KR20170000105A - Antibacterial chitosan-polyurethane nanofiber - Google Patents

Abstract

The present invention relates to a chitosan-polyurethane nanofiber comprising silver sulfadiazine. The chitosan-polyurethane nanofiber comprising silver sulfadiazine has a wide range of antibacterial activity and shows excellent mechanical strength, thereby being widely used as an antibacterial wound dressing material.

Description

Antibacterial chitosan-polyurethane nanofiber {Antibacterial chitosan-polyurethane nanofiber}

The present invention relates to a chitosan-polyurethane nanofiber comprising silver sulfadiazine.

Dressing is a method used to improve the speed of healing by covering the wound surface, which is a skin defect area caused by burns, wound, pressure sores, and trauma. In the dressing method, a dry dressing method for drying a wound surface by using gauze or the like has been generalized. However, in 1962 Winter, Wet dressing has been developed and released since the announcement that it is more than twice as fast as the environment, and there are various ways of treating wounds.

The conditions required for the wet dressing material include a wet environment composition in which the epithelial cells can smoothly spread the wound surface, a prevention of infection which can prevent the bacterial invasion from the outside and proliferation because of the rapid growth of the bacteria in the wet environment, And a protective action that can prevent damage due to external physical impact.

On the other hand, according to recent reports, resistance to bacteria caused by abuse of various antibiotics is very serious. As a representative example, MRSA (Methicillin Resistant Staphylococcus Aureus, methicillin resistant Staphylococcus aureus) or VRE (Vancomycin-resistant enterococcus) is an antibiotic-resistant pathogenic bacterium and most of them appear as skin infections. If this weak person develops into bloodstream infections, bone infections, pneumonia, meningitis, etc., it can have fatal consequences. More than one-third of the population has bacteria such as staphylococci in the skin and nasal mucosa. Since these bacteria are contagious to other people, they are more likely to be infected with such bacteria if they are injured by burns, wounds or trauma . However, if these bacteria are resistant to antibiotics, they can cause considerable problems in wound healing.

In this connection, a dressing product using silver (Ag) which provides a wide range of antibacterial and antifungal effects has been developed. Silver is particularly excellent in antibacterial action against bacteria and viruses in the case of nano-sized particles, and exhibits an excellent effect on anti-allergy and environmental hormone inhibition. In addition, silver nanoparticles are also known as substances that do not cause any adverse effects on the human body. The silver nanoparticles in the form of colloids can easily penetrate into the cell of bacterial and viral bacteria, stop the enzyme action, inhibit cell wall breakage and metabolism of the bacteria, and thus can sterilize various bacteria, viruses and allergic diseases . Colloidal silver in aqueous solution is an FDA approved antibiotic and is used in actual clinical applications such as cotton or bandages because of its scientific evidence of its efficacy in burns, infections, inflammation and other microbial infections.

Techniques have been provided for producing antimicrobial nanomaterials or fibers using the properties of such silver nanoparticles. For example, Korean Patent Laid-Open Publication No. 10-2006-0003233 discloses a method for bonding nano silver to gauze using a water-soluble starch or a three-component hot melt type main component comprising an ethylene-vinyl acetate copolymer, a tackifier resin and a wax Process. Korean Patent Laid-Open Publication No. 10-2005-0075905 discloses a method of dispersing silver particles having a size of 20 to 70 nm in the island component of a sea-island composite fiber to chemically elute the silver particles, Lt; RTI ID = 0.0 > fiber. ≪ / RTI >

It is an object of the present invention to provide a chitosan-polyurethane nanofiber comprising silver sulfadiazine and a method for producing the same.

In one aspect of the present invention, the present invention provides a chitosan-polyurethane nanofiber comprising silver sulfadiazine.

The chitosan-polyurethane nanofiber comprising silver sulfadiazine of the present invention exhibits antimicrobial activity.

In the present invention, the term "silver sulfadiazine" means a compound represented by the following formula (1)

[Chemical Formula 1]

Sulfadiazine is approved for use in Korea by the Korea Food and Drug Administration. Infection of the following pathogens caused by 2-3 degree burns, various skin ulcers (pressure sores, leg ulcers, radiation ulcers, diabetic gangrene, skin wounds, etc.): Pseudomonas aeruginosa, , Clevesiella spp., Staphylococcus spp., Hemolytic streptococci, Candida spp., FDA approved indications. It is known to have excellent antimicrobial activity as a compound having efficacy and efficacy of burn.

The sulfadiazine, chitosan or polyurethane of the present invention may be commercially purchased or used by a person skilled in the art by known methods, but is not limited thereto.

The nanofibers of the present invention are produced by electrospinning. Illustratively, the nanofibers of the present invention can be prepared by preparing a mixture of chitosan, polyurethane and sulfadiazine, and electrospinning the mixture (FIG. 1).

The electrospinning in the present invention can be carried out by a method known in the art, for example, by connecting a spinning solution to an electrospinning nozzle using a feeding device, using a high voltage generating device between the nozzle and the current collector, (High electric field, 10 kV to 100 kV). The size of the electric field is related to the distance between the nozzle and the current collector, and the relationship between them is used in combination to facilitate electrospinning. As the electrospinning device to be used at this time, those generally used in the art can be used, and an electro-bronzing method, a centrifugal electrospinning method, or the like can also be used.

In one specific embodiment, the chitosan is dissolved in a mixed solvent of TFA / DCM and the polyurethane is added to the solution. Subsequently, sulfadiazine was added to prepare a mixture containing chitosan, polyurethane and sulfadiazine, and the mixture was electrospun to prepare nanofibers.

The chitosan and polyurethane nanofiber prepared as described above are biocompatible. The biocompatibility in the present invention preferably means that it does not need to be removed after complete absorption or is compatible with the human body in particular. The chitosan and polyurethane nanofibers of the present invention may not induce or less induce an immune response, Or integrate with a particular cell type or tissue.

In addition, the chitosan and polyurethane nanofiber of the present invention have excellent mechanical strength and excellent antimicrobial activity.

In the present invention, the term "antimicrobial" includes not only inhibiting the growth of pathogenic microorganisms such as bacteria and fungi but also sterilizing by inhibiting its survival. The chitosan and polyurethane nanofibers containing sulfadiazine silver of the present invention can be used for antibacterial treatment of all pathogenic microorganisms in the living environment. Candida, Cryptococcus, Such as Aspergillus, Trichophyton, Trichomonas, Chaetomium, Gliocladium, Aureobasidium, Penicillium, Rhizopus, ), Fungi such as Cladosporium, Mucor, Pullularia, Trichoderma, Fusarium, Myrothecium, and Memnoniella And Escherichia, Bacillus, Pseudomonas, Chetonium, Staphylococcus, Klebsiella Legionella, Salmonella, Vibrio, Leake Oh (Rickettsia) may be mentioned bacteria such as, but not limited to, microorganisms.

Preferably, the chitosan-polyurethane nanofibers comprising the sulfadiazine silver of the present invention are antimicrobial to P. aeruginosa, S. aureus and methicillin-resistant S. aureus.

In a specific embodiment, the morphology of nanofibers comprising chitosan and polyurethane was compared with chitosan single nanofibers by SEM, and as a result, it was found that the nanofibers containing polyurethane were more flat and had a uniform diameter It was confirmed that it represents the shape.

In addition, in one specific embodiment, the nanofiber comprising chitosan and polyurethane exhibited excellent elasticity and elongation, and improved mechanical properties.

In a specific embodiment, the fiber sheet itself containing no sulfadiazine and chitosan and polyurethane has no bactericidal growth inhibitory effect, but chitosan and polyurethane nanofiber sheet containing sulfadiazine have excellent bacteria Growth inhibitory effect was confirmed.

As described above, the nanofiber of the present invention has a wide range of antibacterial activity due to the inclusion of sulfadiazine, and simultaneously exhibits excellent mechanical strength including chitosan-polyurethane. Therefore, the nanofiber can be firmly fixed on the wound surface, Can be utilized as a material for dressing wound.

Accordingly, the present invention provides a material for wound dressing for antimicrobial use comprising the nanofiber. Preferably, the antimicrobial wound dressing material of the present invention may be made of a film, a sheet, a nonwoven fabric, a foam, a rope, a pellet, a powder, a fabric or the like.

The chitosan-polyurethane nanofiber containing sulfadiazine silver of the present invention has broad antibacterial activity and exhibits excellent mechanical strength at the same time, and thus can be widely used as an antimicrobial wound dressing material.

1 is a schematic view of a method for producing a chitosan-polyurethane nanofiber comprising sulfadiazine silver of the present invention,
2 shows SEM image pickup results of (a) polyurethane (PU), (b) chitosan (CTS) and (c) chitosan-polyurethane (CTS / PU) fiber sheet,
3 shows the stress-strain curves of chitosan (CTS) nanofibers (dark gray), polyurethane (PU) nanofibers (black) and chitosan-polyurethane (CTS / PU) fiber sheets (black)
4 shows FT-IT spectra of chitosan (CTS) nanofibers (dark gray), polyurethane (PU) nanofibers (black) and chitosan-polyurethane (CTS / PU) fiber sheets (dark blue)
5 shows the EDX spectrum of a chitosan-polyurethane (CTS / PU / AgSD) (100: 1) fiber sheet comprising (a) chitosan-polyurethane (CTS / PU) and (b) sulfadiazine silver ,
FIG. 6 is a photograph of a chitosan-polyurethane (CTS / PU / AgSD) fiber sheet (100: 1, dark red) containing a chitosan-polyurethane (CTS / PU) fiber sheet (dark blue) and sulfadiazine silver TGA curve,
7 shows SEM and TEM images of a chitosan-polyurethane (CTS / PU / AgSD) fiber sheet comprising electrospun chitosan-polyurethane (CTS / PU) fiber sheet (dark blue color) and sulfadiazine silver CTS / PU: AgSD = 1000: 1 The fiber sheet is composed of (a), (d) (c) and (f)
Fig. 8 shows the results of (a) CTS / PU fiber sheet, (b) CTS / PU: AgSD = 1000: 1 fiber sheet, and (c) CTS / PU: AgSD = 100: 1 MRSA, S. aureus and P aeruginosa The results of the antimicrobial test are shown.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided to further understand the present invention, and the present invention is not limited by the examples.

<Experimental Method>

material

The medium and low molecular weight chitosan, silver sulfadiazine (98%), and trifluoroacid (ReagentPlus ^® , 99%) were purchased from Sigma-Aldrich (St. Louis, Mo.). Polyurethane (Bionate 75A) was purchased from Resomer ^® (Ingelheim, Germany). Dichloromethane (99.0% +), N, N-dimethylformamide (99.5%) and tetrahydrofuran (99.5%) were purchased from JUNSEI (JUNSEI CHEMICAL Co. Ltd., Japan). Sodium hydroxide for neutralizing the electrospun CTS / PU / AgSD (chitosan / polyurethane / sulfadiazine) fiber sheet was purchased from YPC (Yakuri Pure Chemicals CO., LTD.). Methyl alcohol (MeOH) was purchased from Dae Jung Chemical & Metals Co. Ltd. Korea. Deionized-distilled water (DDW) was produced by ultrapure water system (Puris Ro800; Bio Lab Tech., Korea). Membrane tubes for dialysis were purchased from Spectrum Spectra (Spectra / Por 4 dialysis tubing, 12-14 K MWCO, 45 mm flat width, 100-foot length). Bacterial strains P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and methicillin-resistant S. aureus ATCC 700787 were obtained from the American Type Culture Collection (Manassas, Va.). All chemical solvents used were analytical grade and were used without further purification.

Preparation of purified CTS (chitosan)

To obtain pure CTS, a 2 wt.% Solution was prepared by completely dissolving 1.0 g of CTS (Sigma-Aldrich) in 50 mL of 1% acetic acid at 4 ° C. The solubilized solution was vacuum filtered using filter paper for solids removal. The solution was then poured into 25 mL of 1 N NaOH solution. The pure CTS was then washed with DDW until the pH reached 7. The resulting materials were dialyzed and lyophilized.

ELSP  CTS / PU / AgSD  Formation of fiber sheet

Various blends (AgSD = 0, 0.1 and 1 wt.%) Of CTS, PU, CTS / PU and CTS / PU / AgSD fiber sheets were molded with ELSP (electrospinning). To prepare the ELSP solution, pure CTS was dissolved in a mixed solvent system of TFA / DCM (7: 3). PU was then added to the CTS solution at a ratio of CTS / PU (80/20) with a total solids concentration of 7% (w / v). Various concentrations of AgSD (0, 0.1 and 1 wt.%) Were then added to the CTS / PU solution, respectively. This process was used to prepare CTS / PU / AgSD ELSP solutions. Electrospinning CTS and CTS / PU fiber sheets were prepared using TFA / DCM (7: 3) as the solvent system and PU nanofibers alone were molded using a mixture of DMF / THF (5: 5). ELSP was performed according to the following conditions. The ELSP solution was loaded into a luer-lock syringe fitted with a 20 gauge metal blunt tip needle. The collector was an aluminum foil covered with a rotating mandrel. The machine operated at a flow rate of 1 ml / h at 20 kV. The distance between the tip of the needle and the collector was 15 cm. For removal of the residual solvent, the obtained CTS / PU / AgSD fiber sheet was dried overnight at room temperature under vacuum. To neutralize the acid, the dry CTS / PU / AgSD fiber sheet was soaked in 40 ml of 3.2 M NaOH / MeOH (neutralization solution) and washed with DDW until the wash reached pH 7. The wet CTS / PU / AgSD fiber sheets were then freeze-dried.

Physical and chemical analysis (stress strain, TGA )

Tensile strength was measured using a mechanical tester (Instron 44646, Instron, Mass.) To evaluate the mechanical properties of CTS, PU and CTS / PU fiber sheets. For this purpose, test specimens (20 × 5 mm 2) were loaded, stressed until broken and tested at a crosshead speed of 10 mm / min at room temperature. In addition, TGA was performed using TGA Q5000IR (TA Instruments, USA) to investigate the thermal decomposition properties of CTS / PU and CTS / PU / AgSD fiber sheets. The decomposition property of each fiber sheet sample was measured in a nitrogen atmosphere at a heating rate of 10 ° C / min in a temperature range of 25 ° C to 800 ° C.

EDX  (Energy- Dispersive  X-Ray Spectroscopy)

EDX analysis was performed on a Stereo-scan 440 (Leica, United Kingdom) equipped with a Phoenix EDX attachment. The EDX spectra of each peak was recorded in a spot profile mode by aligning the electron beam to the gold surface-coated CTS / PU and CTS / PU / AgSD fiber sheets, respectively.

CTS / PU / AgSD  Morphological Analysis of Fiber Sheet SEM , TEM )

The shape of the electrospun fiber sheet was SEM (Scanning Electron Microscope, Hitachi S-2300, Japan) at 15 kV. All samples were dried at room temperature and gold coated on carbon tape for 10 minutes. Each sample was loaded onto a copper-grid before shooting. The single fibers were also observed by performing each sample at 200 volts using JEM-2100F (JEOL, USA) using transmission electron microscopy (TEM).

Fourier Transform-Infrared Spectroscopy (FT-IR)

The surface molecular structure of CTS, PU and CTS / PU fiber sheets was confirmed by FT-IR spectrometer (Spectrum ^TM One System, Perkin-Elmer). The transmittance of each sample was recorded in 10 scans at a resolution of 4 cm ^-1 between 4000 and 500 cm ^-1 .

Bacterial inhibition test

Three types of bacteria: P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and methicillin-resistant S. aureus ATCC 700787 were aerobically grown in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) At 35 ° C for 18 hours . The disk diffusion test was performed according to the CLSI (Clinical and Laboratory Standards Institute) performance standard for the antimicrobial disk susceptibility test (version 9, standard M2-A9 approval) using a Mueller-Hinton Agar (Difco). Briefly, the Mueller-Hinton agar (Difco) surface was inoculated using a swab immersed in a bacterial cell suspension adjusted to 0.5 McFarland standard turbidity and dried for about 5 minutes. The CTS / PU / AgSD fiber sheet disk was placed on the agar and the susceptible plates were incubated at 35 [deg.] C for 16-18 hours. The inhibition zone formed around the disc was measured in millimeters (mm).

<Experimental Results>

The qualities of CTS, PU, and CTS / PU fiber sheets ( SEM , FT-IR, tensile test)

Each test CTS, PU, CTS / PU fiber sheets were successfully molded into ELSP as in FIG. In order to confirm the surface morphology of each nanofiber, each fiber sheet was identified by SEM. As shown in Figs. 2 (a) and 2 (b), the PU nanofibers were flat-formed and had a very uniform diameter in good shape. CTS nanofibers exhibited a more polydispersed morphology with random pore diameters. As shown in Fig. 2 (c), the CTS / PU fiber sheet (ratio of CTS / PU = 80/20) was well formed and showed a good shape. These results indicate that PU added to CTS nanofibers improves morphology compared to electrospun CTS alone.

The improvement of mechanical properties of CTS / PU fiber sheet compared to CTS alone was confirmed by tensile strength test. As can be seen in Figure 3, there was a significant difference in tensile properties between CTS, PU and CTS / PU fiber sheets. PU nanofibers exhibited excellent flexibility and good elasticity with a Young's modulus of 8.2 MPa, which extended to 118% before rupture. CTS nanofibers had poor flexibility with a Young's modulus of 1.6 MPa and ruptured with a 12% elongation. However, the CTS fiber sheet mixed with PU exhibited a Young's modulus of 5.2 MPa and was elongated about 51% until the bursting point. These results demonstrate that tensile strength and Young's modulus were increased by incorporating the PU polymer into the CTS. This means that the CTS / PU fiber sheet has improved mechanical properties and a cleaner form than the CTS nanofibers alone. This property implies that the two polymers are well mixed and have a molecular level of interaction to improve mechanical formation.

In order to confirm the chemical properties of the CTS / PU mixed fiber sheet, the surface was irradiated with FT-IR. Each sample (CTS, PU, and CTS / PU) fiber spectrum is shown in FIG. Characteristic peaks of CTS were observed at about 905 cm ^-1 and 1157 cm ^-1 , corresponding to the saccharide structure. Additional peaks were observed at 1650 cm ^-1 (C = O stretching), 1540 cm ^-1 and 1320 cm ^-1 (NH, CN stretching peaks), which correspond to amide bonds I, II and III, respectively. The infrared spectra of the PUs were 3320 cm ^-1 (NH), 2960 cm ^-1 (CH), 1710 cm ^-1 (C═O), 1530 cm ^-1 (C═C), 1220 cm ^-1 (CC) cm &lt; ^-1 &gt; (CO) and 777 cm &lt; ^-1 &gt; (CH). Based on these results, the infrared spectrum of the CTS / PU mixture was analyzed. The absorption band around 3390 cm ^-1 in CTS / PU was wider than the pure PU around 3320 cm ^-1 . This means the interaction between the hydroxyl and amino groups and the polyurethane group (NH) in CTS. In addition, the amide bond absorption of CTS at 1650 cm ^-1 (amide bond I) and 1320 cm ^-1 (amide bond II), the PU group at 1530 cm ^-1 (C = C), 1220 cm ^-1 (CC) CTS / PU spectra. These peaks were observed in CTS / PU but not in pure CTS and PU alone. The results indicate that CTS and PU are well mixed in the CTS / PU fiber composite.

CTS / PU / AgSD  Physicochemical Analysis of Fiber Sheet EDX , TGA )

AgSD physical mixtures with various drug loaded CTS / PU fiber sheets were selected. The mixed solution was molded into ELSP to prepare a CTS / PU / AgSD fiber sheet. The primary amino and carboxyl groups of CTS have the ability to chelate with silver ions, allowing for improved dispersion and controlled release. Therefore, AgSD containing silver ions was expected to chemically bond with CTS in the ELSP solution. The molded CTS / PU and CTS / PU / AgSD fiber sheets were identified by EDX to confirm that the fiber sheets contained and bound AgSD. As can be seen from Fig. 5 (a), the spectrum of the CTS / PU fiber sheet showed no Ag content at all. On the other hand, it was clearly confirmed that the CTS / PU / AgSD fiber sheet contains silver in an amount of 0.59 wt% as shown in Fig. 5 (b). These results indicate that Ag is well integrated in the CTS / PU / AgSD fiber sheet.

Each fiber sheet (CTS / PU and CTS / PU / AgSD) was tested with TGA to confirm the temperature stability of the fiber sheets. As shown in FIG. 6, the first weight change curve around 50-60 ° C was due to moisture loss, and the second weight change curve at 230-280 ° C was due to the degradation of the polymer. CTS / PU and CTS / PU / AgSD nonwoven mats exhibited similar degradation at these temperatures. However, above 470 ° C there was a constant weight difference between CTS / PU and CTS / PU / AgSD. This means the presence of undissociated silver in the CTS / PU / AgSD sample. The results also indicate that the fiber sheet is thermally stable.

CTS / PU / AgSD  Surface shape of fiber sheet ( TEM , SEM )

To confirm the surface morphology of the fiber sheet samples, SEM analysis was performed. The SEM showed that the CTS / PU fiber sheets containing various ratios of AgSD (AgSD content = 0, 0.1 and 1 wt.%) Were well formed by ELSP technique, respectively (Figs. 7 (a) - (c)). Although the CTS / PU / AgSD fiber sheets contained various AgSD contents, they were observed to have a similar shape.

TEM analysis of the individual fibers confirmed whether each fiber contained AgSD (Fig. 7 (d) - (f)). AgSD was not observed in the CTS / PU fiber sheet, but AgSD was observed in the fiber in the CTS / PU / AgSD fiber sheet. Interestingly, as the content of AgSD gradually increased from 0.1% to 1%, more AgSD was observed in the fibers (Fig. 7 (e) - (f)).

CTS / PU / AgSD  Bacteria inhibition test of fiber sheet

To determine if CTS / PU / AgSD fiber sheets are effective in preventing bacterial growth, three bacterial antimicrobial tests were performed: P. aeruginosa, S. aureus and MRSA. In the experiment, CTS / PU and CTS / PU / AgSD fiber sheets were used as control and comparison groups, respectively. As shown in FIG. 8 (c), the CTS / PU: AgSD (100: 1) fiber sheet showed a bacterial growth inhibition zone around the fiber disc.

It was also found that the CTS / PU: AgSD (100: 1) fiber sheet had a higher effect on S. aureus and MRSA than P. aeruginosa. As can be seen in FIG. 8 (b), the CTS / PU: AgSD (1000: 1) fiber sheet had the lowest amount of AgSD but showed growth inhibition for all bacteria, Showed a smaller suppression radius than one fiber sheet. As shown in FIG. 8 (a), in the absence of AgSD, the CTS / PU fiber sheet itself had no bactericidal growth inhibitory effect. The bactericidal effect was dose dependent on AgSD content. From the above results, it was confirmed that CTS / PU / AgSD fiber sheet can be useful as an antimicrobial wound dressing agent.

Claims (5)

A chitosan-polyurethane nanofiber comprising silver sulfadiazine. The nanofibers according to claim 1, wherein the nanofibers are antimicrobial. A wound dressing material comprising the nanofiber of claim 1. The method of claim 1, comprising the steps of preparing a mixture of chitosan, polyurethane and sulfadiazine, and electrospinning the mixture. 5. The method according to claim 4, wherein the nanofibers are antimicrobial.

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