultraviolet irradiation instrument for irradiation for 5 hours, and generating nano silver particles after the irradiation is finished.
4) Preparing a double-layer wound dressing of a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer: and (3) casting the mixed solution obtained in the step (3) on a mould on the stretched wax-coated silk fibroin fabric, placing the mould into a freezer at the temperature of-20 ℃ for pre-freezing, placing the mould into a freezer at the temperature of-80 ℃ for freezing overnight after one hour, and immediately placing the mould into a freeze drying box for freeze drying after freezing mould making and molding. After freeze-drying, obtaining a double-layer wound dressing of the wax-coated silk fibroin fabric layer and the regenerated silk fibroin silver-loaded medicine layer, and putting the double-layer wound dressing into a methanol solution with the concentration of 90% v/v for treatment for 30 min; after the excess methanol is washed by ultrapure water, the mixture is put into a constant-temperature drying oven at 60 ℃ for drying and preservation for later use.
The dressing obtained by the method has the tensile modulus of 245.2MPa and the elongation of 30.9%. After 12h of adhesion to partial and full layer of wound, the number of cell adhesion was 3X 104And 5X 104Taking the area of partial and whole layer of wound as 2.5cm2The dressing was attached to the wound and the wound area was reduced to 2.0 and 1.7cm on day 32Day 7, scaling to 1.5 and 1.0cm2On day 14, only 0.2 and 0.1cm of wound area remained2。
Example 3
1) Preparing a wax-coated silk fibroin fabric: carnauba wax was dissolved in 0.1% w/v morpholine solution. Stretching the silk fibroin fabric at room temperature, soaking in a wax solution for 20min, and then airing overnight to obtain the wax-coated silk fibroin fabric.
2) Preparation of silk fibroin aqueous solution: taking out 17 silkworm cocoons, cutting each silkworm cocoon into 8 pieces, placing the silkworm cocoons into a beaker, adding 200ml of ultrapure water into the beaker, placing the beaker in an ultrasonic cleaning instrument for shaking and washing for 5min at 100Hz, taking out the silkworm cocoons after the shaking and washing is finished, and washing with ultrapure water to remove insoluble substances on the silkworm cocoons. After repeating the above operation 3 times, the mixture was dried in a constant temperature oven at 60 ℃ and weighed to 6g after the drying. Preparing 0.02M sodium carbonate solution with 2L large-sized beaker, heating to boil, adding dried Bombyx Bombycis, and degumming for 60min while stirring with glass rod to fully degum. After degumming, taking out the silk fibroin, washing with ultrapure water to remove the attached sericin, and after washing, putting the silk fibroin into a 60 ℃ constant temperature drying oven for thorough drying, wherein the weight of the silk fibroin is weighed as 4 g. Preparing 16ml of 9.3M lithium bromide solution, mixing with dry silk fibroin, and dissolving in a 60 ℃ constant-temperature water bath kettle for 6 hours. A dialysis bag of 17cm in length was cut, the silk fibroin solution obtained by dissolution was poured into it, and placed in 2L of ultrapure water for dialysis for 48 hours to remove lithium bromide, during which time a magnetic stirrer was continuously stirred, and the dialysate (ultrapure water) was changed at 1 st, 4 th, 8 th, 12 th, 24 th, 36 th, 40 th hours. After dialysis was completed, 45ml of crude silk fibroin aqueous solution was obtained, which was subjected to refrigerated centrifugation at 9000rpm at 4 ℃ for 20min, and repeated twice. Removing precipitate, collecting supernatant to obtain 40ml refined silk fibroin aqueous solution, and storing in refrigerator at 4 deg.C for use.
3) Preparing a silk fibroin-nano silver mixed solution: taking the refined silk fibroin aqueous solution prepared in the step 2), dissolving silver nitrate powder into ultrapure water to form silver nitrate mother liquor with a known concentration, mixing the silver nitrate mother liquor with the silk fibroin aqueous solution, and slightly stirring to fully and uniformly mix the silver nitrate mother liquor and the silk fibroin aqueous solution; ultra-pure water was added for titration to give a final silk fibroin concentration of 6% and a final silver nitrate concentration of 0.08%. And (3) placing the mixed solution below an ultraviolet irradiation instrument for irradiation for 6 hours, and generating nano silver particles after the irradiation is finished.
4) Preparing a double-layer wound dressing of a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer: and (3) casting the mixed solution obtained in the step (3) on a mould on the stretched wax-coated silk fibroin fabric, placing the mould into a freezer at the temperature of-20 ℃ for pre-freezing, placing the mould into a freezer at the temperature of-80 ℃ for freezing overnight after one hour, and immediately placing the mould into a freeze drying box for freeze drying after freezing mould making and molding. After freeze-drying, obtaining a double-layer wound dressing of the wax-coated silk fibroin fabric layer and the regenerated silk fibroin silver-loaded medicine layer, and putting the double-layer wound dressing into a methanol solution with the concentration of 90% v/v for treatment for 30 min; after the excess methanol is washed by ultrapure water, the mixture is put into a constant-temperature drying oven at 60 ℃ for drying and preservation for later use.
The dressing obtained by the method has the tensile modulus of 278.2MPa and the elongation of 33.7%. After 12h of adhesion to partial and full layer of wound, the number of cell adhesion is 2X 104And 4X 104Taking the area of partial and whole layer of wound as 2.5cm2The dressing was attached to the wound and the wound area was reduced to 1.7 and 1.5cm on day 32Day 7, scaling to 1.2 and 0.8cm2The wound healed almost completely on day 14.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (8)
1. A preparation method of a double-layer wound dressing containing a wax-coated silk fibroin fabric layer and a regenerated silk fibroin silver-loaded medicine layer is characterized by comprising the following steps:
step 1: preparing a wax-coated silk fibroin fabric: dissolving carnauba wax in morpholine solution, stretching silk fibroin fabric at room temperature, soaking in the obtained wax solution for 10-30min, and air-drying overnight to obtain wax-coated silk fibroin fabric;
step 2: preparation of silk fibroin aqueous solution: taking the silkworm cocoons, shearing the silkworm cocoons into sheets, putting the sheet-shaped silkworm cocoons into a container, adding water, washing by ultrasonic oscillation, taking out the silkworm cocoons and washing the silkworm cocoons after the washing is finished, and repeating the operation for a plurality of times; drying the silkworm cocoon; preparing a sodium carbonate solution, heating to boil, adding dried silkworm cocoons, and degumming under a stirring condition; after degumming, taking out the silk fibroin, washing with water to remove the attached sericin, and completely drying after washing; preparing a lithium bromide solution, mixing the lithium bromide solution with dried silk fibroin, and heating for dissolving; pouring the obtained silk fibroin solution into a dialysis bag for dialysis for 40-50h to remove lithium bromide, continuously stirring the solution in the dialysis bag, and replacing dialysate; after dialysis, freezing and centrifuging the obtained crude silk fibroin aqueous solution, removing precipitates, taking supernatant to obtain refined silk fibroin aqueous solution, and refrigerating for later use;
and step 3: preparing a silk fibroin-nano silver mixed solution: preparing silver nitrate mother liquor, mixing the silver nitrate mother liquor with the refined silk fibroin aqueous solution, uniformly stirring, adding water for dilution, and irradiating the obtained mixed solution under ultraviolet light to generate nano silver particles to obtain a silk fibroin-nano silver mixed solution;
and 4, step 4: preparing a double-layer wound dressing: and (3) casting the silk fibroin-nano silver mixed solution obtained in the step (3) on a mould on a stretched wax-coated silk fibroin fabric, placing the mould into a mould for pre-freezing at the temperature of minus 25 to minus 15 ℃, freezing at the temperature of minus 85 to minus 75 ℃ overnight after 0.5 to 1.5 hours, immediately freezing and drying after freezing and molding to obtain a double-layer wound dressing, placing the double-layer wound dressing into a methanol solution for treatment, washing the residual methanol with water, and then drying and storing at the temperature of 55 to 65 ℃.
2. The method of claim 1, wherein: in the step 1, the concentration of the morpholine solution is 0.025-0.1% w/v.
3. The method of claim 1, wherein: in the step 2, 13-17 silkworm cocoons are taken, each silkworm cocoon is cut into 4-8 pieces, and 200ml of water is added; the ultrasonic oscillation power is 80-120Hz, and the washing is carried out for 3-7 min.
4. The method of claim 1, wherein: in the step 2, the drying temperature of the silkworm cocoons is 55-65 ℃; the concentration of the sodium carbonate solution is 0.01-0.03M; degumming for 15-60 min; the concentration of the lithium bromide solution is 9.0-9.5M, the heating and dissolving temperature is 55-65 ℃, and the dissolving time is 4-6 h.
5. The method of claim 1, wherein: in the step 2, the length of the dialysis bag is 13-17 cm; the freezing and centrifuging conditions are 8000-10000rpm, 1-5 ℃, 15-25min, and the steps are repeated twice.
6. The method of claim 1, wherein: pretreatment is carried out before the dialysis bag is used: preparing an EDTA-disodium solution with a concentration of 1mM, and adjusting the pH =7.8-8.2 with a 0.1M sodium hydroxide solution; preparing 2% w/v sodium bicarbonate solution; uniformly mixing the two solutions according to the volume ratio of 1:1, heating to boil, putting the dialysis bag into the solution, boiling for 5-15min, taking out, and then thoroughly cleaning with ultrapure water; after the cleaning, the mixture is put into the EDTA-disodium solution to be boiled for 5-15min, taken out and put into ultrapure water to be soaked for standby.
7. The method of claim 1, wherein: in the step 3, the concentration of the silk fibroin after being diluted by water is 3-6% g/ml; the concentration of silver nitrate is 0.02-0.08% g/ml; the wavelength of ultraviolet light is 365nm, and the irradiation time is 4-6 h.
8. The method of claim 1, wherein: in step 4, the concentration of the methanol solution is 85-95% v/v, and the treatment time is 20-40 min.
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