CN115105645B - Preparation method of composite microsphere and wound repair dressing - Google Patents
Preparation method of composite microsphere and wound repair dressing Download PDFInfo
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- CN115105645B CN115105645B CN202210750820.7A CN202210750820A CN115105645B CN 115105645 B CN115105645 B CN 115105645B CN 202210750820 A CN202210750820 A CN 202210750820A CN 115105645 B CN115105645 B CN 115105645B
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
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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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- 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
- 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
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
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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
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a preparation method of composite microspheres and wound repair dressing. The preparation method comprises the following steps: obtaining a silk fibroin solution; mixing a silk fibroin solution with a polylysine solution to obtain a silk fibroin-polylysine mixed solution; adding the silk fibroin-polylysine mixed solution into an organic volatile solvent to obtain silk fibroin-polylysine composite microsphere suspension with positive charges on the surface, and then separating, purifying and drying to obtain silk fibroin-polylysine composite microsphere powder; adding the silk fibroin-polylysine composite microsphere powder into a buffer solution of polydeoxyribonucleotide to adsorb polydeoxyribonucleotide with negative charge, thus obtaining silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension. The composite microsphere has antibacterial effect, can slowly release polydeoxyribonucleotide to achieve the effects of anti-inflammation and skin repair, and has the functions of biocompatibility, antibacterial activity and wound healing promotion.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a preparation method of composite microspheres and wound repair dressing.
Background
The skin plays an irreplaceable role in excreting, feeling, preventing moisture evaporation, regulating body temperature, immunizing, etc., is the largest organ of the human body, and serves as a barrier between the body and the external environment. Common skin wound repair includes a hemostatic phase, an inflammatory phase, a cell proliferation phase, and a tissue remodeling phase. However, excessive inflammation of the wound surface of diabetes is easy to cause bacterial infection, angiogenesis of the wound surface is damaged, and wound healing still faces a great challenge. Large-area skin defect is caused by burn, wound, diabetic chronic ulcer and other reasons, and a wound surface with a diameter of more than 4 cm cannot be completely repaired. Especially when bacterial infection happens, staphylococcus aureus on the surface of the wound can seriously damage the immune system, aggravate inflammation, aggravate tissue injury caused by long-time inflammatory reaction and delay wound healing. Therefore, there is an urgent need for multifunctional composite materials with bioactivity such as antibacterial, anti-inflammatory, pro-angiogenic, etc. to improve the current therapeutic effects of diabetic wounds.
Disclosure of Invention
The invention aims to provide a preparation method of a silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere with biocompatibility, antibacterial activity and wound healing promotion.
It is a further object of the present invention to provide a wound repair dressing that is convenient to use.
In particular, the invention provides a preparation method of composite microspheres, which comprises the following steps:
obtaining a silk fibroin solution;
mixing the silk fibroin solution with a polylysine solution to obtain a silk fibroin-polylysine mixed solution;
adding the silk fibroin-polylysine mixed solution into an organic volatile solvent to obtain silk fibroin-polylysine composite microsphere suspension with positive charges on the surface, and then separating, purifying and drying to obtain silk fibroin-polylysine composite microsphere powder;
and adding the silk fibroin-polylysine composite microsphere powder into a buffer solution of polydeoxyribonucleotide to absorb polydeoxyribonucleotide with negative charge by utilizing electrostatic action, thereby obtaining silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension.
Optionally, the preparation method further comprises the following steps:
and separating, purifying and drying the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension to obtain silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere powder.
Optionally, in the step of mixing the silk fibroin solution with the polylysine solution to obtain a silk fibroin-polylysine mixed solution, the mass concentration ratio of the silk fibroin solution to the polylysine solution is any one of 3:7-3:42.
Optionally, the mass of the polydeoxyribonucleotide in the buffer solution of polydeoxyribonucleotide is any value in the range of 1-16 mg.
Optionally, the volume ratio of the organic volatile solvent to the silk fibroin solution is any one of 5-10:1.
Optionally, the buffer solution is a TE buffer.
Optionally, the obtaining a silk fibroin solution comprises the steps of:
degumming silkworm cocoons to obtain silk fibroin fibers;
and adding the silk fibroin fibers into a ternary system for dissolution, and obtaining the silk fibroin solution through dialysis, filtration and concentration.
Optionally, the ternary system is a system of calcium chloride, ethanol and water, wherein the molar ratio of calcium chloride, ethanol and water is any one of 1:1-3:7-9.
In particular, the invention also provides a preparation method of the wound repair dressing, which comprises the following steps:
dispersing the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere powder in a sodium hyaluronate solution to obtain the wound repair dressing.
Optionally, the density ratio of the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere and the sodium hyaluronate solution is any one of 1:2-4.
According to the scheme of the embodiment of the invention, the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere has good antibacterial effect, can slowly release polydeoxyribonucleotide to achieve the effects of resisting inflammation and repairing skin, has the functions of biocompatibility, antibacterial activity and promoting wound healing, is very simple in preparation method, and has green and environment-friendly preparation process, thereby laying a good foundation for the application of the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere in vivo injection biomedical microspheres.
The above, as well as additional objectives, advantages, and features of the present invention will become apparent to those skilled in the art from the following detailed description of a specific embodiment of the present invention when read in conjunction with the accompanying drawings.
Drawings
Some specific embodiments of the invention will be described in detail hereinafter by way of example and not by way of limitation with reference to the accompanying drawings. The same reference numbers will be used throughout the drawings to refer to the same or like parts or portions. It will be appreciated by those skilled in the art that the drawings are not necessarily drawn to scale. In the accompanying drawings:
FIG. 1 shows a schematic flow chart of a method of preparing composite microspheres according to one embodiment of the invention;
FIG. 2 shows a schematic structural flow diagram of a method of preparing composite microspheres according to one embodiment of the invention;
FIG. 3 shows a transmission electron microscope image of the surface morphology of particles after beta-overlap induced by ethanol in a 3% SF solution according to one embodiment of the present invention;
FIG. 4 shows a transmission electron micrograph of the surface morphology of a composite microsphere after EPL modification of 3% SF and ethanol induced beta-stacking according to one embodiment of the present invention;
FIG. 5 shows a transmission electron microscope image of the surface morphology of a composite microsphere loaded with PDRN after EPL modification of 3% SF and ethanol-induced overlap according to one embodiment of the present invention;
FIG. 6 shows a picture of the number of group blanks;
FIG. 7 is a photograph showing the number of bacteria incubated with a silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension;
FIG. 8 shows a statistical plot of the numbers of the blank and silk fibroin-polylysine-polydeoxyribonucleotide complex microsphere group bacteria;
FIG. 9 is a graph showing statistics of potential data of a silk fibroin-polylysine mixed solution prepared by taking 0, 280mg, 350mg, and 420mg of polylysine in step S200, respectively, according to one embodiment of the present invention;
FIG. 10 shows a potential histogram of SF-EPL-PDRN microspheres obtained when the mass of polydeoxyribonucleotide in step S400 is taken as 0, 4mg, 10mg, 14mg and 16mg, respectively, according to an embodiment of the present invention;
FIG. 11 shows the size of diabetic skin wound wounds on day one and day seven of the blank;
FIG. 12 shows the size of diabetic skin wound wounds on the first and seventh days of the SF-EPL-PDRN composite microsphere sets;
figure 13 shows a statistical plot of skin relative wound size for the blank and silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere treatment groups.
Detailed Description
Other advantages and effects of the present application will become apparent to those skilled in the art from the present disclosure, when the following description of the embodiments is taken in conjunction with the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, of the embodiments of the present application. The present application may be embodied or carried out in other specific embodiments, and the details of the present application may be modified or changed from various points of view and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, are intended to be within the scope of the present application.
Fig. 1 shows a schematic flow chart of a method of preparing composite microspheres according to an embodiment of the invention. Fig. 2 shows a schematic structural flow chart of a method of preparing composite microspheres according to an embodiment of the present invention. As shown in fig. 1 and 2, the preparation method comprises:
step S100, obtaining a silk fibroin solution;
step S200, mixing a silk fibroin solution with a polylysine solution to obtain a silk fibroin-polylysine mixed solution;
step S300, adding the silk fibroin-polylysine mixed solution into an organic volatile solvent to obtain silk fibroin-polylysine composite microsphere suspension with positive charges on the surface, and then separating, purifying and drying to obtain silk fibroin-polylysine composite microsphere powder;
step S400, adding silk fibroin-polylysine composite microsphere powder into a buffer solution of polydeoxyribonucleotide to absorb polydeoxyribonucleotide with negative charge by utilizing electrostatic action, thus obtaining silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension.
According to the scheme of the embodiment of the invention, the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere has good antibacterial effect, can slowly release polydeoxyribonucleotide to achieve the effects of resisting inflammation and repairing skin, has the functions of biocompatibility, antibacterial activity and promoting wound healing, is very simple in preparation method, and has green and environment-friendly preparation process, thereby laying a good foundation for the application of the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere in vivo injection biomedical microspheres.
In the step S100, the method for obtaining the silk fibroin solution includes the following steps: degumming silkworm cocoons to obtain silk fibroin fibers; and adding silk fibroin fibers into a ternary system for dissolution, and obtaining silk fibroin solution through dialysis, filtration and concentration.
In the step of degumming the cocoons, the cocoons are placed in boiling water of sodium carbonate for degumming. In a specific embodiment, 5-5.5g of silk cocoons are put into 2L of boiling water containing 4.2g of sodium carbonate, stirring is continued, the silk cocoons are taken out and washed for a plurality of times after a period of time, such as 30min, ultrapure water is taken out and washed for 2-4h to remove sericin and ions, and after degumming, the silk cocoons are put into a drying oven at 45 ℃ to be dried to obtain silk fibroin fibers. And (3) adding the silk fibroin fibers into a ternary system for dissolution, and obtaining silk fibroin solution through dialysis, filtration and concentration, wherein the ternary system is a system of calcium chloride, ethanol and water, and the molar ratio of the calcium chloride to the ethanol to the water is any one of 1:1-3:7-9, for example, 1:2:8, 1:3:9 and 1:1:7. In one embodiment, after dissolving the silk fibroin fibers in a ternary system, dissolving the silk fibroin fibers sufficiently at 65-75 ℃, centrifuging the solution at a high speed such as 9500r/min for 8-12min, putting the obtained solution into a dialysis bag, dialyzing the dialysis solution in deionized water for 2-4 days, centrifuging the dialysis solution to remove impurities to obtain a silk fibroin solution, and finally sterilizing the silk fibroin solution at a high temperature and high pressure to obtain a sterile Silk Fibroin (SF) solution.
In the embodiment of the invention, the SF microsphere prepared from natural SF has controllable particle size, can be used as a main material of a cell carrier, can simulate the protein component of an extracellular matrix to be suitable for penetrating different transmission barriers in organisms, and has lower antigenicity than other common biodegradable polymers such as high-molecular polylactic acid and the like.
In step S200, the mass concentration ratio of the silk fibroin solution to the polylysine (EPL) solution is any one of 3:7 to 3:42. In several examples, 140mg, 280mg, 350mg and 420mg of polylysine may be dissolved in 2ml of ultrapure water, respectively, and the solution may be added dropwise to 10ml of 1% -6% SF solution, respectively, and stirred at room temperature for two or more hours to obtain a silk fibroin-polylysine (SF-EPL) mixed solution. The final SF-EPL composite microspheres in the several examples are different in size, and the different sizes of the microspheres affect the surface electrical properties. The ratio of SF to EPL mass concentration is 3:7-3:42, and the composite microsphere with the positively charged surface can be obtained within the range of the embodiment of the invention.
In step S300, the organic volatile solvent may be, for example, ethanol, methanol, or n-propanol. The SF-EPL composite microsphere suspension obtained in the step has uniform size.
In step S400, the mass of the Polydeoxyribonucleotide (PDRN) in the buffer solution is any one of 1-16mg, for example, 1mg, 2mg, 4mg, 6mg, 8mg, 10mg, 14mg, 15mg or 16mg. The buffer may be, for example, TE buffer, which is prepared from Tris and EDTA, and is mainly used for dissolving nucleic acids, and can stably store DNA and RNA. Solutions resistant to pH changes when small amounts of acid or base are added.
This step S400 is followed by the following steps: and (3) separating, purifying and drying the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension to obtain silk fibroin-polylysine-polydeoxyribonucleotide (SF-EPL-PDRN) microsphere powder. The SF-EPL-PDRN microsphere powder obtained by the step has smaller particle size, uniform distribution, biocompatibility, antibacterial activity and wound healing promoting performance.
In particular, the invention also provides a preparation method of the wound repair dressing, which comprises the following steps: dispersing silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere powder in a sodium hyaluronate solution to obtain the wound repair dressing.
In one embodiment, the density ratio of silk fibroin-polylysine-polydeoxyribonucleotide composite microspheres to sodium hyaluronate solution is any one of 1:2-4, e.g. can be 1:2, 1:3 or 1:4.
The following is a detailed description of specific embodiments:
embodiment one:
in this embodiment, step S100 specifically includes: 5g of silkworm cocoons are put into 2L of a silk cocoon containing 4.2g of Na 2 CO 3 Continuously stirring, taking out and washing 3 times after 30min, and soaking in ultrapure water for 4h to remove sericin and ions. And (5) after degumming, drying the silk in a baking oven at 45 ℃ to obtain degummed silk. Adding degummed silk into ternary system CaCl 2 :C 2 H 5 OH:H 2 In O, caCl 2 :C 2 H 5 OH:H 2 The molar ratio of O is 1:2:8, the solution is fully dissolved at the temperature of 70 ℃, and is centrifuged for 10min at 9500r/min, the obtained solution is put into a dialysis bag (3500 kDa) and dialyzed in deionized water for 3 days, the dialyzed solution is centrifuged (the rotating speed is 9500r/min, the time is 20 min), the impurities are removed to obtain SF solution, and finally the SF solution is sterilized at high temperature and high pressure to obtain sterile SF solution.
The step S200 specifically includes: 420mg of EPL was dissolved in 2ml of ultrapure water, and added dropwise to 10ml of a 3% SF solution, followed by stirring at room temperature for 2 hours to obtain a SF-EPL mixed solution.
The step S300 specifically includes: and slowly dripping the SF-EPL mixed solution into 50ml of ethanol solution to prepare SF-EPL composite microsphere suspension with uniform size, centrifuging, washing with water, and freeze-drying to obtain the SF-EPL composite microsphere.
The step S400 specifically includes: and (3) weighing 20mg of SF-EPL composite microspheres, dispersing in 1ml of ultrapure water, dissolving 14mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL nano composite microsphere heavy suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, washing with water, and obtaining the SF-EPL-PDRN composite microsphere suspension.
And step S400, freeze-drying the SF-EPL-PDRN composite microsphere suspension to obtain SF-EPL-PDRN composite microspheres with smaller particle size, uniform distribution, biocompatibility, antibacterial activity and wound healing promotion.
In this embodiment, there is also provided a method for preparing a wound repair dressing, the method comprising the steps of: 10mg/ml SF-EPL-PDRN composite microspheres are uniformly dispersed in 30mg/ml sodium Hyaluronate (HA) solution to prepare the wound repair dressing.
Embodiment two:
the difference between the second embodiment and the first embodiment is that step S200 is different from step S400, and in the second embodiment, step S200 is specifically: 140mg of EPL was dissolved in 2ml of ultrapure water, respectively, and added dropwise to 10ml of a 3% SF solution, followed by stirring at room temperature for 2 hours to obtain a SF-EPL mixed solution.
The step S400 specifically includes: weighing 20mg of SF-EPL composite microspheres, suspending and dispersing in 1ml of ultrapure water, dissolving 4mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Embodiment III:
the difference between the third embodiment and the second embodiment is that step S200 is different, and in the third embodiment, step S200 is specifically: 280mg of polylysine was dissolved in 2ml of ultrapure water, respectively, and added dropwise to 10ml of a 3% SF solution, followed by stirring at room temperature for 2 hours to obtain a SF-EPL mixed solution.
Embodiment four:
the fourth embodiment differs from the second embodiment in that step S200 is different, and in the fourth embodiment, step S200 specifically includes: 350mg of EPL was dissolved in 2ml of ultrapure water, and added dropwise to 10ml of a 3% SF solution, followed by stirring at room temperature for 2 hours to obtain a SF-EPL mixed solution.
Fifth embodiment:
the fifth embodiment differs from the first embodiment in that step S400 is different, and in the fifth embodiment, step S400 specifically includes: weighing 20mg of SF-EPL composite microspheres, suspending and dispersing in 1ml of ultrapure water, dissolving 4mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Example six:
the difference between the sixth embodiment and the second embodiment is that step S200 is different, and in the sixth embodiment, step S200 is specifically: 840mg of EPL was dissolved in 2ml of ultrapure water, and added dropwise to 10ml of a 3% SF solution, followed by stirring at room temperature for 2 hours to obtain a SF-EPL mixed solution.
Embodiment seven:
the seventh embodiment differs from the first embodiment in that step S400 is different, and in the seventh embodiment, step S400 specifically includes: weighing 20mg of SF-EPL composite microspheres, suspending and dispersing in 1ml of ultrapure water, dissolving 10mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Example eight:
the difference between this embodiment eight and the first embodiment is that step S400 is different, and in this embodiment eight, step S400 is specifically: weighing 20mg of SF-EPL composite microspheres, suspending and dispersing in 1ml of ultrapure water, dissolving 16mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Example nine:
the difference between the ninth embodiment and the first embodiment is that step S400 is different, and in the ninth embodiment, step S400 is specifically: weighing 18mg of PDRN and dissolving in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere heavy suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Example ten:
the tenth embodiment differs from the first embodiment in that step S400 is different, and in the tenth embodiment, step S400 is specifically: weighing 20mg of SF-EPL composite microspheres, suspending and dispersing in 1ml of ultrapure water, dissolving 20mg of PDRN in 4ml of TE buffer, uniformly mixing the SF-EPL composite microsphere suspension and the PDRN solution to obtain a 5ml mixed system, stirring for 12 hours, centrifuging, and washing with water to obtain the SF-EPL-PDRN composite microsphere suspension.
Fig. 3 shows a transmission electron microscope picture of the surface morphology of particles after the beta-overlap induced by ethanol in a 3% sf solution according to an embodiment of the present invention. Figure 4 shows a transmission electron micrograph of the surface morphology of a composite microsphere after EPL modification of 3% sf with ethanol induced β -folding according to one embodiment of the present invention. From fig. 3 and 4, it can be seen that EPL enables SF to self-assemble more tightly, forming smaller, clearer, more spherical composite microspheres, demonstrating successful SF modification.
Fig. 5 shows a transmission electron microscope picture of the surface morphology of PDRN loaded composite microspheres after EPL modification of 3% sf and ethanol induced overlap according to an embodiment of the present invention. From fig. 5, it can be seen that a thin film is formed on the surface of the composite microsphere, and the particle size is slightly increased, which indicates that the PDRN loading is successful.
Fig. 6 shows a picture of the number of bacteria in the blank group. FIG. 7 is a photograph showing the number of bacteria incubated with a silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension. From fig. 6 and 7, it can be seen that the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere has a good antibacterial effect.
FIG. 8 shows a statistical plot of the numbers of the blank and silk fibroin-polylysine-polydeoxyribonucleotide complex microsphere group bacteria. From FIG. 8, it is clear that the bacteriostasis rate of the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere reaches 90%.
FIG. 9 is a graph showing statistics of potential data of a silk fibroin-polylysine mixed solution prepared by taking 0, 280mg, 350mg, and 420mg of polylysine in step S200, respectively, according to one embodiment of the present invention. From FIG. 8, it is clear that the addition of 420mg polylysine maximizes the potential of the silk fibroin-polylysine composite microsphere, allowing more PDRN to be loaded.
FIG. 10 shows a potential histogram of SF-EPL-PDRN microspheres obtained when the mass of polydeoxyribonucleotide in step S400 is taken as 0, 4mg, 10mg, 14mg and 16mg, respectively, according to an embodiment of the present invention. The successful loading of PDRN was seen from the potential change in FIG. 9, and the 14mg PDRN loaded SF-EPL showed weak electropositivity and better adhesion to cells, so that 14mg PDRN was selected for the experiment.
Fig. 11 shows the size of diabetic skin wound wounds on the first and seventh days of the blank.
Figure 12 shows the size of diabetic skin wound wounds on the first and seventh days of SF-EPL-PDRN composite microsphere sets. From FIGS. 11 and 12, it is understood that the SF-EPL-PDRN composite microsphere group has better skin repair performance than the blank group.
Figure 13 shows a statistical plot of skin relative wound size for the blank and silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere treatment groups. From fig. 13, it is clear that the SF-EPL-PDRN composite microsphere set has good skin repair performance, and can improve the therapeutic effect of diabetic wounds.
By now it should be appreciated by those skilled in the art that while a number of exemplary embodiments of the invention have been shown and described in detail herein, many other variations or modifications that are consistent with the general principles of the invention may be directly determined or derived from the disclosure of the invention without departing from the spirit and scope of the invention. Accordingly, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.
Claims (9)
1. The preparation method of the composite microsphere is characterized by comprising the following steps:
obtaining a silk fibroin solution;
mixing the silk fibroin solution with a polylysine solution to obtain a silk fibroin-polylysine mixed solution, wherein the mass concentration ratio of the silk fibroin solution to the polylysine solution is any one of 3:35-3:42;
adding the silk fibroin-polylysine mixed solution into an organic volatile solvent to obtain silk fibroin-polylysine composite microsphere suspension with positive charges on the surface, and then separating, purifying and drying to obtain silk fibroin-polylysine composite microsphere powder;
and adding the silk fibroin-polylysine composite microsphere powder into a buffer solution of polydeoxyribonucleotide to absorb polydeoxyribonucleotide with negative charge by utilizing electrostatic action, thereby obtaining silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension.
2. The method of manufacturing according to claim 1, further comprising the step of:
and separating, purifying and drying the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere suspension to obtain silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere powder.
3. The method according to claim 1, wherein the mass of the polydeoxyribonucleotide in the buffer solution of polydeoxyribonucleotide is any value in the range of 1-16 mg.
4. The method of claim 1, wherein the volume ratio of the organic volatile solvent to the silk fibroin solution is any one of 5-10:1.
5. The method of claim 1, wherein the buffer solution is TE buffer.
6. The method of any one of claims 1-5, wherein the obtaining a silk fibroin solution comprises the steps of:
degumming silkworm cocoons to obtain silk fibroin fibers;
and adding the silk fibroin fibers into a ternary system for dissolution, and obtaining the silk fibroin solution through dialysis, filtration and concentration.
7. The method of claim 6, wherein the ternary system is a system of calcium chloride, ethanol, and water, wherein the molar ratio of calcium chloride, ethanol, and water is any one of 1:1-3:7-9.
8. A method of preparing a wound repair dressing, comprising the steps of:
dispersing the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere powder prepared by the preparation method of any one of claims 2-7 in a sodium hyaluronate solution to obtain the wound repair dressing.
9. The method according to claim 8, wherein the density ratio of the silk fibroin-polylysine-polydeoxyribonucleotide composite microsphere and the sodium hyaluronate solution is any one of 1:2-4.
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| AU2015201538A1 (en) * | 2009-03-04 | 2015-04-16 | Trustees Of Tufts College | Silk fibroin systems for antibiotic delivery |
| JP6362540B2 (en) * | 2011-11-09 | 2018-07-25 | トラスティーズ・オブ・タフツ・カレッジＴｒｕｓｔｅｅｓ Ｏｆ Ｔｕｆｔｓ Ｃｏｌｌｅｇｅ | Injectable silk fibroin particles and uses thereof |
| CN104356395B (en) * | 2014-11-12 | 2016-08-17 | 苏州大学 | A kind of cationization fibroin albumen, preparation method and applications |
| KR20170060599A (en) * | 2015-11-24 | 2017-06-01 | (주)한국비엠아이 | Composition for injection of hyaluronic acid comprising cross-linked hyaluronic acid derivative and DNA fraction, and use thereof |
| CA3026239A1 (en) * | 2016-06-01 | 2017-12-07 | 3-D Matrix, Ltd. | Hemostatic powders with self-assembling peptide hydrogels |
| WO2019040850A1 (en) * | 2017-08-24 | 2019-02-28 | The Regents Of The University Of Colorado, A Body Corporate | Topical silk compositions and methods of using |
| CN107982239B (en) * | 2017-12-08 | 2020-02-21 | 中国医学科学院生物医学工程研究所 | Protein-based non-spherical microcapsule with hydrophobic drug crystal as template and preparation method thereof |
| RU2749803C1 (en) * | 2018-01-10 | 2021-06-17 | Джи2Джибио, Инк. | Polycaprolactone microspheric filler containing collagen peptide and method for its production |
| WO2022035078A1 (en) * | 2020-08-13 | 2022-02-17 | 한지성 | Marine algae-derived polydeoxyribonucleotides and polynucleotides having neovascularization and cell regeneration effects, and extraction method therefor |
| AU2020102982A4 (en) * | 2020-10-23 | 2020-12-24 | Wuhan Youngsen Biotech Co., Ltd. | Anticoagulant coating liquid containing heparin, coating microsphere, preparation method and application for artificial blood vessels |
| CN112933214A (en) * | 2021-02-05 | 2021-06-11 | 王吉成 | Composition for promoting wound healing |
| CN113368054A (en) * | 2021-06-16 | 2021-09-10 | 常州碧研医疗科技有限公司 | Preparation method of poly-deoxyribonucleotide flexible nano-liposome |
| CN114113579B (en) * | 2022-01-26 | 2022-04-08 | 丹娜(天津)生物科技股份有限公司 | New crown RBD protein fluorescent microsphere composite preparation |
| CN114642606B (en) * | 2022-04-18 | 2024-02-13 | 山东禾三千医疗科技有限公司 | Composition with skin barrier repair function and preparation method and application thereof |
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