CN111840629A - Preparation method of antibacterial silkworm cocoon-based wound dressing - Google Patents
Preparation method of antibacterial silkworm cocoon-based wound dressing Download PDFInfo
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Abstract
The invention provides a preparation method of an antibacterial silkworm cocoon-based wound dressing, which is characterized in that natural silkworm cocoons are used as starting materials, firstly, a soft, transparent and malleable silkworm cocoon membrane with a natural structure of the silkworm cocoons reserved is obtained, then chloroauric acid is reduced in situ through the reducibility of sericin to obtain the silkworm cocoon membrane loaded with nano-gold, then, the specific action between the nano-gold and sulfydryl is utilized, and the silkworm cocoon membrane obtains antibacterial performance through the self-assembly of the nano-gold and 2-sulfydryl-1-methylimidazole (MMT), and finally, the transparent and soft silkworm cocoon membrane with stable antibacterial property is obtained. The preparation method is simple and suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of medical dressings, and particularly relates to a preparation method of an antibacterial silkworm cocoon-based wound dressing.
Background
Wound healing is one of the most basic and important things in human history, but currently, infection of wounds by bacteria is still an extremely problematic issue in clinical settings. On the one hand, wound infection can delay wound healing; on the other hand, wound infections are also a significant cause of morbidity and mortality in patients with a high probability. Therefore, the problem of wound infection is not easy to solve. The wound dressing is used as an important material in the wound healing process, and has the important functions of covering the wound, protecting the wound from being infected by external bacteria, releasing medicaments and accelerating the wound healing in the wound healing process. Therefore, the preparation of the antibacterial wound dressing becomes an important way for solving the problem of wound infection.
At present, products for treating wound healing are still mainly traditional wound dressings, including absorbent cotton gauze, bandages, cotton and the like. The dressings have the advantages of protective capability on wounds, certain absorption capability of wound exudates, rich content, simple preparation, low price and suitability as protective materials for mild wounds; the defects are that the dressing has large aperture and high liquid evaporation speed, leads to dry and water-deficient local environment, is easy to be bonded with a wound, causes pain to a patient when changing medicine, has poor hemostatic effect and is easy to permeate microorganisms. In order to prevent the adjacent tissues of the wound from being affected, gauze dressings need to be replaced frequently, the cost is increased, the new tissues are easy to damage during dressing change, and the wound healing delay caused by secondary injury of the wound is caused. Therefore, there is a need to develop new wound dressings to solve the problems of the conventional dressings.
An ideal wound dressing would have the following characteristics: bleeding is prevented, and the wound is kept moist; secondly, the antibacterial property is achieved, and the wound is protected from bacterial infection; the material has good biocompatibility; and fourthly, the adhesive tape has flexibility and can be attached to wounds. To make wound dressings antimicrobial, there are three main classes of antimicrobial substances that have been used in wound dressing materials to eliminate infection: natural antibacterial agents, organic antibacterial agents and inorganic antibacterial agents.
Natural antibacterial agents are effective active ingredients having antibacterial action extracted from substances existing in the natural world. Natural antibiotics can be classified from the sources into animal-derived antibacterial agents, plant-derived antibacterial agents, and microbial-derived antibacterial agents. Taking chitosan as an example, it is an animal-derived antibacterial agent with good film forming ability, good biocompatibility and antibacterial activity. The antibacterial mechanism of chitosan includes two mechanisms, one is that amino group in chitosan molecular chain is protonated and then has positive charge, and can produce electrostatic effect with negatively charged anions, such as electrically charged phosphate ester, etc. to limit the freedom of microbe and inhibit its metabolism and propagation, and the other is that low molecular weight bright polysaccharide can invade into microbe cell to inhibit the transcription of microbe genetic code from DNA to RNA and inhibit the growth and propagation of microbe. Many researches show that the chitosan has obvious inhibition effect on escherichia coli, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and the like. By utilizing the characteristic, Radhakumary and the like prepare the temperature-sensitive antibacterial hydrogel dressing. However, the poor solubility of chitosan in neutral solutions leads to difficulties in mass production, limiting its application in biomedicine.
The organic antibacterial agent is the most widely used antibacterial agent for human, mainly comprises alcohols, phenols, quaternary ammonium salts, biguanides, organic acids and the like, has an antibacterial mechanism of affecting the development of microorganisms and causing the death of bacteria by hindering the energy metabolism of cells and the synthesis of proteins and cell walls, and has the advantages of immediate antibacterial effect and wide antibacterial spectrum. Fan et al prepared a quaternary ammonium salt chitosan/polyvinyl alcohol/polyoxyethylene composite hydrogel by a gamma radiation method, and the hydrogel had an obvious inhibiting effect on staphylococcus aureus and escherichia coli. However, the safety of organic antibacterial agents has not been clarified yet, and there are disadvantages of poor chemical stability, thermolabile property and large toxicity, and moreover, the organic antibacterial agents are liable to cause drug resistance in bacteria and have a short period of validity.
The inorganic antibacterial agent is mainly developed in a novel photocatalysis type and a carrier type, wherein the photocatalysis type inorganic antibacterial agent depends on strong oxidizing free radicals excited by light to play a role in sterilization. The carrier type inorganic antibacterial agent is prepared by combining antibacterial components and compounds thereof or nano metal powder thereof with inorganic materials, and has the characteristics of high safety, heat resistance and durability. The antibacterial mechanism of such antibacterial agents varies depending on the antibacterial element and the carrier material. The antibacterial agent represented by silver system belongs to a contact reaction antibacterial mechanism, when trace silver ions reach microbial cells, the silver ions are charged with negative charges and are firmly adsorbed by coulomb attraction, so that the electrolyte balance of the microbial cells is destroyed, and the microbial cells are killed due to the damage of cell walls; the disadvantages are high price and slow-acting antibacterial property, and can not kill bacteria as quickly as organic antibacterial agents.
Aiming at the problems of the existing antibacterial agent, the research and development of antibacterial wound dressings focus on the aspects of antibacterial effect, drug resistance, toxicity and the like. Meanwhile, the physical and chemical properties of the wound dressing are comprehensively considered, and the similar microstructure and appearance of the wound dressing can be considered to be constructed so as to improve the wound healing effect of the wound dressing. In addition, the ideal wound dressing material has the characteristics of stability, flexibility and biocompatibility on the basis of having antibacterial property.
Disclosure of Invention
Studies find that sericin in silkworm cocoon can reduce Ag+The gold nanoparticles (AuNPs) have no antibacterial performance, but can be specifically combined with sulfydryl, and have stronger antibacterial activity when being combined with 2-sulfydryl-1-methylimidazole (MMT). Based on the above, the invention provides a preparation method of an antibacterial silkworm cocoon-based wound dressing, which is characterized in that 2-mercapto-1-Methylimidazole Micromolecule (MMT) is self-assembled by reducing chloroauric acid in situ, so that the antibacterial 3D multi-cavity silkworm cocoon-based wound dressing is obtained.
Except for special description, the percentages are mass percentages.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of an antibacterial silkworm cocoon-based wound dressing comprises three steps of preparation of a silkworm cocoon membrane, and self-assembly of the silkworm cocoon membrane loaded with nanogold and 2-mercapto-1-methylimidazole, and is characterized in that: the method for loading the nanogold on the silkworm cocoon membrane comprises the steps of adding a chloroauric acid solution into deionized water to prepare a chloroauric acid solution with the mass fraction of 1/9000 g/ml-1/1000 g/ml, adding the silkworm cocoon membrane into every 30ml of the chloroauric acid solution per sheet of the silkworm cocoon membrane, and then reacting for 5-30 minutes at 60-100 ℃ under a sealed condition to obtain the nanogold-loaded silkworm cocoon membrane.
Researches show that the concentration and the reaction temperature of the chloroauric acid solution influence the effect of loading the nanogold on the silkworm cocoon membrane, and once the control is not good, the problems of the change of the silkworm cocoon shape, such as the swelling of the silkworm cocoon surface due to water absorption, the rotten silkworm cocoon and the small amount of purple spots on the silkworm cocoon surface, and the loading of the nanogold, such as less or more reduced nanogold, uneven distribution, and the like, can occur. Preferably, the concentration of the chloroauric acid solution is 1/3000g/ml, and the reaction temperature under sealed conditions is 100 ℃. Further, in the above method, the reaction temperature under a sealed condition is 100 ℃ and the reaction time is 10 minutes.
In the method, the self-assembly of the nanogold and the 2-mercapto-1-methylimidazole is to put the nanogold-loaded silkworm cocoon membrane into a 2-mercapto-1-methylimidazole solution, and react for 5-30 minutes at 60-100 ℃ under a sealed condition to obtain the self-assembled antibacterial silkworm cocoon-based wound dressing of the nanogold and the 2-mercapto-1-methylimidazole.
In order to improve the antibacterial performance of the silkworm cocoon-based wound dressing, in the method, the molar concentration ratio of the chloroauric acid to the 2-mercapto-1-methylimidazole (MMT) is 1: 10-50.
During the preparation process, the addition of chloroauric acid and 2-mercapto-1-methylimidazole is found to weaken the transparency of the silkworm cocoon membrane to some extent. In order to reduce the influence of chloroauric acid and 2-mercapto-1-methylimidazole on the transparency of the silkworm cocoon membrane and ensure the antibacterial performance of the silkworm cocoon-based wound dressing, in the method, the concentration of the 2-mercapto-1-methylimidazole solution is preferably 0.0005-0.005 g/ml, the reaction temperature is 80-100 ℃, and the reaction time is 5-15 minutes. Further, the concentration of the 2-mercapto-1-methylimidazole solution was 0.005g/ml, and the reaction time was 15 minutes.
In the method, the preparation of the silkworm cocoon membrane comprises the steps of completely soaking silkworm cocoons without silkworm chrysalis in a ternary solution, pouring the ternary solution after the silkworm cocoons are completely soaked, refilling the ternary solution into a filter flask, then performing suction filtration on the filter flask filled with the ternary solution and the silkworm cocoons on a suction filtration machine, subpackaging the silkworm cocoons and the ternary solution in a wide-mouth bottle after the suction filtration is completed, then placing the wide-mouth bottle in a water bath kettle at 55-60 ℃ for water bath until the silkworm cocoons become soft and transparent, taking out the wide-mouth bottle, and washing the ternary solution with deionized water to obtain the soft and transparent silkworm cocoon membrane; the ternary solution is water in molar ratio: calcium chloride: mixed solution of 8:1:2 alcohol.
In the method, the preparation method of the ternary solution comprises the following steps: firstly, 648ml of deionized water is added into 500g of calcium chloride, and after the calcium chloride solution is cooled and the calcium chloride is completely dissolved, 525ml of alcohol is added, and the calcium chloride-containing water-based paint is obtained after the solution is cooled again.
A preparation method of an antibacterial silkworm cocoon-based wound dressing comprises the following steps:
a) preparing a silkworm cocoon membrane:
firstly, 648ml of deionized water is added into 500g of calcium chloride, 525ml of alcohol is added after the calcium chloride solution is cooled and the calcium chloride is completely dissolved, the preparation of the ternary solution is completed after the solution is cooled again, then the silkworm cocoons without silkworm chrysalis are completely soaked in the ternary solution to soak the surfaces of the silkworm cocoons, after the ternary solution is completely soaked, the ternary solution is poured out, the ternary solution is re-filled into a filter flask, and the amount of the ternary solution is based on the fact that the silkworm cocoons are completely submerged; then, carrying out suction filtration on a suction filtration bottle filled with the ternary solution and the silkworm cocoons on a suction filtration machine, after the suction filtration is finished, subpackaging the silkworm cocoons and the ternary solution in wide-mouth bottles, putting 5-10 silkworm cocoons into 250ml of the ternary solution, then placing the wide-mouth bottles in a water bath kettle at 58 ℃ for water bath until the silkworm cocoons become soft and transparent, taking out the wide-mouth bottles, and washing the ternary solution with deionized water to obtain a soft and transparent silkworm cocoon membrane;
b) nano gold loaded on silkworm cocoon membrane
Adding 1ml of chloroauric acid solution with the mass fraction of 0.01g/ml into 29ml of deionized water to prepare a chloroauric acid solution with the mass fraction of 1/3000g/ml, then adding a piece of soft and transparent silkworm cocoon film prepared in a), namely, adding a piece of soft and transparent silkworm cocoon film into each 30ml of chloroauric acid solution with the mass fraction of 1/3000g/ml, sealing the chloroauric acid solution and the transparent silkworm cocoon film by using a preservative film, putting the sealed chloroauric acid solution and the transparent silkworm cocoon film into a water bath kettle with the temperature of 100 ℃ for reaction for 10 minutes, taking out the silkworm cocoon film after 10 minutes, and washing off chloroauric acid remained on the surface to obtain the silkworm cocoon film loaded with nanogold;
c) self-assembly of nanogold and 2-mercapto-1-methylimidazole
Firstly, 0.1006g of 2-mercapto-1-methylimidazole is dissolved in 30ml of deionized water to obtain a 2-mercapto-1-methylimidazole solution with the mass fraction of 0.0033 g/ml; and (b) putting the silkworm cocoon membrane loaded with the nanogold obtained in the step b) into a 2-sulfydryl-1-methylimidazole solution, sealing the surfaces with a preservative film, putting the sealed surfaces together into a water bath kettle at 100 ℃ for reacting for 15 minutes, and taking out the silkworm cocoon membrane after 15 minutes to obtain the antibacterial silkworm cocoon-based wound dressing.
Has the advantages that:
the invention provides a preparation method of an antibacterial silkworm cocoon-based wound dressing, which is characterized in that natural silkworm cocoons are used as starting materials, firstly, a soft, transparent and malleable silkworm cocoon membrane with a natural structure of the silkworm cocoons reserved is obtained, then chloroauric acid is reduced in situ through the reducibility of sericin to obtain the silkworm cocoon membrane loaded with nano-gold, then, the specific action between the nano-gold and sulfydryl is utilized, and the silkworm cocoon membrane obtains antibacterial performance through the self-assembly of the nano-gold and 2-sulfydryl-1-methylimidazole (MMT), and finally, the transparent and soft silkworm cocoon membrane with stable antibacterial property is obtained. The antibacterial silkworm cocoon membrane prepared by the invention has good shape, good flexibility and transparency and excellent antibacterial performance, and is suitable for being developed into antibacterial silkworm cocoon-based wound dressing. The preparation method is simple and suitable for industrial production.
Drawings
FIG. 1 is a structural diagram of a cocoon membrane, which is a macroscopic view of the cocoon membrane, a scanning electron microscope microscopic view (100 μm) of the cocoon membrane, and a scanning electron microscope microscopic view (50 μm) of the cocoon membrane, respectively, from left to right;
FIG. 2 is a graph showing the results of transparency of different silkworm cocoon membranes;
FIG. 3 is a diagram of an animal model wound;
FIG. 4 is a graph of the effect of histological analysis of wounds;
FIG. 5 is a graph showing the results of cell viability of cells treated with different silkworm cocoon membranes.
Detailed Description
The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure.
Firstly, preparation of antibacterial silkworm cocoon-based wound dressing
Example 1
A preparation method of an antibacterial silkworm cocoon-based wound dressing comprises the following steps:
a) preparation of silkworm cocoon membrane-using ternary solution (molar ratio; water: calcium chloride: alcohol 8:1:2) water bath treatment of silkworm cocoons to obtain soft and transparent silkworm cocoon films;
firstly, 648ml of deionized water is added into 500g of calcium chloride, 525ml of alcohol is added after the calcium chloride solution is cooled and the calcium chloride is completely dissolved, and the preparation of the ternary solution is completed after the solution is cooled for the second time. And then, completely soaking the silkworm cocoons without silkworm pupas in the ternary solution, namely pouring a proper amount of the ternary solution into a filter flask filled with the silkworm cocoons, then shaking the filter flask to soak the surfaces of the silkworm cocoons, pouring the ternary solution after the silkworm cocoons are completely soaked, and refilling the ternary solution into the filter flask, wherein the amount of the ternary solution is based on the fact that the silkworm cocoons are completely submerged. And then, carrying out suction filtration on the suction filtration bottle filled with the ternary solution and the silkworm cocoons on a suction filtration machine. Carrying out suction filtration for three times, wherein the solution is boiled during suction filtration; after the filtration, the cocoons and the ternary solution are respectively filled in a wide-mouth bottle (5-10 cocoons are put in 250ml of ternary solution), then the wide-mouth bottle is placed in a water bath at 58 ℃ until the cocoons become soft and transparent, and then the wide-mouth bottle is taken out, and the ternary solution is washed away by deionized water to obtain the soft and transparent cocoons membrane.
b) The silkworm cocoon membrane is loaded with the nanogold, namely the nanogold-loaded silkworm cocoon membrane is obtained by reducing chloroauric acid in situ by utilizing silk fibroin in the silkworm cocoon.
1ml of chloroauric acid solution with the mass fraction of 0.01g/ml is added into 29ml of deionized water to prepare the chloroauric acid solution with the mass fraction of 1/3000 g/ml. Then adding a piece of the soft and transparent silkworm cocoon membrane prepared in the step a) into the solution. The method comprises the steps of putting a soft and transparent silkworm cocoon film into each 30ml of 1/3000g/ml chloroauric acid solution, sealing the two films by using preservative films, and putting the sealed films into a 100 ℃ water bath kettle for reaction for 10 minutes. And after 10 minutes, taking out the soft and transparent silkworm cocoon membrane, and washing off residual chloroauric acid on the surface to obtain the silkworm cocoon membrane loaded with the nanogold.
c) Self-assembly of nano-gold and 2-mercapto-1-methylimidazole to prepare antibacterial silkworm cocoon membrane, namely, the antibacterial performance of soft and transparent silkworm cocoon membrane is endowed by utilizing the electrostatic action of nano-gold and 2-mercapto-1-methylimidazole
The invention adopts the following condition parameters and operation steps: firstly, 0.1006g of 2-mercapto-1-methylimidazole is dissolved in 30ml of deionized water to obtain a 2-mercapto-1-methylimidazole solution with the mass fraction of 0.0033 g/ml; and then putting the nano-gold loaded silkworm cocoon film obtained in the step b) into a 2-sulfydryl-1-methylimidazole solution, sealing the film by using a preservative film, and putting the film and the solution into a water bath kettle at 100 ℃ for reaction for 15 minutes. After 15 minutes, the cocoon membrane was taken out to obtain an antibacterial soft and transparent cocoon membrane (antibacterial cocoon-based wound dressing of the present invention).
The antibacterial soft and transparent cocoon film prepared in example 1 was freeze-dried, and observed by a scanning electron microscope, and the microscopic and macroscopic structures thereof were as shown in fig. 1, which revealed that the cocoon film had a multi-cavity structure.
The addition of chloroauric acid and 2-mercapto-1-methylimidazole can weaken the transparency of the silkworm cocoon membrane to some extent. The soft and transparent silkworm cocoon membrane having antibacterial property prepared in example 1 (antibacterial silkworm cocoon-based wound dressing of the present invention) was subjected to absorbance examination at different wavelengths, and the silkworm cocoon membrane was placed on a 96-well plate, and its optical transparency was measured by an microplate reader, and as a result, as shown in fig. 2, the self-assembly between the nano-gold and the load and between the nano-gold and 2-mercapto-1-methylimidazole affected the transparency of the silkworm cocoon to a certain extent.
The results of examining the influence of the concentration of the chloroauric acid solution and the reaction temperature on the loading of nanogold on the silkworm cocoon film with reference to example 1 are shown in table 1.
TABLE 1 influence of the concentration of chloroauric acid solution and reaction temperature on loading of nanogold on silkworm cocoon membrane
Referring to example 1, the concentration of 2-mercapto-1-methylimidazole (MMT) solution and the water bath treatment time were examined for the effect on the antibacterial performance of the cocoon membrane, and the results are shown in table 2. The concentration of chloroauric acid was determined to be 3/10000g/ml (MMT concentration was calculated based on the molar ratio of chloroauric acid to MMT of 1:10, 1:30, 1:50, respectively).
TABLE 2 influence of MMT solution concentration and water bath treatment time on the antibacterial property of silkworm cocoon membrane
(II) investigation of antibacterial performance of antibacterial silkworm cocoon-based wound dressing
The wound healing ability of the soft and transparent silkworm cocoon film (the antibacterial silkworm cocoon-based wound dressing of the present invention) having antibacterial properties prepared in example 1 was examined by establishing an animal model. After shaving the backs of the rabbits, three circular wounds of 2 cm diameter were cut in the skin on the spine with a stainless steel scalpel. Then, a suspension of Staphylococcus aureus (200. mu.L, 1X 10)7Colonies) were dropped onto all wounds and then covered with gauze and bandage, and 24h after infection, the wounds were covered with a beauty patch and a silkworm cocoon film for comparison. Finally, the rabbits were euthanized after days 5, 10, and 15, respectively, and their wounds were photographed, as shown in fig. 3. As a result: at day 15, the remaining wounds, except for the control group, healed almost completely. The healing conditions of the wound healed by the silkworm cocoon membrane and the beautiful skin patch are equivalent, and the latter has better performance in promoting wound contraction.
And finally, after 5, 10 and 15 days, respectively, euthanizing, taking the wound and the surrounding intact skin of the rabbit, fixing the intact skin with 10% formaldehyde solution, staining the intact skin with hematoxylin-eosin wound collagen fibers, and performing histological observation and analysis, wherein the specific structure is shown in fig. 4.
In the rabbit back full-skin infected wound surface model, the soft, transparent silkworm cocoon film prepared in example 1 having antibacterial properties also promoted healing of infected wound surfaces, compared to commercial dressings. On day 10 of wound healing, it was clearly observed that all wounds appeared to contract significantly. After the dressing completely fell off (15 days), the cocoon membrane group recovered substantially the same as the commercial dressing group, and the recovery was better than the control group. This shows that the antibacterial cocoon-based wound dressing of the present invention has excellent antibacterial properties.
Histology was performed on identically treated wounds with H & E staining at different times. On day 5, all wounds scabbed (triangle mark). Among them, the soft and transparent silkworm cocoon film-treated wounds having antibacterial properties prepared in example 1 were observed to have new epidermal tissues (arrow marks) formed. On day 10, mild inflammation was still visible under the new epidermis in the blank group (diamond marks). In contrast, in the wounds treated with the soft, transparent silkworm cocoon film having antibacterial properties and the commercial dressing prepared in example 1, generation of a new sub-epidermal fibrous tissue (hexagonal marks) was observed. Example 1 preparation of antibacterial soft, transparent silkworm cocoon film treated wounds new epidermis and skin appendages (oval marks) were also observed on day 15. While the neoepidermis in the blank did not heal completely. The epidermal growth of the wound treated with the commercial dressing was nearly complete, but no skin appendages were seen, indicating that the healing effect of the soft, transparent silkworm cocoon film with antibacterial properties prepared in example 1 was slightly better than that of the commercial dressing, probably due to its higher antibacterial activity and biocompatibility.
The antibacterial effect of different silkworm cocoon membranes prepared by different proportions of MMT and chloroauric acid is considered, the cell survival rate is tested by using an MTT method, and the result is shown in figure 5. Wherein, the first group: a transparent, soft silkworm cocoon film; second group: a cocoon membrane loaded with nanogold; third group: an antibacterial silkworm cocoon membrane with the molar concentration ratio of chloroauric acid to MMT being 1: 10; and a fourth group: an antibacterial silkworm cocoon membrane with the molar concentration ratio of chloroauric acid to MMT being 1: 30; and a fifth group: the molar concentration ratio of the chloroauric acid to the MMT is 1: 50. The results show that the higher the specific gravity of MMT and chloroauric acid addition, the greater the effect on the cells and the lower the survival rate of the cells.
Claims (10)
1. A preparation method of an antibacterial silkworm cocoon-based wound dressing comprises three steps of preparation of a silkworm cocoon membrane, and self-assembly of the silkworm cocoon membrane loaded with nanogold and 2-mercapto-1-methylimidazole, and is characterized in that: the method for loading the nanogold on the silkworm cocoon membrane comprises the steps of adding a chloroauric acid solution into deionized water to prepare a chloroauric acid solution with the mass fraction of 1/9000 g/ml-1/1000 g/ml, adding the silkworm cocoon membrane into every 30ml of the chloroauric acid solution per sheet of the silkworm cocoon membrane, and then reacting for 5-30 minutes at 60-100 ℃ under a sealed condition to obtain the nanogold-loaded silkworm cocoon membrane.
2. The method of claim 1, wherein: the concentration of the chloroauric acid solution is 1/3000g/ml, and the reaction temperature is 100 ℃ under the sealed condition.
3. The method of claim 2, wherein: the reaction temperature was 100 ℃ and the reaction time was 10 minutes under sealed conditions.
4. The method of any one of claims 1-3, wherein: the self-assembly of the nano-gold and the 2-mercapto-1-methylimidazole is to put the silkworm cocoon membrane loaded with the nano-gold into a 2-mercapto-1-methylimidazole solution, and react for 5-30 minutes at 60-100 ℃ under a sealed condition to obtain the self-assembled antibacterial silkworm cocoon-based wound dressing of the nano-gold and the 2-mercapto-1-methylimidazole.
5. The method of claim 4, wherein: the molar concentration ratio of the chloroauric acid to the 2-mercapto-1-methylimidazole is 1: 10-50.
6. The method of claim 4, wherein: the concentration of the 2-mercapto-1-methylimidazole solution is 0.0005-0.005 g/ml, the reaction temperature is 80-100 ℃, and the reaction time is 5-15 minutes.
7. The method of claim 6, wherein: the concentration of the 2-mercapto-1-methylimidazole solution is 0.005g/ml, and the reaction time is 15 minutes.
8. The method of any one of claims 1-3 or 5-7, wherein: the preparation method of the silkworm cocoon membrane comprises the steps of completely soaking silkworm cocoons without silkworm chrysalis in a ternary solution, pouring the ternary solution after the silkworm cocoons are completely soaked, filling the ternary solution into a filter flask again, carrying out suction filtration on the filter flask filled with the ternary solution and the silkworm cocoons on a suction filtration machine, subpackaging the silkworm cocoons and the ternary solution in a wide-mouth bottle after the suction filtration is finished, then placing the wide-mouth bottle in a water bath kettle at 55-60 ℃ for water bath until the silkworm cocoons become soft and transparent, taking out the wide-mouth bottle, and washing the ternary solution with deionized water to obtain the soft and transparent silkworm cocoon membrane; the ternary solution is water in molar ratio: calcium chloride: mixed solution of 8:1:2 alcohol.
9. The method of claim 8, wherein: the preparation method of the ternary solution comprises the following steps: firstly, 648ml of deionized water is added into 500g of calcium chloride, and after the calcium chloride solution is cooled and the calcium chloride is completely dissolved, 525ml of alcohol is added, and the calcium chloride-containing water-based paint is obtained after the solution is cooled again.
10. A preparation method of an antibacterial silkworm cocoon-based wound dressing comprises the following steps:
a) preparing a silkworm cocoon membrane:
firstly, 648ml of deionized water is added into 500g of calcium chloride, 525ml of alcohol is added after the calcium chloride solution is cooled and the calcium chloride is completely dissolved, the preparation of the ternary solution is completed after the solution is cooled again, then the silkworm cocoons without silkworm chrysalis are completely soaked in the ternary solution to soak the surfaces of the silkworm cocoons, after the ternary solution is completely soaked, the ternary solution is poured out, the ternary solution is re-filled into a filter flask, and the amount of the ternary solution is based on the fact that the silkworm cocoons are completely submerged; then, carrying out suction filtration on a suction filtration bottle filled with the ternary solution and the silkworm cocoons on a suction filtration machine, after the suction filtration is finished, subpackaging the silkworm cocoons and the ternary solution in wide-mouth bottles, putting 5-10 silkworm cocoons into 250ml of the ternary solution, then placing the wide-mouth bottles in a water bath kettle at 58 ℃ for water bath until the silkworm cocoons become soft and transparent, taking out the wide-mouth bottles, and washing the ternary solution with deionized water to obtain a soft and transparent silkworm cocoon membrane;
b) nano gold loaded on silkworm cocoon membrane
Adding 1ml of chloroauric acid solution with the mass fraction of 0.01g/ml into 29ml of deionized water to prepare a chloroauric acid solution with the mass fraction of 1/3000g/ml, then adding a piece of soft and transparent silkworm cocoon film prepared in a), namely, adding a piece of soft and transparent silkworm cocoon film into each 30ml of chloroauric acid solution with the mass fraction of 1/3000g/ml, sealing the chloroauric acid solution and the transparent silkworm cocoon film by using a preservative film, putting the sealed chloroauric acid solution and the transparent silkworm cocoon film into a water bath kettle with the temperature of 100 ℃ for reaction for 10 minutes, taking out the silkworm cocoon film after 10 minutes, and washing off chloroauric acid remained on the surface to obtain the silkworm cocoon film loaded with nanogold;
c) self-assembly of nanogold and 2-mercapto-1-methylimidazole
Firstly, 0.1006g of 2-mercapto-1-methylimidazole is dissolved in 30ml of deionized water to obtain a 2-mercapto-1-methylimidazole solution with the mass fraction of 0.0033 g/ml; and (b) putting the silkworm cocoon membrane loaded with the nanogold obtained in the step b) into a 2-sulfydryl-1-methylimidazole solution, sealing the surfaces with a preservative film, putting the sealed surfaces together into a water bath kettle at 100 ℃ for reacting for 15 minutes, and taking out the silkworm cocoon membrane after 15 minutes to obtain the antibacterial silkworm cocoon-based wound dressing.
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| CN101498061A (en) * | 2009-03-05 | 2009-08-05 | 浙江大学 | Method for improving antibiosis of silk fibroin material |
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