KR20160126252A

KR20160126252A - Preparing method of AgNPs-chitosan nanofiber

Info

Publication number: KR20160126252A
Application number: KR1020150057112A
Authority: KR
Inventors: 권일근; 이상진; 허동녕; 이동현
Original assignee: 경희대학교 산학협력단
Priority date: 2015-04-23
Filing date: 2015-04-23
Publication date: 2016-11-02

Abstract

The present invention relates to a producing method for a silver nanoparticle-chitosan nanofiber, a silver nanoparticle-chitosan nanofiber produced by the method, and wound dressing materials comprising the nanofiber.

Description

Preparation method of silver nanoparticles-chitosan nanofibers {Preparing method of AgNPs-chitosan nanofiber}

The present invention relates to a method for producing silver nanoparticle-chitosan nanofibers, silver nanoparticle-chitosan nanofibers prepared by the above method, and wound dressing materials comprising the nanofibers.

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, Since the announcement that it is more than twice as fast as the environment, wet dressing has been continuously developed and released, and methods of treating wounds have been developed variously.

BACKGROUND ART [0002] Recently, active research and application have been made on electrospinning, in which a polymer melt is produced by using an electric field of high voltage to adjust the diameter of a fiber from several tens nm to several hundreds nm. Nanofibers produced by electrospinning are nanofibers at the same time as nanotubes are made into three-dimensional nonwoven fabrics. Therefore, it is possible to use wound dressing materials and medical fibers It is getting bigger. Korean Patent Registration No. 10-0588228 discloses wound dressing using a hydrophilic nanofiber in which a protein component is contained in a polyester nanofiber. Patent No. 10-0791039 discloses a method of manufacturing a dressing using nanofibers containing an antioxidant Lt; / RTI > In the case of the above-mentioned patents, the use of nanofibers makes it possible to improve the specific surface area by volume and to improve the recovery speed of wounds by the function of proteins and antioxidants when healing wounds. However, It is difficult to anticipate the antibacterial action, and it is difficult to have sufficient mechanical strength, and there is a problem that the strength is lowered due to absorption of exudates.

On the other hand, silver is known to control more than 650 kinds of bacteria, viruses and molds harmless to human body. Silver is attached to the -SH group of the protein cysteine which is constituted by bacteria and viruses and converted into a sulfur compound so that it acts as a catalyst for promoting the oxidation reaction by inhibiting propagation or attaching to an enzyme that acts on oxygen, (Digestion, respiration) in the specific part of the part involved in the antibacterial activity is known as a semi-permanent antibacterial agent.

Recently, there have been reported applications of silver nanoparticles loaded on various nanofiber scaffolds using such antimicrobial properties. As the polymer made of nanofibers, PHBV, PMMA, PTBAM, PVP, polyradamine and mixtures such as CTS / PVA, CTS / PEO and CTS / gelatin are used.

It is an object of the present invention to provide a method for producing silver nanoparticle-chitosan nanofibers, silver nanoparticle-chitosan nanofibers prepared by the above method, and wound dressing materials comprising the nanofibers.

In order to accomplish the above object, the present invention provides a method for producing a silver nanoparticle-chitosan complex, comprising the steps of: adding silver nitrate to chitosan and then adding a reducing agent to form a silver nanoparticle-chitosan complex; And a second step of electrospunning the composite to produce silver nanoparticle-chitosan nanofibers.

The method of manufacture of the present invention is schematically illustrated in Fig. The first step is a step for forming silver nanoparticles in chitosan, which includes a process of chemically reducing silver nitrate.

The chitosan of the present invention is a natural polymer derived from chitin (poly-N-acetyl-D-glucosamine) wherein a substantial portion of the N-acetyl groups are removed by hydrolysis. The degree of deacetylation is preferably in the range of 50 to 95%, more preferably 85%. The degree of deacetylation indicates that 85% of the amino groups are acetylated. The chitosan of the present invention comprises repeating monomer units of formula (I)

Wherein n is an integer, and further, the amino group is acetylated in m units. The sum of n + m represents the polymerization degree, that is, the number of monomer units in the chitosan chain.

The chitosan of the present invention can be purchased commercially and used, for example, from Sigma Aldrich or Nova Matrix.

As the reducing agent for reducing silver nitrate, a reducing agent known to those skilled in the art can be freely used, and preferably sodium borohydride can be used.

It is preferable that the first step of adding silver nitrate to the chitosan and then the reducing agent is performed under a light-shielded condition, that is, under a light-shielding condition. This is to prevent changes in the suspension color.

In a specific embodiment, the chitosan is dissolved in acetic acid, silver nitrate is added under light shielding, and sodium borohydride is added to synthesize silver nanoparticles. To obtain a chitosan / silver nanoparticle (CTS / AgNPs) complex, the solution was poured into NaOH to solidify the CTS / AgNPs solution and obtain a CTS / AgNPs complex.

The second step is a step of electrospunning a silver nanoparticle-chitosan complex to produce silver nanoparticles-chitosan nanofibers.

In a specific embodiment, the silver nanoparticle-chitosan complex is dissolved in a TFA / DCM (7: 3) solvent to prepare a solution, which is then electrospun at a discharge rate of 1 ml / h, a spinning distance of 15 cm, a voltage of 23 kV, &Lt; / RTI >

In another specific embodiment, it has been found that as the content of silver nanoparticles increases, the nanofiber diameter of the silver nanoparticle-chitosan complex gradually decreases. This may be due to a decrease in the viscosity of the silver nanoparticle-chitosan complex. The silver nanoparticle content in the silver nanoparticle-chitosan affects the diameter of the electrospun nanofiber and produced fiber beads due to the surface tension enhancement of the electrospinning solution.

2 is a TEM image of a silver nanoparticle-chitosan composite according to an embodiment of the present invention. TEM images show that silver nanoparticles of various sizes are well dispersed inside the silver nanoparticle-chitosan complex.

Also, FIG. 6 shows a TEM image of CTS and CTS / AgNPs processed by electrospinning, and silver nanoparticles present on the surface of the processed nanofibers were confirmed.

The present invention provides silver nanoparticle-chitosan nanofibers prepared by the above-described method.

According to the present invention, silver nanoparticles impart antimicrobial properties to chitosan nanofibers and can produce antimicrobial nanofibers by electrospinning a solution containing antimicrobial agents (silver nanoparticles).

In a specific embodiment, the antimicrobial activity of the chitosan nanofiber composite according to the content of silver nanoparticles was confirmed by the area inhibition test of P. aeruginosa and MRSA in agar medium. As a result of comparing the antimicrobial activities of chitosan nanofiber composites prepared with different concentrations of silver nanoparticles at different concentrations of 0 wt%, 2 wt%, 1.3 wt%, and 0.7 wt%, the antimicrobial effect was more excellent when the content of silver nanoparticles was larger .

Therefore, the silver nanoparticle-chitosan nanofibers of the present invention can be used as a wound dressing material, and can be preferably used as an antibacterial wound dressing material.

The present invention provides a method of manufacturing silver nanoparticle-chitosan nanofibers simply by chemical reduction and electrospinning, and the silver nanoparticle-chitosan nanofibers produced by the above-described method have antibacterial properties and are usefully used as wound dressings .

FIG. 1 is a diagram illustrating a process for producing CTS / AgNPs nanofibers.
FIG. 2 (a) is a TEM image of silver nanoparticles in the composite solution, and FIG. 2 (b) is a size distribution diagram of silver nanoparticles in the composite.
Fig. 3 shows the UV-Vis spectra of silver nanoparticles reduced with NaBH4 in silver nitrate contained in the CTS solution.
Figure 4 is a graph showing the thermogram curves (blue) and the thermograms (red) of the CTS / AgNPs complexes.
Figure 5 shows SEM images after CTS and CTS / AgNPs electrospinning.
FIG. 6 shows TEM images of CTS and CTS / AgNPs processed by electrospinning, wherein (a) - (e) show neutralization and (f) - (j) show neutralization: (a) CTS nanofiber, (b) CTS / AgNPs nanofiber, (c) CTS: CTS / AgNPs = 50:50, (d) CTS: CTS / AgNPs = 67:33, (c) CTS: AgNPs = 83:17.
Figure 7 shows the results of antimicrobial zone inhibition tests against MRSA (dark yellow) and P. aeruginosa (green).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, these embodiments are provided only for the purpose of easier understanding of the present invention, and the scope of the present invention is not limited by the following description.

1. Experimental Method

1-1. material

Alginate (Sigma-Aldrich, St. Louis, Mo.), chitosan powder (average MW, 370 kDa; deacetylation degree 85%), silver nitrate (ACS reagent ≥99.0%), and trifluoroacetic acid (ReagentPlus ^® , MO). Dichloromethane (Extra Pure, 99/0% ⁺ ) was purchased from Junsei (Junsei Chemical Co. Ltd, Japan). Sodium borohydride (NaBH ₄ ) was purchased from TCI (Tokyo Chemical Industry Co., Ltd., Japan). Sodium hydroxide (NaOH) was purchased from YPC (Yakuri Pure Chemicals Co., Ltd. Japan), and methyl alcohol (MeOH) was purchased from DaeJung Chemical & Metals Co. Ltd., Korea. Deionized-distilled water (DDW) was produced with an Ultra Pure Water System (Puris-Ro800; Bio Lab Tech., Korea). Membrane tubes for CTS / AgNPs complex dialysis were Spectrum Spectra (Spectra / Por4dialysis tubing, 12-14K MWCO, width: 45 mm width, length: 100-ft). P. aeruginosa ATCC 27853 and Methicillin-resistant S. aureus ATCC 700787 were obtained from the American Type Culture Collection (Manassas, Va.). All other reagents and solvents were analytical and used without further purification.

1-2. Chitosan / Silver nanoparticle (CTS / AgNPs ) Preparation of complex

4.0 g of chitosan (CTS) was dissolved in 200 ml of 1% acetic acid to form a solution of 2 wt.% At 4 占 폚. After completely dissolving the CTS, the solution was stored at room temperature for 24 hours. The solution was vacuum filtered at room temperature using filter paper (about 10 [mu] m) to remove impurities. To this solution was added 200 ml of 10 mM silver nitrate in DDW with stirring for 2 hours. This procedure was performed to protect the suspension from light and to prevent changes in color. To synthesize silver nanoparticles (AgNPs) in chitosan solution, 80 ml of 40 mM sodium borohydride in DDW was added to chitosan (CTS) solution containing silver nitrate and stirred for 1 hour. To obtain the chitosan / silver nanoparticle (CTS / AgNPs) complex, the CTS / AgNPs solution was poured into 1N NaOH in 100 ml of DDW solution. As a result, the CTS / AgNPs solution solidified. The synthesized CTS / AgNPs complex was washed with DDW until the pH reached 7, lyophilized after dialysis.

1-3. Electrospinning ( Electrospinning , ELSP ) Through the chitosan / Silver nanoparticle (CTS / AgNPs) Processing of nanofibers

The nanofibers were prepared by mixing 5 wt.% CTS and CTS / AgNPs solutions at 100: 0, 0: 100, 50:50, 67:33, 83:17 (AgNPs content = 0.7, 1.3, wt.%). < / RTI > ELSP was performed according to the following conditions. Briefly, a 5 wt.% Solution was prepared by dissolving the CTS / AgNPs complex in a mixed TFA / DCM (7: 3) solvent. For ELSP, the polymer solution was loaded into a luer-lock syringe attached to a metal blunt needle (22G, Kovax-needle Korea Vaccine Co., Ltd., Korea), and a feed rate of 1 ml / h , KDS-200, KD Scientific Inc.) and a high-voltage DC power supply (Nano NC, Korea) at a distance of 15 cm from the needle to the collector using a mandrel covered aluminum foil rotating at 23 kV. The resulting CTS / AgNPs nanofibers were dried under vacuum overnight to remove residual solvent. For neutralization, the dried CTS / AgNPs nanofibers were immersed in 50 ml of 3.2 M NaOH / MeOH (neutralizing solution) for 10 minutes and then washed with DDW until pH reached 7. The CTS / AgNPs nanofibers were then lyophilized.

1-4. UV-visible spectroscopy (UV)

To confirm the presence of AgNPs in the CTS / AgNPs solution, ultraviolet-visible spectroscopy was performed using UV1650PCS (Shimadzu, Japan). The ultraviolet-visible spectrum was measured over a wavelength range of 300-700 nm. CTS solution containing silver nitrate (10 mM) was used as a reference blank.

1-5. X-ray diffraction ( XRD )

To identify the crystalline structure of AgNPs in the CTS / AgNPs complexes, X'pert PRO (PANalytical) with CuKα (λ = 0.154 nm) radiation in the 2θ range of 10 ° to 70 ° at a voltage of 45 kV and a current of 20 mA , USA) was used to record XRD patterns for each of the dry CTS and CTS / AgNPs nanocomposites. The measurement was repeated three times.

1-6. Thermogravimetric analysis ( thermogravimetric analysis, TGA )

TGA was performed with TGA-50 (Shimadzu, Japan) to confirm the thermal decomposition profile of the CTS and CTS / AgNPs complexes. The CTS or CTS / AgNPs complex dried 5 mg sample was measured at a nitrogen flow rate of 50 ml / min and a heating rate of 10 ° C min ^-1 at 25 ° C to 800 ° C. The measurement was repeated three times.

1-7. Viscosity measurement Viscometry , Viscometer)

In order to investigate the viscosity of the ELSP solution, the solution was measured with a sine-wave Vibro Viscometer (SV-10, A & D Company, Ltd., Japan) at a vibration frequency of 30 Hz and a range of 0.3 mPas to 10,000 mPas. All measurements were performed at room temperature. A solution sample was prepared according to the following conditions. Briefly, a 5 wt.% Solution was prepared by dissolving a complex of CTS and CTS / AgNPs (AgNPs content 0, 4, 2, 1.3 and 0.7 wt.%) In a mixed TFA / DCM (7: 3) solvent. The measurement was repeated three times.

1-8. Transmission electron microscopy (TEM) TEM )

In order to verify the amount and presence of AgNPs in the CTS / AgNPs nanofibers, TEM observation was carried out by TEM using an H-7100 (Hitachi, Japan) at room temperature and an acceleration voltage of 100 kV. CTS nanofibers alone or in varying proportions of CTS / AgNPs nanofiber specimens were applied to a 200 mesh copper lattice coated with Formvar / carbon (Catalog No. 01801, Ted Pella Inc., USA) and dried at room temperature before imaging.

1-9. Electronic scanning microscope (Scanning electron microscopy, SEM )

The surface morphology of CTS nanofibers and CTS / AgNPs nanofibers was confirmed by scanning electron microscopy (SEM, Hitachi S-2300, Japan) at an accelerating voltage of 15 kV. All samples were dried at room temperature and sputter-coated with gold by IB-3 (Eiko, Japan) for 10 minutes. Matrix morphology and fiber diameter validation were performed with image analysis software (Eyeview-Analyzer (Digiplus, Korea)).

1-10. P. aeruginosa And antimicrobial activity against MRSA

P. aeruginosa ATCC 27853 and MRSA ATCC 700787 were obtained from the American Type Culture Collection (Manassas, Va.). They were grown in aerobic conditions of Mueller-Hinton broth at 35 ° C for 18 hours. The disk diffusion method was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006) using Mueller-Hinton Agar (Difco). Briefly, the agar surface was inoculated using a dipped swap with a turbidity adjusted bacterial cell suspension of 0.5 McFarland standard units and dried for about 5 minutes. CTS-containing disks (8 mm) were placed and susceptibility plates were incubated at 35 ° C for 16-18 hours. The inhibition zone around the disc was measured in millimeters using a ruler.

2. Results

2-1. Synthetic chitosan / Silver nanoparticle (CTS / AgNPs ) Solution Identification (UV, TEM )

The CTS / AgNPs solution was obtained by reduction of silver nitrate using sodium borohydride in CTS solution. In the first step, silver nitrate was added to the CTS solution. Silver ions in silver nitrate bound to amine groups in CTS molecules by chelate bonds. In the second step, a chemical reducing agent, sodium borohydride, was added to the CTS solution containing silver nitrate. After addition, the color of the solution immediately changed from light yellow to dark brown. These results indicate that AgNPs were synthesized rapidly in CTS solution. The synthesized solution becomes a CTS / AgNPs complex after recrystallization purification. As shown in Figure 2a, the presence of AgNPs in the CTS / AgNPs solution was confirmed by TEM for a dry CTS / AgNPs sample solution on a copper-lattice plate. The TEM image demonstrated that the AgNPs were well dispersed in the CTS / AgNPs solution. It also showed that the resulting particles formed a nearly spherical shape. For the measurement of average diameter, 100 specimens of AgNPs were measured. As a result, the sizes of 10 nm and 11 nm were the most common, and the average diameter of AgNPs was 10 ± 2 nm. This result is similar to previous studies in which silver nitrate interacts with CTS and is reduced by sodium borohydride. This result is also clearly demonstrated in the UV-Vis absorption spectra peak. Absorption band was observed in the range of 325-475 nm. The narrow and sharp wavelength band was also observed at 408 nm, indicating the formation of small diameter AgNPs in the CTS solution. These distinctive distinct peaks indicate strong plasma excitation of AgNPs.

2-2. Synthetic chitosan / Silver nanoparticle (CTS / AgNPs Properties of Composites XRD , TGA )

After recrystallization, the CTS / AgNPs solution was made a CTS / AgNPs complex for use as an ELSP material. XRD was performed to confirm the crystal structure of CTS and AgNPs in the lyophilized CTS / AgNPs complex. The pure CTS and CTS / AgNPs complexes have strong peaks at 20 °, which means the degree of crystallinity due to the main component chain of CTS. On the other hand, CTS / AgNPs showed four different peaks compared to the CTS complex. CTS / AgNPs showed peaks at 38.0 °, 44.3 °, 64.5 ° and 77.2 °, respectively, indicating (111), (200), (220) and (311) of the plane centered cubic structure of AgNPs. These results indicate that AgNPs are well dispersed in the CTS / AgNPs complex. TGA analysis was performed to evaluate the content of AgNPs in CTS / AgNPs. As shown in Figure 4, all samples showed three distinct weight loss in the 25-800 < 0 > C range. The first weight loss interval was due to drying of the water molecules in the composite matrix at 36 ° C and the second weight loss was due to the decomposition of the polymer at 239 ° C. The third weight loss was due to the decomposition of the CTS complex at 573 ° C, and the CTS / AgNPs complex was decomposed at 513 ° C. These other degradations were influenced by the residues of AgNPs. At 600-800 ℃, the amount of CTS / AgNPs complex showed that the residues of AgNPs remained intact even at high temperatures without any polymer. For this reason, we have demonstrated that AgNPs bind not only to CTS but also to CTS / AgNPs complex at about 4 wt%.

2-3. CTS / AgNPs Viscosity Property of Solution

As shown in Table 1 below, the viscosities of the CTS and CTS / AgNPs solutions were 424, 252, 281, 364 and 416 mPs for each of the AgNPs contents of 0, 4, 2, 1.3 and 0.7 wt%. The viscosity of the ELSP solution was observed to decrease with increasing CTS / AgNPs complex content. Viscosity measurements correlate with solution surface tension. Solution surface tension or viscosity plays an important role in determining the diameter of the ELSP fibers. Various ratios of processed fibers showed variety in diameter and bead formation. The AgNPs in the 4 wt% solution had the lowest viscosity, which is the highest surface tension compared to the other specimens. As shown in FIG. 5b, AgNPs having a content of 4 wt% were observed to have beads which can contribute to the low viscosity of the solution and the high surface tension due to the increased silver content. The solution viscosity affects fiber morphology. The high viscosity solution has a longer stress action or a longer relaxation time, which can avoid cracking of the ejected jet during ELSP. On the other hand, low viscosity solutions have an adverse effect on fiber processing during ELSP. This is because the spray jet breaks small beads to produce fiber mixtures and distinct beads.

CTS: CTS / AgNPs Content of AgNPs (wt%) Diameter (nm) Viscosity (mPas) (a) 100: 0 0 460 ± 80 424 (b) 0: 100 4 126 ± 28 252 (c) 50:50 2 238 ± 46 281 (d) 67:33 1.3 337 ± 49 364 (e) 83:17 0.7 349 ± 56 413

2-4. CTS / AgNPs Morphological Qualification of Nanofibers (Characterization) SEM , TEM )

5 shows SEM micrographs of various nanofibers, respectively. Previously, CTS and CTS / AgNPs nanofibers were obtained through ELSP technology. The reason for choosing this method as a method of mixing the complex is because it is difficult to quantitatively measure AgNPs, and it is too small to accurately weigh. As shown in Figs. 5A and 5E, homogeneous nanofiber structures were observed in a single form. On the other hand, FIG. 5B shows that 4 wt% AgNPs in the nanofibers have a rough surface shape in which beads are wound. As shown in FIG. 5 and Table 1, the average diameters of CTS and CTS / AgNPs nanofibers were 460 ± 80 nm, 126 ± 28 nm and 238 ± 46 nm for AgNPs content angles 0, 4, 2, 1.3 and 0.7 wt% , 337 49 nm and 349 56 nm. It was observed that the diameter of the nanofibers decreased as the content of AgNPs increased. Due to the presence of AgNPs in the CTS / AgNPs solution, the charge was increased and conductive, which allowed the fibers to have smaller diameters. The micrographs of Figure 5f-j were obtained after neutralization of CTS and CTS / AgNPs nanofibers. Each of these fibers was slightly swollen compared to the non-neutralized nanofibers. However, these neutralized fibers did not show any significant differences in fiber diameter and morphology compared to the fibers shown in Figs. 5a-e. The TEM image (Figure 6) clearly shows whether the nanofibers comprise AgNPs. Although no particles were present in the pure CTS nanofibers shown in FIG. 6A, it was observed that the CTS / AGNPs rod nanofibers shown in FIG. 6B-e had well dispersed AgNPs in the fibers. In addition, it was observed that the amount of AgNPs decreased to e in FIG. 6B indicating that the AgNPs content gradually decreased. Likewise, in the SEM image, the neutralized nanofibers of the TEM image were not different in their morphology with respect to the non-neutralized image shown in Figs. 6a-e.

2-5. P. aeruginosa And zone inhibition test of MRSA antimicrobial activity

Bacterial inhibition tests of CTS and CTS / AgNPs against P. aeruginosa and MRSA were performed. To determine whether the CTS / AgNPs nanofibers had antibacterial activity, pure CTS nanofibers were used as negative control and CTS / AgNPs nanofibers were used as control samples in various ratios. As shown in Figure 7, both P. aeruginosa (green plate) and MRSA (dark yellow plate) all clearly show bacterial growth around the CTS / AgNPs nanofiber. The diameter of each sample is shown in the graph. The pure CTS nanofibers did not show any inhibition on the bacteria. On the other hand, CTS / AgNPs nanofibers exhibited slightly higher bacterial growth inhibitory effect on P.areuginosa than MRSA. This demonstrated that bacterial growth was inhibited by the presence of AgNPs in the fiber. Antimicrobial tests of CTS and CTS / AgNPs against P. aureuginosa were performed on CTS / AgNPs = 100: 0, 50:50, 67:33, 83:17 (AgNPs content = 0, 2, 1.3, 0.7 wt% 0 mm, 16.73 mm, 16.53 mm, and 16.07 mm. The MRSA inhibition zone was 0 mm, 15.75 mm, 15.42 mm, respectively for the CTS / AgNPs = 100: 0, 50:50, 67:33, 83:17 (AgNPs content = 0,2, 14.9 mm. This result implies that the amount of AgNPs is increased in the fiber to enhance the antimicrobial effect. This means that the amount of AgNPs can be adjusted to control the antimicrobial effect. Bacteria are affected by AgNPs particles. First, AgNPs bind negatively to the surface of cell membranes that are charged. This phenomenon leads to loss of osmolality and affects the respiratory chain. Second, AgNPs penetrate cell bodies and damage cells because they interact with sulfur and phosphorus found in physiological components such as DNA. Finally, AgNPs lead to cell lysis and cell leaks leading to apoptosis. In the case of the present invention, the CTS / AgNPs nanofiber acts as a vehicle for transferring silver ions. As all previous results show, CTS / AgNPs nanofibers have good potential for use as biocompatible and antimicrobial wound dressings.

Claims

A first step of adding silver nitrate to chitosan and then adding a reducing agent to form a silver nanoparticle-chitosan complex; And a second step of electrospunning the composite to produce silver nanoparticle-chitosan nanofibers.

The method according to claim 1, wherein the chitosan of the first step has a deacetylation degree of 85%.

The process according to claim 1, wherein the reducing agent is sodium borohydride.

The method according to claim 1, wherein the first step is performed under light-shielding conditions.

A silver nanoparticle-chitosan nanofiber produced by the production method of any one of claims 1 to 4.

A wound dressing material comprising silver nanoparticle-chitosan nanofibers prepared by the method of any one of claims 1 to 4.

7. The wound dressing material of claim 6, wherein the wound dressing material is antimicrobial.