WO2015130096A1

WO2015130096A1 - Nitrogen oxide-releasing wound treatment film and preparation method therefor

Info

Publication number: WO2015130096A1
Application number: PCT/KR2015/001864
Authority: WO
Inventors: 유진욱
Original assignee: 부산대학교 산학협력단
Priority date: 2014-02-28
Filing date: 2015-02-26
Publication date: 2015-09-03

Abstract

The present invention relates to a nitrogen oxide-releasing wound treatment film and a preparation method therefor. A nitrogen oxide-releasing film containing S-nitroglutathione, which is a nitrogen oxide donor to be spontaneously formed in the human body, has mechanical characteristics enabled to be applied to the human body, slowly releases nitrogen oxide and inhibits a pathogen, which is the main cause of wound infection, and can quickly heal a wound, and thus the film can be useful for treating a wound.

Description

Nitric oxide releasing wound treatment film and preparation method thereof

The present invention relates to a nitric oxide-releasing wound treatment film and a method for producing the same, which can inhibit pathogens which are the main cause of wound infection and can cure wounds promptly.

The skin that surrounds our bodies is exposed to various dangerous physical damages in our daily lives. Therefore, many factors around us, such as mechanical damage to the skin, bruises, burns, wounds occur. A wound is a state in which the anatomical continuity of a human tissue has lost its original continuity by external action. For example, our skin is composed of epidermis, dermis, and subcutaneous fat, and the loss of continuity such as epidermis, dermis, and subcutaneous fat by trauma such as cut or falling is called wound.

In general, dressing is used to effectively treat wounds of the skin such as wounds and traumas. Wound dressings require the ability to maintain adequate moisture at the contact surface with wounds, the ability to control wound secretions, ease of attachment and removal of dressings to wounds, and air and vapor penetration between external wounds. Sex, insulation of the wound area against the outside, resistance to the invasion of bacteria, non-toxic to the human body, excellent mechanical properties are required.

Currently, the most popular wound healing methods are ointment. The most popular products for promoting skin wound healing include Madecassol (Dongkuk), Sentica (Samjin Pharm) and Tinadex (Chong Kun Dang).

Prior art of the present invention is disclosed in Korean Patent Laid-Open No. 2002-0066024.

It is an object of the present invention to provide a wound healing film containing s-nitrosoglutathione, which is a nitric oxide donor spontaneously formed in the human body, and a method of manufacturing the same.

In order to achieve the above object, the present invention provides a nitric oxide releasing wound treatment film, characterized in that the nitric oxide donor is encapsulated in a polymer.

In addition, the nitric oxide donor may be selected from the group consisting of s-nitrosoglutathione, diaeniumdiolate, organic nitrate and iron-nitrosyl complexes. .

In addition, the polymer may be a biocompatible polymer selected from the group consisting of chitosan, silicone, polyurethane and calcium alginate.

In addition, the film may contain 2.5 to 30% by weight of a nitrogen oxide donor in 100% by weight of the film.

According to another aspect of the invention, dissolving the polymer in a solvent, adjusting the pH, and then adding a plasticizer to prepare a polymer solution; Adding a nitric oxide donor to the polymer solution and stirring to prepare a reactant; And casting the reactant in the form of a film and drying in a dark room dehumidified state.

In addition, the polymer may be selected from the group consisting of chitosan, silicone, polyurethane and calcium alginate.

In addition, the plasticizer may be selected from the group consisting of polyethylene glycol, sorbitol, and glycerol.

In addition, the step of preparing a polymer solution may be adjusted to pH 4.0 to pH 6.0 using 0.1 M acetate buffer solution.

In addition, the nitrogen oxide donor may be added in an amount of 2.5 to 30 parts by weight based on 100 parts by weight of the polymer solution.

In addition, the drying may be dried for 1 to 2 days at 25 to 37 ℃.

According to the present invention, the nitric oxide releasing film containing s-nitroglutathione (GSNO), a nitric oxide donor spontaneously formed in the human body, not only has mechanical properties applicable to the human body, but also gradually releases nitric oxide. In addition, the film can be usefully used for wound healing because it can inhibit pathogens which are the main cause of wound infection and can heal wounds quickly.

1 is a scanning electron microscope (SEM) photograph showing the surface shape of the chitosan film (a) and the GSNO film (b),

Figure 2 shows the thermal properties of the GSNO film 10, chitosan film (B), chitosan (C), GSNO (G) and a mixture (GC) of a mixture of GSNO and chitosan,

3 shows the NO release profile from the GSNO film,

Figure 4 shows the antimicrobial and anti-biofilm effect of the NO release film for Gram-positive bacteria and Gram-negative bacteria,

Figure 5 shows the macroscopic changes in the wound over time according to the application of NO release film,

Figure 6 shows the wound size reduction effect of the application of NO release film,

Figure 7 shows the epithelialization rate according to the application of NO release film,

FIG. 8 shows histopathological findings of wounds following application of NO release film (A-C: gauze control, D-F: NO 0 mg; G-I: NO 10 mg).

NO release film was prepared using a solvent evaporation method. That is, chitosan was dissolved in acetone buffer (100 mM) and the pH of the solution was adjusted to 4.4 using acetic acid. Glycerol was added to the chitosan solution to a final concentration of 1% (w / w). 10 mg GSNO was added to the solution and stirred for 20 minutes. The resulting solution was cast in Petri dishes and dried in a dark room at 37 ° C. for 2 days in a dryer equipped with a dehumidifier. The film thus prepared (hereinafter 'CS / NO film') was stored at 7 ° C. under vacuum and stored in the dark until use. At this time, a blank film containing no GSNO was prepared as a control.

In particular, the film was dried under dark and dehumidification conditions to promote evaporation. When the film is dried under dark and dehumidifying conditions, a reddish, transparent and homogeneous film having a loading efficiency of 47% nitric oxide donor can be obtained. In case of drying in a drying oven without dehumidification conditions, the loading efficiency was less than 20%.

On the other hand, in order to compare the effects of wound healing in vivo, polyvinyl alcohol (polyvinyl alcohol; PVA, Sigma Adlrich, MO, USA), which used a polymer other than chitosan, was used as a polymer and heat was applied to 1% (w / w). ) PVA solution was prepared, and a film was prepared by the same process as the CS / NO film (hereinafter 'PVA / NO film').

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

The inventors have found that the nitric oxide releasing film containing s-nitroglutathione (GSNO), a nitric oxide donor spontaneously formed in the human body, not only has mechanical properties applicable to the human body, but also gradually releases nitric oxide and wounds. The present invention has been accomplished by finding out that it is possible to suppress pathogens which are the main cause of infection and to cure wounds promptly.

Accordingly, the present invention provides a nitric oxide releasing wound treatment film, characterized in that the nitric oxide donor is encapsulated in a polymer.

The nitric oxide donor may be selected from the group consisting of s-nitrosoglutathione, diaeniumdiolate, organic nitrate and iron-nitrosyl complexes, In particular, it is more preferable to use s-nitrosoglutathione which is spontaneously formed in the human body and has high stability in the film manufacturing process and storage process.

The polymer may be a biocompatible polymer selected from the group consisting of chitosan, silicone, polyurethane, and calcium alginate, but more preferably chitosan, which absorbs wound secretions and maintains the surface of the wound while maintaining its own wound healing effect. .

It may contain 2.5 to 30% by weight of the nitrogen oxide donor in 100% by weight of the film. Nitric oxide donors are substances that release nitric oxide ('NO') after being mixed with a polymer solution and dried, and when the nitric oxide donor is added at less than 2.5% by weight, the amount of NO released is low and the wound healing effect is continuously decreased. When it is not possible to release NO, and in excess of 30% by weight, the overall efficiency of the manufacture of the wound care film is reduced and the mechanical properties and stability may be reduced.

The present invention also provides a method for producing a nitric oxide releasing wound treatment film by a solvent evaporation method. More specifically, the manufacturing method comprises the steps of dissolving the polymer in a solvent, adjusting the pH, and then preparing a polymer solution by adding a plasticizer; Adding a nitric oxide donor to the polymer solution and stirring to prepare a reactant; And casting the reactants in a film form and drying in a dark room dehumidified state.

According to one embodiment, the manufacturing method comprises the steps of dissolving a polymer such as chitosan in a solvent, adjusting the pH with an acidifying agent, and preparing a polymer solution by adding glycerol; Preparing a reactant by adding and stirring a nitric oxide donor such as s-nitrosoglutathione (hereinafter 'GSNO') to the polymer solution; Casting the reactant in the form of a film, and drying in a dark room dehumidified state.

In preparing the polymer solution, acetic acid buffer solution may be adjusted to pH 4.0 to pH 6.0 using acetic acid buffer solution of 0.1 M.

When the nitric oxide donor is mixed by maintaining the polymer solution with a weak acid, GSNO may be homogeneously mixed in the polymer solution, and does not affect the stability of the GSNO.

The plasticizer may be selected from the group consisting of polyethylene glycol, sorbitol, and glycerol, and the glycerol may increase mechanical properties by increasing solubility of the wound healing film.

The nitrogen oxide donor may be added in an amount of 2.5 to 30 parts by weight based on 100 parts by weight of the polymer solution, and then stirred for 10 minutes to 60 minutes, preferably 20 minutes. If the nitrogen oxide donor is added in excess of the content range, phase separation may occur, which may cause problems such as appearance problems and deterioration of mechanical properties.

The drying may be dried for 1 to 2 days at 25 to 37 ℃. If dried outside the temperature range, problems may occur in film transparency and stability of GSNO.

In particular, when the cast film is dried under the dark room and dehumidification conditions, the loading efficiency of the nitric oxide donor is 47%. On the other hand, when drying under dehumidification conditions, the loading efficiency of the nitric oxide donor is 20% or less. May be caused.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

<실시예 1> GSNO 합성Example 1 GSNO Synthesis

GSNO was synthesized according to a known method (Tetrahedron Lett, 1985; 26: 2013 ?? 6). That is, 1.005 g of reducing L-glutathione (Sigma Adlrich, MO, USA) was dissolved in 2M HCl (Daejung) at 4 ° C to prepare a solution having a final concentration of 0.625 mM. To the solution was added 220.6 mg of sodium nitrate (Sigma Adlrich, Mo., USA) and stirred on an ice bath for 30 minutes. After 40 minutes the solution so obtained was precipitated with cold 80% acetone and stirred for 20 minutes. The stirred solution was centrifuged at 4 ° C., 20,000 g for 30 minutes. The obtained precipitate was centrifuged by adding 20 mL of cold 100% acetone under the same conditions. The obtained precipitate was centrifuged by adding 20 mL of 100% dimethyl ether under the same conditions. The pink powder GSNO thus obtained was lyophilized for 24 hours, stored at −20 ° C., and used for later experiments.

<실시예 2> NO-방출 필름 제조Example 2 NO-Release Film Preparation

<실시예 3> NO-방출 필름의 특성 분석Example 3 Characterization of the NO-releasing Film

1. film thickness

The thickness of the film prepared in Example 2 was measured at five different locations (center and four corners) using a digital outer micrometer (Bluebird Multinational Co.). The average value of five positions was used as the thickness of the film.

As a result, the average thickness of each of the center and four corners of the blank film and the NO emitting film was 61.2 ± 4.5 μm and 64.6 ± 8.6 μm, confirming that the thickness of the film did not change with the addition of GSNO.

2. Scanning electron microscope (SEM) analysis

The surface shape of the CS / NO film prepared in Example 2 was investigated using SEM (FE-SEM, S4800, Hitachi, Japan). A 1.5 x 1.5 cm ² sample was placed on a double sided carbon tape and coated with platinum for 2 minutes under vacuum. The samples thus prepared were observed under FE-SEM at an acceleration voltage of 1-5 kV.

As shown in FIG. 1, the blank film exhibited a homogeneous and smooth surface shape, and there was no change in the surface shape of the film by the insertion of GSNO.

3. Thermal Characteristics

Thermal properties of pure GSNO, blank film and CS / NO film were measured using differential scanning calorimetry (DSC; N-650, SCINCO, Korea). Each sample was heated in a fully sealed aluminum pan at a rate of 5 ° C./min with varying temperatures from 30 ° C. to 200 ° C. under a dynamic nitrogen atmosphere.

As shown in FIG. 2, GSNO showed a strong endothermic peak at 195 ° C., and chitosan showed a weak endothermic peak compared to GSNO. However, the endothermic peak of GSNO was not observed in the CS / NO film, and it was confirmed that GSNO completely dissolved in the film and disappeared molecularly. Broad endothermic peaks of both films were observed below 140 ° C., which was due to moisture evaporation.

4. Mechanical Properties

In order to analyze the mechanical properties of the CS / NO film prepared in Example 2, the tensile strength (TS) and elongation at break were evaluated using a tensile tester (Instron 3345, Instron, Norwood, MA). First, the prepared film was cut into specific dogbone shapes (80 mm long, 30 mm wide). All tests were performed at an elongation of 10 mm / min, and the film thickness was measured using a caliper just before the test. Elongation at break (E%) was calculated from the difference between the initial length of the sample (30 mm) and the elongation at break.

As shown in Table 1, the elongation at break was not affected by the addition of NO. The blank film was drawn to 134.4 ± 11.5% relative to the original length, but the NO release film was only recorded at 129.9 ± 18.1%. However, the addition of GSNO reduced the tensile strength and Young's modulus.

Table 1

	Tensile Strength (MPa)	Elongation at Break (%)	Young's modulus (MPa)
Blank	7.87 ± 1.02	134.4 ± 11.5	7.18 ± 0.53
NO 10 mg	6.53 ± 0.70	129.9 ± 18.1	5.48 ± 0.31

5. NO emission analysis

After cutting into square models corresponding to 50 mg of CS / NO film, it was immersed in 10 mL of phosphate buffer (PBS, pH 7.4) at 37 ° C. NO released from the film was analyzed using Griess reagent. 100 μl of PBS was taken at defined time intervals and replaced with fresh PBS. After proper dilution, 100 μl of the reaction solution obtained by reacting the Griess reagent with dilute PBS at a ratio of 1: 1 was added to a 96 well plate and stored at room temperature for 30 minutes. The amount of nitrite was measured using a microplate reader at 540 nm.

As shown in FIG. 3, NO was shown as a first-order exponential graph from the NO emitting film.

NO showed a slow release pattern until the first hour and then released according to the first order equation. The initial slow release is due to the time required for the film to hydrate. Since the CS / NO film had to swell for NO to be released, a delay was expected before the NO was released for the first time, and the CS / NO film had to release enough NO during the wound healing period. Of the CS / NO films included, CS / NO films containing 20% by weight of GSNO were found to consistently release the highest amount of NO for 48 hours.

6. Antimicrobial and Antibiofilm Effect Review

Figure 4 shows the antimicrobial and anti-biofilm effect of the NO release film for Gram-positive bacteria and Gram-negative bacteria.

For this experiment, P. aeruginosa PAO1 (wild type prototroph, Peerson et al. , 1997), Staphylococcus aureus strain RN4220 (Kreiswirth et al. , 1983), or Methicillin-resistant Staphylococcus aureus (Methicillin-resistant) Staphylococcus) was used. Staphylococcus aureus was grown overnight with vigorous shaking in 3 mL of LB broth. These cells were seeded on a cover glass of a 12 well plate and incubated in 2 mL LB broth medium containing 2 μl antibiotic at 37 ° C. Thereafter, the blank film and the CS / NO film were cut to a size of 1.3 cm x 1.3 cm and added to each well. The cover glass was lightly washed with sterile water at predetermined time intervals to remove unstained Staphylococcus aureus, and then observed under a fluorescence microscope. For Pseudomonas aeruginosa, 2 mL of a medium containing 25 μl antibiotic and plasmid inducer were dispensed onto a 12 well plate containing cover glass. 40 μl Pseudomonas aeruginosa was inoculated onto the cover glass of each well, and then a film was added to each well. The cover glass was lightly washed with sterile water at predetermined time intervals to remove unattached Pseudomonas aeruginosa, and observed with a fluorescence microscope.

In Pseudomonas aeruginosa, 8 hours after GSNO-encapsulated CS / NO film showed less biofilm than chitosan film, and after 24 hours, biofilm on CS / NO film surface was much less than chitosan film.

In addition, it was confirmed that even after 24 hours, the CS / NO film significantly reduced the number of bacteria in Staphylococcus aureus compared to the chitosan film.

7. in vivoin vivo Review of wound healing effects

In order to examine the effect of NO release film on wound healing in vivo , 250-280 g of male SD rats were used as animal models. Before creating a wound or the like of the rats before the sol retil 50 ^® (Til Alpharetta Min / Jolla jepam) and rompeon ^® (xylazine hydrochloride) 5: using a volume ratio of 2 were anesthetized animals. After the hairs of rats and the like were removed with an electric razor, the back of the skin was incised to make a full layer wound (1.5 cm x 1.5 cm). 50 μL of P. aeruginosa (3.2 × 1088888 cells / mL solution) was added to each wound and left for 24 hours to make an infectious wound.

The medipom ^® a PVA / NO film or CS / NO film prepared in Example 2 (Mundie Pharma, control group), each wound were applied. Each material was applied and then fixed using an elastic adhesive tape (Micropore, 3M Consumer Health Care, St Paul, MN, USA). All dressings treated on wound lesions were replaced with new ones at appropriate times.

All rats were stored in separate cages and digital images of each lesion were collected every three days using a digital camera. After wounding, macroscopic changes in the wound were observed using images obtained on

days

4, 7, 10, 13 and 16. Wound size reduction and epithelialization rate were calculated based on the following equation.

Epithelialization rate (%) = E _t / (W _t + W ₀ ) x 100

E _t : epithelialized area per hour t

W _t : wound area per hour t

W ₀ : initial wound area

Figure 5 shows the macroscopic observation results according to the temporary change after surgery, very severe inflammation was observed in the film except the CS / NO film on the fourth day of surgery, while the CS / NO film was confirmed that the wound is closed very quickly .

Figure 6 shows the wound size reduction effect according to the application of the NO release film, CS / NO film showed a significantly faster wound healing effect compared to Medifoam confirmed that the wound almost healed after 10 days. Another NO-releasing film, PVA / NO film, showed a faster healing effect than Mediform, but showed a slower healing effect than CS / NO film.

In addition, Figure 7 shows the rate of epithelialization, CS / NO film applied wounds showed epithelialization at a faster rate than the Mediform control on the fourth day of surgery, in particular, wounds with CS / NO releasing film applied epithelialization at the fastest rate. The wounds with CS / NO releasing film after 7 to 10 days post surgery showed epithelialization at a higher rate compared to wounds with Mediform control.

Therefore, the NO release film showed better wound healing ability, and the CS / NO film using chitosan as a substrate showed a better healing effect than the PVA / NO film using PVA as a substrate. The results show that chitosan has excellent ability to encapsulate NO and release it stably, and the antibacterial action and wound healing action of chitosan itself have led to synergy.

8. Review histopathological findings

1) Histopathological Procedure

Whole skin wound tissues of the skin, including the dermis and subcutaneous tissue, were collected and one or two sites, possibly central areas, for the wound sample based on granulation tissue were cut out. The cut skin was fixed with 10% neutral buffered formalin. After paraffin embedding, 3-4 μm fragments were prepared. Each fragment was stained with hematoxylin and eosin (H & E) for light microscopy or Masson's trichrome for collagen fibers. Thereafter, each skin was observed histopathologically under an optical microscope (E400, Nikon, Japan).

2) Organization type measurement

To identify more detailed histopathological changes, the epithelial skin was peeled off using a computer equipped with an automated digital image analyzer ( i Solution FL ver 9.1, IMT i- solution Inc., Quebec, Canada) on each of the cross-cut skin samples. Area (mm), number of microvessels in granulation tissue (vessel number in field / mm ² ), number of infiltrating inflammatory cells in granulation tissue (% / mm ^{2 of} field) and area of granulation tissue (mm ² / cross cut) Central area). Eight wounds from each experimental group were analyzed by a histopathologist to perform a blind test. In addition, the re-epithelialization rate was calculated by changing a previously known method (Biol Pharm Bull 2007 Dec; 30 (12): 2406-2411).

Re-epithelialization rate (%) = [total length of wound (10mm)-bare epithelial area (mm)] / wound total length X 100

3) results

The histopathological findings are shown in FIGS. 8 and 2, and histopathological and histological measurements were significantly reduced in epithelial tissues peeled from the film-applied experimental group, and the re-epithelialization rate was significantly increased in the film-applied experimental group. . In particular, the NO release film application group showed significantly faster wound healing effects than the blank film or gauze application, and showed rapid reconstruction of granulation tissue, lower inflammatory cell infiltration and lower angiogenesis. In addition, it was confirmed that NO release film in the experimental group significantly accelerated collagen fiber regeneration.

TABLE 2

Organization type measurement	Gauze control	Blank film	CS / NO film
Stripped epithelial area (mm)	2.93 ± 0.68	1.44 ± 0.22	0.61 ± 0.1 ^ce
Re-epithelialization rate (%)	70.66 ± 6.79	85.58 ± 2.19	93.91 ± 0.98 ^ce
Microvascular	154.24 ± 28.36	66.38 ± 16.15 ^a	30.25 ± 11.72 ^ab
Invasive inflammatory cell count	441.13 ± 122.71	139.00 ± 21.53 ^c	79.75 ± 14.27 ^ce
Collagen Production Tissue	34.32 ± 8.88	49.55 ± 7.19 ^a	62.34 ± 6.41 ^ab
Granulation tissue area (mm ² )	6.39 ± 1.58	4.25 ± 0.55 ^c	2.46 ± 0.46 ^ce

The values are expressed as the average of 8 S.D let wounds.

^a p <0.01 compared to gauze control by LSD test

^b p <0.01 Comparison with NO 0 mg by LSD test

Comparison with gauze control by ^c p <0.01 and ^d p <0.05 MW test

compared to 0 mg NO by ^e p <0.01 MW test

As mentioned above, although this invention was demonstrated by the limited embodiment and drawing, this invention is not limited by this, The person of ordinary skill in the art to which this invention belongs, Of course, various modifications and variations are possible within the equivalent scope of the claims to be described.

Claims

A nitric oxide releasing wound healing film comprising a nitric oxide donor encapsulated in a polymer.
The method of claim 1, wherein the nitric oxide donor is in the group consisting of s-nitrosoglutathione, diaeniumdiolate, organic nitrate and iron-nitrosyl complexes Nitric oxide releasing wound treatment film, characterized in that selected.
The nitric oxide releasing wound treatment film of claim 1, wherein the polymer is a biocompatible polymer selected from the group consisting of chitosan, silicone, polyurethane, and calcium alginate.
The method of claim 1, wherein the nitric oxide releasing wound treatment film, characterized in that it contains 2.5 to 30% by weight of the nitrogen oxide donor in 100% by weight of the film.
Dissolving the polymer in a solvent, adjusting the pH, and then preparing a polymer solution by adding a plasticizer;

Adding a nitric oxide donor to the polymer solution and stirring to prepare a reactant; And

Casting the reactants into a film form and drying in a dark room dehumidified state

Method for producing a nitric oxide releasing wound treatment film comprising a.
The method of claim 5, wherein the nitric oxide donor is in the group consisting of s-nitrosoglutathione, diaeniumdiolate, organic nitrate and iron-nitrosyl complexes Method for producing a nitric oxide releasing wound treatment film, characterized in that selected.
The method of claim 5, wherein the polymer is a biocompatible polymer selected from the group consisting of chitosan, silicone, polyurethane, and calcium alginate.
The method of claim 5, wherein the plasticizer is selected from the group consisting of polyethylene glycol, sorbitol, and glycerol. 6.
The method for preparing a nitric oxide-releasing wound treatment film according to claim 5, wherein the preparation of the polymer solution is performed at pH 4.0 to pH 6.0 using 0.1 M acetate buffer.
The method of claim 5, wherein the nitrogen oxide donor is added in an amount of 2.5 to 30 parts by weight based on 100 parts by weight of the polymer solution.
The method of claim 5, wherein the drying is performed at 25 to 37 ° C. for 1 to 2 days.