KR20040011786A - Wound dressing of silk fibroin nanofibers nonwoven and its preparation - Google Patents

Abstract

PURPOSE: A cover material for cut injury made of silk-fibroin nano-fiber and having structure of a non-woven fabric is provided to obtain high closing ability and air-permeation and advantage of cell re-generation of damaged tissue. CONSTITUTION: The cover material comprises the silk fibroin obtained by removing sericin from the silk and other additives such as plastic agent, softening agent, anti-bacterial agent, anti-microorganism agent, cells, enzymes, antibodies, pigments and other mixture. The material is prepared by freezing and drying the silk-fibroin solution; dissolving the silk fibroin in an electrically radiational solvent; then radiating the solution. The radiational solvent is formic acid or water solution of the formic acid.

Description

Wound coating material in the form of non-woven fabric made of silk fibroin nanofibers and its manufacturing method {WOUND DRESSING OF SILK FIBROIN NANOFIBERS NONWOVEN AND ITS PREPARATION}

The present invention relates to a wound dressing and a method of manufacturing the same, more specifically, wound fibrous obtained by removing the sericin from the silk fibers, wound wound having a structure in which the nanofibers of the silk fibroin entangled with each other in the form of a non-woven fabric, and by removing the sericin from the silk fibers It relates to a method for producing a wound coating comprising the step of lyophilizing the obtained silk fibroin solution, dissolving the dried silk fibroin in an electrospinning solvent and then electrospinning.

When the human body develops a defect such as a wound on the skin, the human body has a property of defending the defect and self-healing. In this case, wound dressing is used as an adjuvant to effectively heal the defect and to speed up the healing. The conditions for the wound dressing should be excellent in biocompatibility and there should be no rejection at the defect site, sufficient to absorb the bodily fluids exuded from the wound site, and the decomposition products will not break down even if some of the coating material is immersed in the new tissue. It should not be toxic and the wound and the cladding should be in proper contact. In addition, in order to prevent further infection of the defect site, it should be easy to mix with other medicines that can enhance the function of the wound healing aid and should be less reactive when mixed.

Various types of wound dressings including antibiotics are currently used for the treatment of burn sores due to burns and long bed life or unclean skin care, but are not effective in preventing infection. However, the demand for wound dressings for the treatment of pressure sores is expected to increase rapidly due to the aging of the population. Therefore, it is urgent to conduct R & D and market of products.

Natural silk fiber refers to a fiber made from yarn extracted from cocoon that grows on mulberry, and has been used as a high-quality fiber material since 4,000 years ago due to its high tensile strength, inherent gloss and excellent dyeability. The silk fiber has a structure in which two strands of fibroin are wrapped in a sericin outer membrane. Among them, fibroin (hereinafter referred to as "dog fibroin") is excellent in biocompatibility and does not adversely affect any surrounding tissues, so it is manufactured in various forms such as membranes, powders, gels, aqueous solutions, and the like. It is used in various fields.

In addition, silk fibroin is suitable for the skin, the powder form is useful as a material used for proliferating or activating epidermal cells, and the fine powder of silk fibroin is also used as a filler, a coating material, and a cosmetic material. At this time, the powder used in cosmetics or paint is used to remove sericin from natural silk fibers, drop the molecular weight with alkali and then grind. A method for preparing such powders is known from Korean Patent Laid-Open Publication No. 2001-52075, and it is reported that silk fibroin powder having a diameter of less than 3 micrometers is excellent in hygroscopicity, moisture resistance, and moisture permeability.

Korean Laid-Open Patent Publication No. 1999-7001234 discloses a wound coating material or a method for producing the amorphous film having a crystallinity of less than 10% by mixing silk fibroin and sericin in an appropriate ratio. In addition, Korean Laid-Open Patent Publication No. 2000-51938 discloses a wound wound coating in the form of a sponge. However, in the case of the wound coating material in the form of a film or sponge, since the silk fibroin is directly manufactured by lyophilization, there is a disadvantage in that the treatment effect is inferior due to the lack of flexibility and the adhesion between the skin and the coating material.

In addition, US Pat. No. 6,110,590 proposes a method of obtaining a nonwoven fabric of silk nanofibers by dissolving the silk fibers in hexafluoroisopropanol without pretreatment and then electrospinning them. However, the method has a disadvantage in that biocompatibility is inferior because sericin is not removed from the silk fiber, and in particular, it takes several months to dissolve the silk, and commercialization is difficult because hexafluoroisopropanol used as a solvent is very expensive.

Accordingly, the present inventors endeavored to develop a wound coating material that satisfies all the adhesion to the skin, permeability, drug impregnation such as antibiotics, and ease of use, so that the nanofibers of the silk fibroin obtained by removing sericin from the silk fibers are in the form of nonwoven fabric. The present invention has been completed by confirming that a wound coating having an intertwined structure can satisfy the above requirements.

It is an object of the present invention to provide a wound dressing which satisfies both adhesion to skin, permeability, drug impregnation such as antibiotics, and ease of use.

Another object of the present invention is to provide a wound dressing having a structure in which nanofibers of silk fibroin obtained by removing sericin from silk fibers are entangled with each other in the form of a nonwoven fabric.

Still another object of the present invention is to provide a method for producing a wound coating material comprising lyophilizing a silk fibroin solution obtained by removing sericin from silk fibers, dissolving the dried silk fibroin in an electrospinning solvent, and then electrospinning. .

1 is a schematic diagram showing an example of an electrospinning apparatus used for electrospinning,

Figure 2 is a result of observing magnified 5,000 times the microfiber of silk fibroin prepared in Example 1 of the present invention,

Figure 3 is a graph showing the distribution of the frequency according to the diameter of the silk fibroin ultrafine fiber yarn prepared in Example 1 of the present invention,

4 is a result of measuring the cell adhesion ability of normal skin keratinocytes and oral keratinocytes of normal humans by the silk fibroin fiber aggregate prepared in the present invention,

PS: Polystyrene SF only: Silk fibroin nonwoven

BSA: Bovine serum albumin treatment in SF sample Coll: Type I collagen treatment in SF sample

FN: Skin Ronectin treatment on SF sample LN: Laminin treatment on SF sample

Figure 5 is a photograph showing the result of comparing the wound site after 4 days for the silk fibroin nanofiber aggregate (left) and lyophilized sponge sheet (right).

The wound dressing according to the present invention has a structure in which nanofibers of silk fibroin obtained by removing sericin from silk fibers are entangled with each other in the form of a nonwoven fabric. As used herein, the term "nanofiber (or ultrafine fiber)" refers to a fiber having a nanoparticle diameter, and nanofibers having a particle diameter of 50-1000 nm can be easily manufactured by appropriately adjusting conditions during electrospinning. Nanofibers having a particle diameter of 100-500 nm are preferred in consideration of contact with skin, the ratio of voids in the wound coating, the mechanical strength of the wound coating, and the like. Most preferably 100-300 nm.

The wound dressing of the present invention is a fiber assembly composed of nano-micron diameter microfiber, flexible in nature, having a lot of micro spaces and having a large surface area per unit weight, so that the wound coating material has good adhesion and air permeability. It is excellent and can prevent infection by the invasion of bacteria from the outside, so that cells of damaged tissues can be efficiently regenerated. When the damaged tissues are skin keratinocytes and oral keratinocytes, they show excellent effects. According to an embodiment of the present invention, the non-woven wound coating material made of silk fibroin nanofibers has cell adhesion ability of normal human epithermal keratinocytes (NHEK) and noraml human oral keratinocytes (NHOK). As a result of the measurement, the cell adhesion was significantly improved compared to the conventional sponge-type wound coating material (see FIG. 4 ).

In order to prevent further infection of the defect site, the wound dressing of the present invention may further include an additive additionally used as an additive commonly used in wound dressings, and examples of the additive include plasticizers, softeners, antibiotics, antimicrobial agents, Cells, enzymes, antibodies, pigments and the like.

The present invention also relates to a method for producing a wound dressing, the method comprising lyophilizing a silk fibroin solution obtained by removing sericin from silk fibers, and dissolving the dried silk fibroin in an electrospinning solvent and then electrospinning. The method of the present invention can also reduce the solubility in water and improve the mechanical strength of the fiber by performing recrystallization of the silk fibroin nanofibers. Examples of solvents that can be used for recrystallization include C ₁ to C ₃ alcohols such as methanol, ethanol, propanol, isopropanol or aqueous solutions thereof.

Natural silk fibers extracted from silkworm cocoons have a structure in which two strands of fibroin are wrapped in a sericin envelope, and the step of removing sericin from silk fibers is called refining. Such refining processes are well known to those of ordinary skill in the art. For example, a refining method using proteolytic enzymes such as Asperdillus oryzae, a refining method of boiling in an alkaline aqueous solution such as sodium carbonate or sodium oleate, a high temperature-high pressure refining method using an autoclave, etc. Can be mentioned. Subsequent processes without the removal of sericin will generate a large amount of foam, which may cause a considerable number of problems in subsequent processes.

Sericin-free silk fibroin is dissolved in a suitable solvent to prepare a silk fibroin solution. The method of obtaining a silk fibroin solution is also well known. For example, after adding and dissolving silk fibroin to an aqueous ethanol solution containing neutral salts such as lithium chloride, lithium bromide, sodium iodide, zinc chloride, calcium chloride, and the like, the solution Can be achieved by dialysis using a dialysis membrane such as cellophane membrane to completely remove the neutral salt.

The silk fibroin solution is changed into a sponge by lyophilization. The obtained sponge fibroin is dissolved in an electrospinning solvent and then applied to an electrospinning apparatus to produce a wound coating material having a structure in which nanofibers are entangled with each other in the form of a nonwoven fabric. As used herein, the term "electrospinning solvent" refers to a solvent that can be applied to electrospinning on the premise that it can dissolve the silk fibroin. According to the embodiment of the present invention, it was confirmed that the formic acid or the formic acid aqueous solution satisfies all the above requirements.

The electrospinning device that can be used for electrospinning is not particularly limited, and may be appropriately selected in consideration of the diameter of the nanofibers, the thickness of the nanofibers, and the like. Spinning devices can be widely used.

The diameter of the nanofibers (or ultrafine fibers) is 50-1000 nm, preferably 100-500 nm, most preferably 100 by appropriately adjusting the concentration of the silk fibroin solution, the type of electrospinning apparatus used and the conditions during electrospinning. Can be adjusted appropriately within the range of 300 nm. Such matters will be apparent to those of ordinary skill in the art. According to the specific example of this invention, it is preferable to use 5-20% with respect to the weight of the electrospinning solvent like formic acid or its aqueous solution, and, as for the silk fibroin concentration, 8-10% is more preferable. In addition, the given voltage is preferably performed in the range of 5 to 35 kV, more preferably 15 to 25 kV. As for the distance between a spinneret and an integrated board, 5-30 cm was preferable, More preferably, it was 5-15 cm. However, the concentration of the silk bromine solution, the voltage and the distance between the spinneret and the integrated plate should be determined in consideration of the type of electrospinning apparatus, the required physical properties, the shape of the wound coating material, and the like. The electrospun nanofibers formed a nonwoven form intertwined with each other ( FIGS. 2 and 3 ),

The method of the present invention can also reduce the solubility in water and improve the mechanical strength of the fiber by performing recrystallization of the silk fibroin nanofibers. Examples of solvents that can be used for recrystallization include C ₁ to C ₃ alcohols such as methanol, ethanol, propanol, isopropanol or aqueous solutions thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples.

This embodiment is intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples. Those skilled in the art to which the present invention pertains may make modifications or improvements with reference to the present specification without departing from the scope and object of the present invention.

<Example 1>

The silk fiber, previously washed with hot water, was immersed in water corresponding to 100 times the weight of the fiber. With respect to the amount of the silk fiber, 0.3% of sodium oleate in a weight ratio was further added, treated at 95 ° C. for 120 minutes, washed with water, and then treated with 0.1% sodium oleate at 95 ° C. for 60 minutes. The sericin of silk fiber was removed by neutralizing the solution with aqueous sodium carbonate solution and washing with boiling water several times. The silk fiber from which sericin was removed was added to a mixed solvent having a molar ratio of calcium chloride, distilled water, and anhydrous ethanol 1: 2: 8, respectively, and stirred at 70 ° C. for 4 hours to dissolve. The silk fiber from which sericin was removed was placed in a cellulose dialysis membrane, and dialyzed under distilled water for 3 days to prepare a pure silk fibroin solution in which salt and ethanol were completely removed. The silk fibroin solution from which the salt and ethanol were removed was freeze-dried at -80 ° C and lyophilized for 2 days in a freeze dryer maintained at -4 ° C to obtain a sponge-derived silk fibroin. The silk fibroin was dissolved in formic acid to prepare a 9% solution. Using the electrospinning apparatus of FIG. 1, the distance from the tip of the spinneret to the integrated plate was fixed at 5 cm and a voltage of 15 kV, followed by electrospinning of the silk fibroin nanofibers. A fiber assembly was prepared. The prepared nonwoven fabric of ultrafine fiber yarn was immersed in an aqueous methanol solution for 10 minutes and then crystallized. After completion of the crystallization, methanol and water were removed to prepare a water-insoluble fiber aggregate. An image analyzer (Scope Eye, Korea) was used to analyze the diameter of the ultrafine fibers constituting the fiber aggregate. Figure 2 shows the results, it was confirmed that the number of fibers of the diameter corresponding to the diameter of 150 ~ 300 nm. The fiber assembly prepared above was observed at a magnification of 5,000 times using a scanning electron microscope (Hidachi S-2350, Japan), and the results are shown in FIG. 3. As can be seen from the above results, the fiber aggregate had a structure in which nanofibers having a uniform thickness of 150 to 300 nm were entangled with each other in the form of a nonwoven fabric.

<Example 2>

The same concentration as in Example 1 except for dissolving the silk fibroin in formic acid, the same concentration of 9% solution and the distance from the tip of the spinneret to the integrated plate is 7 cm, and electrospinning by changing the voltage to 20 kV It was performed by the method. As a result of the above, an aggregate of ultrafine fiber yarns having a relatively uniform thickness (210 nm ± 140) was prepared.

<Example 3>

Silk fibroin was dissolved in formic acid and prepared in the same concentration of 9% solution, and the same as in Example 1 except that the electrospinning by changing the distance from the spinneret tip to the integrated plate 7 cm and the voltage to 25 kV It was performed by the method. As a result, an aggregate of ultra-fine fiber yarns having a relatively uniform thickness (230 nm ± 150) was prepared.

Experimental Example 1 Measurement of Cell Adhesion of Wound Fibroin Nanofiber Nonwoven Fabric

In order to measure the cell adhesion ability of normal human skin (NHEK) and oral keratinocytes (NHOK) by the silk fibroin nanofiber aggregate prepared in Example.

Step 1: Preparation of Skin Keratinocytes

NHEKs (normal human epithermal keratinocytes, hereinafter referred to as "NHEKs") were taken from human epidermal tissue and tissue samples were obtained from healthy humans between 1 and 3 years of age. The tissue samples were washed three times with calcium- and magnesium-free Hanks balanced salt solution (CMF-HBSS; GIBCO) containing no calcium and magnesium.

In order to separate epithelial tissues from epidermal tissues, the tissues contained collagenase (collgenase type II, 1.0 mg / ml; sigma Chemical Co.) and dispase grade II (2.4 mg / ml; Boeringer-Mannheim). CMF-HBSS was added and incubated for 90 minutes at 37 ° C. under 95% air and 5% carbon dioxide conditions. NHEK was prepared from the epidermal tissue separated as described above, and cultured in epithelial cell culture (KGM; Clonetics Corp.) containing 0.15mM calcium and growth factor (bulkart). NHEKs were dispensed at 1 × 10 ⁵ cells per 60 mm Petri dishes when the cell density was approximately 70% and incubated until the cell density became approximately 70%. Subculture was performed in this way, and two passages of NHEK were used.

Step 2: Preparation of Oral Keratinocytes

NHOK (normal human oral keratinocytes, hereinafter referred to as "NHOK") was treated with the same method as NHEK except that tissue samples were obtained from gingival tissues of humans and obtained from healthy individuals between the ages of 18 and 30 years.

Step 3: Measuring Cell Adhesion

Prepared by 10 minutes treatment with aqueous methanol solution, 14 mm diameter silk fibroin nanofiber non-woven fabric into a 24-well culture dish, 0.3 ml bovine serum albumin (1 mg / ml), type I collagen ( Extracellular protein (50 μg / ml), fibronectin (1 μg / ml) or laminin (10 μg / ml) was added and incubated for 12 hours at room temperature. ) Was coated. When only silk fibroin nanofiber nonwovens were tested, 0.3 ml of phosphate buffered saline (PBS) was added and treated the same. Extracellular protein was removed and each well was carefully washed once with PBS. In order to block the active sites not coated with the extracellular matrix protein, 0.4 ml of bovine serum albumin (1 mg / ml) was added to each well and incubated at 37 ° C. for 30 minutes. Bovine serum albumin was removed and washed carefully with PBS. 1 x 10 ⁵ cells (0.5 ml) were dispensed per well and incubated at 37 ° C. for 60 minutes under 95% air and 5% carbon dioxide conditions. The culture medium used was cultured in epithelial cell culture (KGM; Clonetics Corp.) containing 0.15 mM calcium and growth factor bullet kit. After the culture was removed, it was carefully washed once with PBS. 0.4 ml of 10% formalin solution (10% formalin in PBS) was added and the cells were fixed by standing at room temperature for 15 minutes. After washing twice with PBS, 0.4 ml of 1% toluidine blue dissolved in 10% formalin solution was added thereto, and left at room temperature for 1 hour. Toluidine blue was removed after standing, and the silk fibroin nanofiber nonwoven fabric was washed three times with distilled water to allow the dye at the bottom to escape. When washing, washed for 1 hour while shaking each time. Silk fibroin nanofiber nonwovens were transferred to a new 24-well culture plate, and 0.2 ml of 2% SDS was added and incubated for 15 minutes to lyse the cells. Cell lysates were diluted 6-fold, 0.1 ml was taken and transferred to a 96 well plate, and the absorbance was measured at 595 nm with a microplate reader Model 550 (Bio-Rad).

Figure 4 shows the results of measuring the cell adhesion of the normal human skin (NHEK) and oral keratinocytes (NHOK) by the silk fibroin nanofiber aggregate prepared in Example, as compared to polystyrene, extracellular matrix protein pretreatment It was confirmed that the cell adhesion ability of the silk fibroin nanofiber nonwoven fabric regardless of whether or not (in Figure 4, PS is the result of treating only polystyrene, SF only is the result of treating only the silk fibroin nonwoven fabric, BSA is a silk fibroin nonwoven sample After the addition of bovine serum albumin to the treatment, Coll is the result of treatment after adding type I collagen to the silk fibroin non-woven sample, FN is the result of treatment after the addition of dermalonectin to the silk fibroin non-woven sample , LN is the result of treatment after addition of laminin to the sample of silk fibroin).

Experimental Example 2 Evaluation of Wound Healing Capacity of Non-woven Fabric of Silk Fibroin for Wound Coatings

A lyophilized silk fibroin sponge sheet, which was in a state before the electrospinning, was made by making two square wounds (skin layer defects) each having a size of about 1 cm × 1 cm on the back of a rat having a weight of about 250 g. 2000-51938), and then suture and fixation, and on the other side, the non-woven silk fibroin nanofiber aggregate was fixed in the same manner. After 4 days, the wound was observed.

5 is a photograph showing the results of comparing the wound site after 4 days with respect to the silk fibroin nanofiber aggregate (left) and the lyophilized sponge sheet (right) of the present invention, the silk fibroin nanofiber nonwoven fabric (right), Compared to the conventional lyophilized sponge sheet (left), the wound was healed at a significantly faster rate, and from these results, it was confirmed that the wound healing ability of the silk fibroin nanofiber nonwoven fabric was significantly superior to the lyophilized sponge sheet.

The wound dressing of the present invention is a fiber aggregate composed of nano-micron diameter microfiber, flexible in nature, has a lot of micro spaces and has a large surface area per unit weight, and thus the adhesion and air permeability to the skin when developed as a wound dressing It is excellent and can prevent infection due to the invasion of bacteria from the outside, has the advantage of efficiently regenerating cells of damaged tissue.

Claims (8)

A wound coating material having a structure in which nanofibers of silk fibroin obtained by removing sericin from silk fibers are entangled with each other in the form of a nonwoven fabric. 2. The wound dressing according to claim 1, wherein the wound dressing further comprises an additive. The wound dressing according to claim 1, wherein the additive is selected from the group consisting of plasticizers, softeners, antibiotics, antimicrobial agents, cells, enzymes, antibodies, pigments, and mixtures thereof. The wound dressing according to claim 1, wherein the wound dressing is applied to skin keratinocytes and oral keratinocytes. A method of producing a wound dressing according to claim 1, comprising lyophilizing a silk fibroin solution obtained by removing sericin from silk fibers, dissolving the dried silk fibroin in an electrospinning solvent, and then electrospinning. The method of claim 5, wherein the electrospinning solvent is formic acid or an aqueous formic acid solution. The method of claim 5, wherein the method further comprises recrystallizing the fibroin nanofibers after electrospinning. 8. A process according to claim 7, wherein said recrystallization is carried out in C ₁ to C ₃ alcohols or aqueous solutions thereof.