KR20000051938A - Preparation of silk fibroin membrane for wound covering materials - Google Patents

Abstract

PURPOSE: A wound skin-covering film consisting of fibroin protein and preparation method thereof are provided. CONSTITUTION: Fibroin is obtained from silkworms by using protease of Aspergillus oryzae. The fibroin film should have a alpha-helix structure to be attached closely on the wound. A fibrin solution for producing the fibrin film is prepared by the steps of: dissolving fibroin obtained from silkworms in hydrogen chloride, hydrogen sulfate, lithium chloride, lithium bromide, sodium iodine, zinc chloride, and calcium chloride; and removing salts by filtration with filter such as cellophane membrane. The fibroin film is prepared by freeze-drying the fibroin solution and the resulting film should have a 0.1 to 10 mm of thickness.

Description

Preparation method of silk fibroin membrane for wound coating {{Preparation of silk fibroin membrane for wound covering materials}

The present invention relates to a novel wound dressing and a method of manufacturing the same, and more particularly, biocompatibility, a part of the membrane is melted by rapidly lyophilizing a fibroin protein solution in the peptide constituting the silk protein in the form of a membrane The present invention relates to providing a wound dressing having excellent adhesion to the upper surface, moisture permeability, and skin regeneration ability.

In general, when a defect occurs in the body (living body), the organism has a property of self-healing and defending the defect to some extent. In particular, when the defect site of the living body corresponds to the skin such as a wound, it is necessary to have a fairly sensitive condition such as strictly requiring the biocompatibility of the coating material used to effectively heal the defect site. There may be various conditions for such wound dressings, but most of all, the biocompatibility is excellent, so there is little rejection at the defect site, and even if a part of the skin is immersed in the new tissue, the decomposition products are not toxic. It is desirable to have a structure that can be tightly adhered to, and also to be able to prevent infection at the defect site and to be less reactive when mixed with other medicines. Therefore, if these conditions are satisfied, the self-healing ability can be further promoted, and further, the development of artificial skin can be taken a step closer.

Currently, some wound dressings including antibiotics and other medicines have been used for the treatment of wounds such as fissures from burns and dirty skin care, but it is still not commercially available to effectively prevent infections. The need for this is recognized. Moreover, due to the aging trend of modern society, the amount of wound dressing required for the treatment of pressure sores is expected to far exceed the demand for the treatment of pressure sores.

Therefore, there is a demand for a material that can more powerfully and effectively cover the defects of the skin.

At present, known materials such as water-containing gelatin (Korean Patent Publication No. 90-6892), chitin-based film (Korean Patent Publication No. 97-5310), synthetic polymers (Cho Soo-soo et al., Korean Polymer Society 1996 Autumn conference). However, these materials still need a lot of evaluation and review for practical use.

Accordingly, the present inventors have searched for other materials that can be used as a wound dressing, and as a result, the silk fibroin is used as a surgical suture because the silk fibroin has excellent biocompatibility and does not affect any surrounding tissue. This study was conducted to use as a wound dressing.

On the other hand, USP 4,818,291 discloses a composition in which human fibrinogen-containing band-aid composition further contains silk fibroin, since human fibrinogen-based band-aid is used as a surgical band-aid. (helix) structure, but it is easily converted into β-sheet structure by the physical force such as stirring and vibration, so that it is insoluble, and the converted β-sheet structure is not converted back to α-helix structure It is used. In other words, silk fibroin of β-sheet structure was used as a binder.

However, in the case of using the β-sheet structure fibroin as the wound dressing desired in the present invention, the insolubility of the β-sheet structure slows absorption to the human body such as skin and muscles, thus exuding the fluid between the wound site and the wound dressing. (exudate) is stuck, the adhesion is bad, there is a problem that is easy to secondary infection by bacteria.

Therefore, the present inventors conducted a study in order to solve the above problems, as a result of rapid freeze-drying the silk fibroin solution to prepare a membrane having an α-helix structure, and when used as a wound coating material exudates from the wound site The present invention was completed by discovering that it can be gradually absorbed into the wound surface, and has good adhesion to the wound surface, prevents secondary infection by bacteria, and at the same time promotes the production of collagen to regenerate skin.

It is therefore an object of the present invention to provide a method for producing a silk fibroin protein membrane for use as a wound dressing.

Another object of the present invention to provide a protein membrane for wound fibroin prepared according to the above-described manufacturing method.

Figure 1 is a photograph of the skin cross-sectional tissue 10 times observed after coating the silk fibroin protein membrane of the present invention on the wound surface of the mouse for 7 days.

A: Cross section of mouse skin tissue in steady state

B: Cross section of mouse skin tissue of the control group that induced the wound

C: Cross section of mouse skin tissue coated with silk fibroin protein membrane on wound surface

Figure 2 is a photograph of the skin cross-sectional tissue 40 times after the silk fibroin protein membrane of the present invention coated on the wound surface of the mouse for 7 days.

A: Cross section of mouse skin tissue in steady state

B: Cross section of mouse skin tissue of the control group that induced the wound

C: Cross section of mouse skin tissue coated with silk fibroin protein membrane on wound surface

In order to achieve the above object, the production method of the present invention is characterized in that the membrane is prepared so that the final membrane thickness is 0.1 to 10 mm by rapid freeze-drying the aqueous solution of protein fibroin.

Hereinafter, the present invention will be described in more detail.

The silkworm cocoon used in the present invention may be used from the highest silkworm cocoons to lower grades of broken sand and waste dogs containing impurities.

The method for obtaining silk fibroin protein from silkworm cocoon is not particularly limited, and known methods such as hot-high pressure refining using protease or autoclave produced from Aspergillus oryzae and Bacillus mutants. Obtained by removing sericin from silkworms by law.

As a method for producing a silk fibroin protein solution from the obtained silk fibroin, a known method can be used, for example, a solution containing a salt such as hydrochloric acid, sulfuric acid, lithium chloride, lithium bromide, sodium iodide, zinc chloride, calcium chloride, or the like. After adding and dissolving silk fibroin to the solution, the solution is dialyzed using a dialysis membrane such as a cellophane membrane to completely remove the salt to obtain a silk fibroin protein solution.

On the other hand, in order to use the silk fibroin protein solution of the present invention as a wound coating should have a water-soluble α-helix structure. In other words, if the silk fibroin protein solution becomes an insoluble β-sheet structure instead of an α-helix structure, the fibroin protein solution does not absorb the exudates from the wound surface when applied to the wound, so that it does not adhere to the wound surface and is secondarily infected by bacteria. Since there is a problem such that it is easy to prolong the healing period, it should have an α-helix structure. Therefore, the method of the present invention is characterized in that the membrane is prepared by rapid freeze drying of the silk fibroin solution so that the silk fibroin has an α-helix structure.

On the other hand, in order to use the prepared protein membrane as a skin coating, the thickness of the membrane should be 0.1 to 10 mm. For example, in a 10 cm diameter curry, about 20 ml of an aqueous solution of silk fibroin is added and rapidly lyophilized. It can manufacture.

The silk fibroin protein membrane prepared according to the manufacturing method of the present invention may have a form of a membrane, a sponge, a nonwoven fabric, or the like.

In addition, the silk fibroin protein membrane of the present invention is a substance known for use as an artificial skin within the range that the α-helix structure of the silk fibroin protein is not converted into a β-sheet structure, for example, chitin, chitosan, gelatin, polyvinyl Antibiotics and medicines such as alcohol (PVA), propolis, silver sulfa diadine, neomycin and the like can be prepared by adding the silk fibroin protein solution.

The silk fibroin protein membrane prepared by the present invention can quickly absorb the exudates from the wound surface, has a strength similar to that of known artificial skins, and when a skin defect occurs, a considerable amount of water per unit area evaporates. Therefore, it is one of the important requirements for wound dressings to prevent the loss of moisture or body fluids and to maintain proper moisture state. The moisture permeation rate is better than that of known artificial skins. Moreover, the silk fibroin protein membrane of the present invention is effective as a wound dressing because it can activate the production of fibroblasts in the skin tissue, promote the production of collagen and regenerate the skin.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited only to these Examples.

Preparation Example 1 Preparation of Silk Fibroin Protein Membrane

1) Flavozyme, a proteolytic enzyme, is added at a rate of 1% (w / w) with respect to the amount of silk, and sufficient nitrogen gas is added for 5 minutes, followed by scouring at 55 ° C for 6 hours. Sericin was removed. The annual deterioration rate is maintained at around 23% to remove almost all sericin surrounding fibroin.

2) 532.8 g of calcium chloride, 692 mL of distilled water, and 560 mL of anhydrous ethanol were added to 70 g of the silk yarn from which sericin was removed, and the silk fibroin protein was heated at 90 ° C. for 5 hours. Thereafter, the silk fibroin solution was placed in a cellulose dialysis tube, and dialyzed with distilled water for 4 days to completely remove the salt.

3) In order to put the silk fibroin solution obtained in the above 2 into the film thickness range of 0.1 to 10 mm, 20 ml of a silk fibroin aqueous solution (about 2.5% solution) was added to a 10 cm diameter round shale, and then a balance plate was attached. After freezing at -90 ° C. or below, the freeze-dried freeze dryer was used to obtain a sponge-type fibroin protein membrane. At this time, the film thickness was about 0.5 mm on average, and the sponge-like film | membrane which was comparatively favorable in the pure white flexibility was obtained.

Test Example 1 Physical Properties and Water Permeability Test

In order to evaluate whether it can be used as a wound coating membrane using the silk fibroin protein membrane prepared in Preparation Example 1, the most important property among the moisture content, the tensile strength value of the dry state and the wet state, and the nature of the wound coating material or artificial skin The water permeability properties that can be examined were investigated.

1) Moisture Content Measurement

After weighing the dry weight of the silk fibroin protein membrane of Preparation Example 1, immersed in 75% methanol for 30 minutes to make the silk fibroin protein membrane insoluble membrane, washed with distilled water, and immersed in water for 24 hours. After that, the weight at the time of measurement was measured.

As a result, the average capacity was 60%. From this, it can be seen that the silk fibroin protein membrane of the present invention can rapidly absorb the exudate from the skin.

2) strength measurement

For the purpose of wound dressings, when the wound is covered, it must exhibit adequate strength. Therefore, after cutting the silk fibroin protein membrane of Preparation Example 1 to a size of 1.5 × 4 cm, the tensile strength values at the time of drying and wetting were measured by Universal Testing Machine (UTM). As a result, the tensile strength at drying was 0.7-1.0 kg / mm 2, and the wet tensile strength was 0,07 kg / mm 2.

This is because the tensile strength at drying of skin is about 1 kg / mm2 and the tensile strength at wetting is about 0.1 kg / mm2, so that the physical properties of the silk fibroin protein membrane of the present invention approach the standard value of artificial skin to some extent. To show.

3) Moisture evaporation measurement

One of the most important factors in the use of wound or artificial skin is the evaporation of moisture. When the skin is damaged, a significant amount of water per unit area evaporates. For example, first-degree burns show approximately the same amount of water evaporation as normal skin, whereas second-degree burns result in water loss of 20-40 times that of normal skin, resulting in dehydration. Can happen. Therefore, it is important to prevent loss of moisture or body fluids and to maintain proper moisture. In other words, if the moisture permeation amount of the wound dressing is too low, the exudates between the burned site and the wound dressing may be poor, resulting in poor adhesion, secondary infection by bacteria, and prolongation of the healing period. Moisture permeability of the is better to have a larger value than normal skin. Therefore, it is possible to promote the treatment while preventing the loss of water or body fluids in the burned area to prevent the threat of life and bacterial infection caused by severe dehydration.

The evaporation of water is measured in terms of the amount of weight transmitted per unit area of the membrane to a device capable of storing a certain amount of water. That is, after measuring the weight of the whole water permeation apparatus, the water permeation amount was calculated from the amount of the weight change in the state of 37 degreeC constant temperature and humidity.

As a result, the water permeation amount of the silk fibroin protein membrane was found to be 550-600 g / m 2 / day on average. Since this value is higher than the water evaporation amount of 200 ~ 500g / ㎡ / day of artificial skin, it can be seen that the silk fibroin protein membrane of the present invention has an excellent moisture permeation amount.

[Test Example 2] Wound coating effect test

The silk fibroin protein membrane of Preparation Example 1 was directly coated on the wound surface of the mouse, and its effect was visually observed with a microscope. That is, the hair of the mouse was removed, shaved and disinfected, and then a portion of the skin tissue of the mouse was artificially cut to a size of about 1 × 1 × 0.3 cm (width × length × depth). Subsequently, the silk fibroin protein membrane of Preparation Example 1 was coated on the surface of the cut skin tissue, and the body was rolled with a thin gauze (for hospital, manufactured by Dong-A Sangsa Co., Ltd.) and fixed to prevent the silk fibroin protein membrane from falling off (coated group). In addition, the wound surface of the mice not coated with the silk fibroin protein membrane protected the wound surface with gauze (control). By observation at the same time every day for 7 days, visual observations were made over the course of the coating days. In addition, normal skin tissue was used as a normal control group.

On the other hand, in order to observe the tissue cross section under a microscope, the tissues of the coated group, the control group and the normal control group were cut off 7 days after the coating, and the tissues were fixed in 15% formalin solution and the fixed skin tissues were removed by H & H. Staining was performed with E staining solution, and observed at a microscope magnification of 10 times (FIG. 1) and 40 times (FIG. 2).

As a result of daily visual observation, in the control group which protected the wound surface only with a gauze on the skin wound, the treatment of effusion solution was not resolved at all. As the days passed, black oxide coagulation appeared on the wound surface. However, when wound wound was coated with a silk fibroin protein membrane at the wound site, it was observed that exudates were gradually absorbed into the silk fibroin protein membrane and adhered firmly to the window surface, thereby exhibiting the flexibility of the skin tissue according to the movement of the mouse.

On the other hand, the observation results of the tissue cross section under the microscope, as can be seen from Figures 1 and 2, when the wound coating with the control (Fig. 1-B and 2-B) and the silk fibroin protein membrane (Fig. 1- Tissue defects were observed in both C and FIG. 2-C). However, in the case of the control group (Fig. 1-B), the skin surface is severely depressed, and the coating group coated with the silk fibroin protein membrane (Fig. 1-C) shows that the surface is relatively smooth. In addition, the tissue defects of the control group is mainly filled with inflammatory granulation tissue (Fibroblast) (Fig. 2-B), collagen (Collagen) is also partially proliferated (natural healing), but most of the vertical magnification. On the other hand, in the skin tissue coated with the silk fibroin protein membrane, some inflammatory fibroblasts remain in the wound area, but abundant collagen production with activated fibroblasts is observed in the dermal layer. In addition, the collagen array is also being regenerated horizontally, confirming that it firmly supports skin defects. In other words, in the case of the coating group coated with the silk fibroin protein membrane, it can be said that the epithelialization (derma) proceeds to the wound area and the restoration process of the skin is progressing normally.

From the above results, it can be seen that the silk fibroin protein membrane dissolves in the exudates from the defects of the skin and is a part of the skin by covering the minute irregularities of the wound surface closely, and absorbing the exudates shown by visual observation and excellent water permeation. It can be seen that the ability and the like worked effectively. Accordingly, when the silk fibroin protein membrane of the present invention is used as a wound dressing, the silk fibroin protein membrane exhibits exudative absorption and excellent water permeability, thereby preventing secondary infection of the wound, activating fibroblasts in the skin tissue, and collagen. Since it promotes the production, it can help the regeneration effect of the skin in the skin tissue and is effective as a wound dressing.

As can be seen from the above description, the silk fibroin protein membrane prepared according to the production method of the present invention is excellent in biocompatibility, skin regeneration ability, physical properties, water permeability, etc., and also activates fibroblast production in skin tissue. It is effective as a wound dressing because it can promote the production of collagen and regenerate skin.

Claims (5)

A method of producing a silk fibroin protein membrane for wound coating, characterized in that the final membrane is 0.1-10 mm thick by rapid freeze drying of aqueous solution of silk fibroin protein. The method of claim 1, wherein chitin, chitosan, polyvinyl alcohol (PVA), and propolis are further added to the silk fibroin protein solution. The method of claim 1, wherein an antibiotic is further added to the silk fibroin protein solution. The silk fibroin protein membrane produced by the method according to any one of claims 1 to 3. 5. The silk fibroin protein membrane according to claim 4, wherein the silk fibroin protein membrane has a form of a membrane, a sponge, or a nonwoven fabric.