Accepted Manuscript Title: Fabrication and durable antibacterial properties of electrospun chitosan nanofibers with silver nanoparticles Author: Yanan Liu Yang Liu Nina Liao Fuhai Cui Mira Park Hak-Yong Kim PII: DOI: Reference:
S0141-8130(15)00399-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.05.058 BIOMAC 5142
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-2-2015 26-5-2015 30-5-2015
Please cite this article as: Y. Liu, Y. Liu, N. Liao, F. Cui, M. Park, H.-Y. Kim, Fabrication and durable antibacterial properties of electrospun chitosan nanofibers with silver nanoparticles, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.05.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
Fabrication and durable antibacterial properties of electrospun chitosan nanofibers with silver nanoparticles
Department of BIN fusion technology, Chonbuk National University, Jeonju 561-756,
cr
a
ip t
Yanan Liua, Yang Liua, Nina Liaob, Fuhai Cuia, Mira Parkc, *, Hak-Yong Kima,* *
b
us
Republic of Korea
Department of Bionanosyestem Engineering, Chonbuk National University, Jeonju
Department of Organic Materials and Fiber Engineering, Chonbuk National
M
c
an
561-756, Republic of Korea
Ac
ce pt
ed
University, Jeonju 561-756, Republic of Korea
* Corresponding author: Tel: +82-63-270-2351, Fax: +82-63-270-4249. E-mail address: [email protected] (M. Park) ** Corresponding author: Tel: +82-63-270-2351, Fax: +82-63-270-4249. E-mail address: [email protected] (H. Y. Kim)
Page 1 of 30
Abstract Non-precipitation Chitosan/silver nanoparticles (AgNPs) in 1% acetic acid aqueous solution was prepared from chitosan colloidal gel with various contents of silver
ip t
nitrate via electron beam irradiation (EBI). Electrospun chitosan-based nanofibers decorated with AgNPs were successfully performed by blending poly(vinyl alcohol).
cr
The morphology of as-prepared nanofibers and the size of AgNPs in the nanofibers
us
were investigated by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The presence of AgNPs in as-obtained
an
nanofibers was also confirmed by ultraviolet-visible spectroscopy (UV), Fourier
M
transform infrared (FT-IR) spectroscopy, EDX spectrum and metal mapping. Silver ion release behavior indicated that these hybrid nanofibers continually release
ed
adequate silver to exhibit antibacterial activity over 16 days. These biocomposite nanofibers showed pronounced antibacterial activity against Staphylococcus aureus (S.
ce pt
aureus) and Escherichia coli (E. coli).
1.
Ac
Keywords: chitosan, silver nanoparticles, electrospinning, antibacterial nanofibers
Introduction
Chitosan (CS), as the second abundance of natural polysaccharide, has been
widely used in numerous applications such as medicine, food and chemical engineering, pharmaceuticals, nutrition, and agriculture [1]. CS-based nanofibers (NFs) have been identified as an excellent biomaterial, especially for tissue scaffold and wound healing because CS possesses renewability, nontoxicity, absorption of
Page 2 of 30
exudates, and antibacterial activity [2, 3], simultaneously nanofibrous architectural morphology is similar to natural extracellular matrix which can promote cell’s attachment, proliferation and differentiation [4]. However, electrospun pristine CS
ip t
NFs is considered extremely difficult due to its high molecular weight, its low stability and its low solubility in common organic solvent. It is essential to manage
cr
molecular weight of CS via depolymerization, chemical or radiation-treatment to
us
improve its solubility [5-8]. CS-based nanofibrous mats are supposed to be fabricated successfully via electrospinning using appropriate chemical structure of CS.
an
Electrospinning is recognized as an efficient technology to prepare micro or nano
M
sized fibers for various applications spreading in different fields, such as environment, energy, electronic and biomaterials [9-11]. The distinct features of electrospun
ed
nanofibers emerge to be high porosity, large surface area, and simple fabrication process. In addition, nanofibers possess high capacity for incorporating of various
ce pt
biological materials or active substances, for instance, drugs, natural remedies, or metal nanoparticles [12, 13]. Moreover, the combination of nanofibers and
Ac
antibacterial substances as a fascinating wound healing material has demonstrated antibacterial efficacy to prevent wound contamination by some bacteria. Owing to AgNPs possess outstanding antibacterial activity toward germs on
contact without the release of toxic biocides [14, 15], utilization of AgNPs to integrate into electrospun NFs has been drawn considerable attention for wound healing, tissue engineering, biological protective materials, and medical devices applications [16-18]. In order to attain hybrid NFs containing AgNPs, reduction of Ag ion to AgNPs has to
Page 3 of 30
be carried out before or after elelctrospinning [19, 20], which can be accomplished by irradiation, thermal or chemical reduction process [21]. Among these treatment method, electron beam irradiation (EBI) is considered as an efficient method for
ip t
large-scale synthesis of AgNPs [22], and has more advantages over others, including easy operation, inexpensive, short irradiation time, and absence of chemical residues
cr
after irradiated. Furthermore, EBI treatment is possible to control the size and
us
suppress the agglomeration of as-obtained AgNPs, which affect the property of nanoparticles [21, 23]. Generally, radiation-induced reduction performs as follows
an
[24],
M
1) Water radiolysis:
(1)
ed
2) Reduction by hydrated electron and hydrogen atoms: (2) (3)
ce pt
Therefore, EBI is a facile, feasible, and highly desirable approach to attain AgNPs-loaded polymer fibers.
Ac
CS/PVA NFs were successfully prepared from biocompatible polymer-solution via EBI and electrospinning technique in our previous work [8]. The advantages of this strategy were non-toxic and environmental-friendly throughout the overall system with no chemical residues for NFs. In spite of the antibacterial property showed from CS, the obtained NFs still need to improve their durably antibacterial activity. Thus, we develop advanced CS-based NFs by adding AgNPs as a reinforced antibacterial agent. It is expected to associate the superiorities of CS/PVA composite with
Page 4 of 30
antibacterial activity of AgNPs. In order to reduce process steps, EBI treatment is managed to diminish molecular weight of CS and fabricate AgNPs from Ag ions synchronously. It is a facile fabrication process to accomplish CS/AgNPs solution.
ip t
Then CS/PVA/AgNPs biocomposite NFs are prepared by blending non-toxic PVA solution to enhance the spinnability of CS/AgNPs solution [4]. AgNPs effect on the
Experimental
us
2.
cr
morphology, diameter and antibacterial properties of NFs is further evaluated.
2.1 Materials (200,000cps,
75-85%
deacetylated),
an
Chitosan
poly(vinyl
alcohol)
M
(Mw=85,000-124,000, 87-89% hydrolyzed) and glyoxal solution (40 wt % in H2O) were purchased from Aldrich Co., USA. Silver nitrate (Showa, Japan) was used as
ed
metal precursor. Acetic acid and phosphoric acid were obtained from Samchun Pure
purification.
ce pt
Chemical Co., Ltd, South Korea. All the chemicals were used without further
2.2 Preparation of CS/AgNPs homogenous solution
Ac
8 g CS powder was dispersed in 92 ml deionized water at 90 °C for 24 h, then Silver nitrate (0, 0.5, 1 and 2 wt % with weight of CS) was added with stirring for 8 h at 25 °C, respectively. CS/AgNO3 gelatinous solid was obtained by pouring 1 mL acetic acid. All the gelatinous CS/AgNPs samples were irradiated with an electron-beam at a dose of 150 kGy to get non-precipitate solutions. 2.3 Fabrication of CS/PVA/AgNPs NFs via electrospinning 10 wt % PVA solution was prepared by dissolving PVA powder in deionized
Page 5 of 30
water at 90 °C for 24 h. The homogeneous solution was obtained by mixing 4g PVA solution and 5g CS/AgNPs solution (AgNPs contents = 0, 0.5, 1, and 2 wt % of CS) under vigorously magnetic stirrer for 4h, respectively. And then 0.06 ml glyoxal
ip t
solution was added as cross-linking agent when the pH values of these system were adjusted to 2~3 by phosphoric acid. In a typical electrospinning experiment, CS/PVA
cr
with various contents of AgNPs (0, 0.25, 0.5 and 1 wt %) NFs were successfully
us
electrospun at 15 kV using a high-voltage DC power supply with a 14 cm tip-to-collector distance and 35% relative humidity, respectively. As-spun
an
CS/PVA/AgNPs nanofibers were collected by Teflon papers. In order to make
M
resulting fibrous mats cross-linking effectively, put them in the heat oven under alcohol vapor at 50 °C for 24 h, subsequently cured at 120 °C for 10 min. A schematic
ce pt
2.4 Characterizations
ed
process of CS/PVA/AgNPs NFs is shown in Fig. 1.
CS/AgNPs solution was performed from CS/AgNO3 gelatinous paste via electron beam accelerator (EBTECH Co., Ltd., Korea, beam energy of 2.5 MeV,
Ac
beam current of 8.5 mA, and dose rate of 6.67 kGy/sec) at 25 °C in an air atmosphere. The chemical structure of CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs was confirmed by Fourier transform infrared spectroscopy (FT-IR, Varian 1000 Scimitar series). Ultraviolet–visible (UV–vis) absorption spectra of as-obtained NFs was measured by UV-2600 UV-Vis Spectrophotometer (Shimadzu, Japan). The surface morphologies and diameters were determined by Field emission scanning electron microscopy (FESEM, S-7400, Hitachi, Japan). The size, shape, and manner of
Page 6 of 30
deposition of AgNPs on the fiber surface were investigated via biological transmission electron microscopy (Bio-TEM, JEM-2010, JEOL, Japan). The metal mapping and EDX spectrum of hybrid NFs mat were recorded with the scanning
ip t
electron microscopy instrument (SEM, JSM-5900JEOL Co.). Mechanical properties were measured with a universal testing machine (AG-5000G, Shimadzu, Japan),
cr
under a crosshead speed of 5 mm/min at room temperature. The samples were
us
prepared in the form of standard dumbbell shapes according to ASTM Standard D 638 via die cutting from the electrospun mats and tested in the machine direction. Each
an
sample was performed at five times.
M
2.5 Silver ion release
Silver ion release behavior of the NFs was determined as followed, 50 mg hybrid
ed
NFs was placed in a conical flask and 50 ml deionized water was added into it as the release medium. The flask was shaken to make sure the NFs was completely
ce pt
immersed, then sealed and incubated at room temperature. The deionized water was collected every 12, 24, 36, 72 or 96 h. Silver ion concentration in the solution was
Ac
measured using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (ICPS-7500, SHIMADZU, Japan). 2.6 Antibacterial Properties Characterizations The antibacterial activities of CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs membranes were investigated by a zone inhibition method. Gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus) cells were used as the model microorganisms. All CS/PVA/AgNPs NFs with different
Page 7 of 30
contents of AgNPs were cut into 6 mm diameter discs and sterilized by UV light prior to bacterial viability test. Then, NFs discs were transferred to E. coli or S. aureus bacterium suspension containing around 106 colony forming units (CFUs)/mL and
diameter of inhibition area around each disc. 3.
Results and Discussions
us
3.1 Preparation of CS/AgNPs solution and electrospinning
cr
ip t
then incubated for 24 h at 37 °C. The zone of inhibition was measured by testing the
One role of EBI treatment was to decrease the molecular weight of CS for
an
dissolving in 1% acetic acid aqueous solution [8], the other role was large scale
M
production of AgNPs according to the procedure described by Kang et al [24]. Interestingly, the AgNPs grow in size with an increase of the absorbed dose. Therefore,
ed
an EBI dose at 150 KGy has effectively transformed CS/Ag+ gelatinous paste to non-precipitation solution. In addition, biocompatible and biodegradable PVA was
ce pt
chosen as a composition to improve the viscosity and spinability of CS/AgNPs solution owing to its easy-to-obtain, its good fiber-forming ability, its non-toxic and
Ac
water-solubility [4]. Finally, the weight ratio of PVA and CS/AgNPs was permanent at 1:1 in order to make NFs contain the high content of antibacterial component on the premise of forming good fiber morphology. To detect the formation of AgNPs in the CS/AgNPs solution, UV spectroscopy was performed to test the characteristic ultraviolet peak of AgNPs. The spectra of as-prepared CS/AgNPs solutions are shown in Fig. 2 (e). For pristine CS solution, one peak appeared at 300 nm, likely due to the absorption peak of chitosan
Page 8 of 30
oligosaccharide [25].The maximum absorption peak was observed at approximately 410 nm, which is characteristic surface Plasmon absorption for AgNPs. UV absorption peak of as-prepared AgNPs was consistent with other researchers recorded
ip t
in the range 410–420 nm [16, 26]. In addition, the intensity of AgNPs absorption peak increased when increasing the concentration of AgNO3 in precursor solution, implying
cr
that the maximum efficiency of large scale producing AgNPs was realized via EBI
us
treatment. 3.2 Morphology of electrospun NFs
an
FESEM images in Fig. 2 (a~d) show the morphology of as-obtained hybrid
M
nanofibrous mats with various contents of AgNPs (0, 0.25, 0.5 and 1 wt %). All electrospun NFs possessed the uniform, smooth, continuously and bead-free
ed
morphology. An average diameter of pristine CS/PVA NFs was 155 nm, the average diameter of hybrid NFs decreased by increasing AgNPs content, 148, 144 and 139 nm,
ce pt
respectively, which were determined by averaging the diameter of 50 random fibers. But this was not significantly difference, because the AgNPs content is very low and
Ac
all the CS/AgNPs solution possessed the similar viscosity (about 25 cps) and conductivity (about 0.7 ms/m). To quantify the average size and distribution of AgNPs and assembling structure of AgNPs on the NFs, TEM images were used to measure the NPs size and distribution of NFs. Fig. 2 (f~h) exhibited that many individual AgNPs with oval and spherical shape distributing on the surface of CS/PVA NFs were observed. The particle size was on the range of 1-30 nm and average size of particles increased with increasing AgNPs content, 14.0, 15.4 and 18.6
Page 9 of 30
nm, respectively. These results were ascribed to the aggregation of AgNPs, which may be caused by constantly attack of the Ag atoms by hydrated electrons and hydrogen atoms generated during the e-beam process at relatively high dose and high
ip t
concentration [21, 24]. To further explore the distribution of AgNPs on the NFs, metal mapping image and EDX of hybrid NFs was taken (Fig. 3), which also confirmed the
cr
deposition of AgNPs on the surface of CS/PVA NFs. In addition, it was obvious that
us
the particles dispersed uniformly on the NFs. The content of AgNPs was 0.02, 0.06, 0.09 %, respectively, which was found to be similar with theoretical calculated value.
an
All of these results implied nanoparticles to the utmost extent to be utilized without
3.3 FT-IR spectra of electrospun NFs
M
loss.
ed
The FT-IR spectra of electrospun CS/PVA/AgNPs NFs are shown in Fig. 4. The frequencies and assignments for the pristine CS/PVA were indicated as follows: C–H
ce pt
asymmetric vibration (2933 cm−1), –OH and –NH2 groups (3700-3000 cm−1), C=O bond (1733 cm−1), C=O bond (1640 cm-1) in NHCO–, and C–C stretching vibration
Ac
(851cm−1) [25, 27]. The presence of AgNPs in the NFs was evidenced by the vibration band of N–C=O group taken place the blue shift from 1534 cm-1 to 1552cm-1 with increasing AgNPs content [28], which indicated that N-H vibration was affected by attachment of the AgNPs. In short, these results implied the existence of AgNPs in the NFs and the interaction force between NPs and –NH2 groups of CS via FT-IR spectra. 3.4 Mechanical properties Typical stress−strain curves for hybrid NFs with varying AgNPs content are
Page 10 of 30
illustrated in Fig. 5. It was observed that the tensile strengths of as-prepared NFs were significantly increased with more AgNPs loaded, from 3.65 MPa of PVA/CS NFs to 3.78 and 4.52 MPa with 0.25 and 0.5 wt % NPs loading, respectively. The possible
ip t
interactions between CS/PVA with AgNPs enhanced by intermolecular interactions (-OH of PVA, Ag, -CH2OH of CS) [29]. When 1 wt % AgNPs loaded to NFs, the
cr
tensile strength of hybrid NFs decreased appreciably to 3.74 MPa probably due to
us
regularity and compactness of NFs affected by the aggregation of AgNPs. Thus, CS/PVA/AgNPs (0.5 wt %) NFs showed the highest tensile strength, this implied that
an
the tensile strengths of NFs membranes were sufficient for practical handling during
M
the wound healing process. 3.5 Silver ion release
ed
In order to evaluate Ag+ release rate for these CS/AgNPs-based antibacterial materials, ICP-AES spectra was used to test Ag+ release behavior. For purpose of
ce pt
providing effective antibacterial properties, it is necessary to keep a steady and prolonged Ag+ release rate at a concentration level at least 0.1 ppb [13, 30]. When the
Ac
hybrid NFs was incubated into aqueous solution, the AgNPs gradually release Ag+ into the water. The release rate is mainly determined by the concentration of NPs and the diffusion distance from inside the fiber to the surface [31]. Ag+ release profiles of hybrid NFs are presented in Fig. 6. It is found that the release rate is relatively high in the first few days and then continuously release over time. For the electrospun hybrid NFs loaded with 0.25, 0.5 and 1 wt % AgNPs, the accumulated released amount of Ag after 16 days were approximately 40, 56 and 36 %, respectively. After 9 days,
Page 11 of 30
AgNPs-loaded hybrid NFs with (0.5 and 1 wt %) still gave a steady at 0.13–0.14 ppm per day, and release rate of NFs with 0.25 % AgNPs went to level off at 0.005 ppm. The results indicated that CS/PVA/AgNPs NFs prepared by EBI and electrospinning
ip t
can release sufficient amounts of silver and have a long lifetime to exhibit sustained antibacterial activity.
cr
3.6 Zone of inhibition testing of antibacterial efficacy (E. coli and S. aureus)
us
AgNPs leads to bacterial cell death by interacting with sulfur-containing proteins in germ membrane and releasing Ag+ to attack the respiratory chain, cell division [32].
an
Furthermore, an extremely large surface area of AgNPs can provide larger action sites
M
for microorganisms to promote bactericidal activity. The antibacterial property of pristine CS/PVA and CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs against E. coli
ed
and S. aureus were performed by the disc diffusion susceptibility test after 24 h incubation. As presented in Fig. 7, both of E. coli and S. aureus clearly showed a zone
ce pt
of inhibited growth of the bacteria around the CS/PVA and CS/PVA/AgNPs NFs. The diameter of inhibition ring for each sample was shown in the schematic diagram.
Ac
Pristine CS/PVA possessed inhibition ring diameter at 7.0 mm owing to the cationic nature of CS as antimicrobial agents [33]. The inhibition zones of CS/PVA/AgNPs (0.25, 0.5 and 1 wt %) NFs against E. coli were 7.7, 13.3 and 9.0 mm, respectively. Furthermore, CS/PVA/AgNPs (0.25 and 0.5 wt %) NFs were exhibited to inhibit the growth of bacteria with slightly higher effectiveness against S. aureus compared with E. coli, the zones of inhibition increased to 9.1 and 14.3 mm, which was in accordance with CS-Ag composite [34, 35]. It was mentioned that the most
Page 12 of 30
antibacterial effected against for both two microorganisms when AgNPs (0.5 wt %) loaded to hybrid NFs. After adding more AgNPs, the antibacterial property of hybrid NFs weakened mainly owing to the aggregation of AgNPs at relative high
ip t
concentration and the potentially devastating effects of AgNPs [15, 36]. Therefore, this work indicated the wonderful antibacterial performance of CS/PVA/AgNPs (0.5
Conclusions
us
4.
cr
wt %) which can be used as excellent materials in biomedical application.
In this study, continuous and uniform CS/PVA/AgNPs NFs were successfully
an
electrospun using eco-friendly solvent. It is a wonderful approach to fabricate
M
CS/AgNPs-based biocomposite NFs by combine EBI and electrospinning technique. The green EBI process was achievable to gain CS solution from CS gel and
ed
large-production of AgNPs from Ag+. It was observed that AgNPs dispersed uniformly in the CS/PVA NFs, which remained utilization of nanoparticles to the
ce pt
utmost extent. Furthermore, the properties of PVA/CS NFs were improved greatly by AgNPs loaded. These hybrid NFs mats showed good mechanical property and
Ac
antibacterial activity toward E. bacteria and S. aureus, and latter bacteria was the more sensitive microbe against antimicrobial disk when the concentration of AgNPs was only 0.5 wt %. The resultant hybrid NFs provide a steady and prolonged silver ion release. This work provides a new direction for efficient CS/AgNPs-based NFs as antibacterial biomedical applications.
Page 13 of 30
Acknowledgements Following are results of a study on the “Basic Research Program through the National Research Foundation of Korea (NRF)” project, funded by the Ministry of Education
ip t
(No.NRF-2014R1A1A2008489). This research was supported by the National Research Foundation of Korea (NRF) Grant funded by Korea Government (MISP)
Figure Captions
an
Fig. 1. A schematic process of CS/PVA/AgNPs NFs.
us
cr
(NO. 2014R1A4A1008140).
M
Fig. 2. FESEM images of CS/PVA NFs (a), FESEM and TEM images of hybrid NFs with contents of AgNPs, 0.25 wt % (b and e), 0.5 wt % (c and f) and 1 wt % (d and g), respectively. Vis-UV spectra of CS solutions with various contents of AgNPs (e).
ed
Fig. 3. SEM metal mapping images and EDX of hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively (a~c).
ce pt
Fig. 4. FTIR spectra of electrospun fibrous mats: (a) CS/PVA, (b~d) hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively. Fig. 5. Stress−strain curves of hybrid NFs with different contents of AgNPs, 0, 0.25,
Ac
0.5 and 1 wt %, respectively (a~d).
Fig. 6. Silver release profiles of hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively (a~c). Fig. 7. Zone of inhibition antibacterial testing against E. coli (left) and S. aureus (right). (a~d) CS/PVA /AgNPs NFs with different contents of AgNPs, 0, 0.25, 0.5 and 1 wt %, respectively. Schematic diagram is shown that measured dimension of inhibited bacteria.
Page 14 of 30
ip t
Fig. 1.
Fig. 3.
Ac
ce pt
ed
M
an
us
cr
Fig. 2.
Page 15 of 30
ip t cr us an M Ac
ce pt
ed
Fig. 4.
Page 16 of 30
Fig. 6.
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 5.
Page 17 of 30
us
cr
ip t
Fig. 7.
Ac
ce pt
ed
M
an
GRAPHICAL ABSTRACT
References
[1] A. Domard, Carbohydr. Polym., 84 (2011) 696-703. [2] D. Archana, B.K. Singh, J. Dutta, P.K. Dutta, Int. J. Biol. Macromol., 73 (2015) 49-57. [3] A.S. Mehta, B.K. Singh, P.K. Dutta, Asian Chitin Journal, 10 (2014) 25-28. [4] M.Z. Elsabee, H.F. Naguib, R.E. Morsi, Mat Sci Eng C-Mater, 32 (2012) 1711-1726.
Page 18 of 30
[5] I. Prasertsung, S. Damrongsakkul, C. Terashima, N. Saito, O. Takai, Carbohydr. Polym., 87 (2012) 2745-2749. [6] C.T. Tsao, C.H. Chang, Y.Y. Lin, M.F. Wu, J.L. Han, K.H. Hsieh, Carbohydr. Res., 346 (2011)
ip t
94-102. [7] H.A. Abd El-Rehim, N.M. El-Sawy, S.A. Hegazy el, S.A. Soliman el, A.M. Elbarbary, Int. J. Biol.
cr
Macromol., 50 (2012) 403-413.
[9] R.J. Wade, J.A. Burdick, Nano Today, 9 (2014) 722-742.
us
[8] Y. Liu, M. Park, H.K. Shin, B. Pant, S.-J. Park, H.-Y. Kim, Mater. Lett., 132 (2014) 23-26.
an
[10] X. Wang, B. Ding, J. Yu, M. Wang, Nano Today, 6 (2011) 510-530.
M
[11] V.J. Babu, S. Vempati, S. Sundarrajan, M. Sireesha, S. Ramakrishna, Solar Energy, 106 (2014) 1-22.
ed
[12] A.J. Meinel, O. Germershaus, T. Luhmann, H.P. Merkle, L. Meinel, Eur. J. Pharm. Biopharm., 81 (2012) 1-13.
ce pt
[13] Q. Shi, N. Vitchuli, J. Nowak, J.M. Caldwell, F. Breidt, M. Bourham, X. Zhang, M. McCord, Eur. Polym. J., 47 (2011) 1402-1409.
Ac
[14] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv., 27 (2009) 76-83. [15] K.-H. Cho, J.-E. Park, T. Osaka, S.-G. Park, Electrochim. Acta, 51 (2005) 956-960. [16] A.M. Abdelgawad, S.M. Hudson, O.J. Rojas, Carbohydr. Polym., 100 (2014) 166-178. [17] Z.C. Xing, W.P. Chae, J.Y. Baek, M.J. Choi, Y. Jung, I.K. Kang, Biomacromolecules, 11 (2010) 1248-1253. [18] H. Deka, N. Karak, R.D. Kalita, A.K. Buragohain, Polym. Degrad. Stab., 95 (2010) 1509-1517. [19] S.-W. Park, H.-S. Bae, Z.-C. Xing, O.H. Kwon, M.-W. Huh, I.-K. Kang, J. Appl. Polym. Sci., 112
Page 19 of 30
(2009) 2320-2326. [20] B. Pant, H.R. Pant, D.R. Pandeya, G. Panthi, K.T. Nam, S.T. Hong, C.S. Kim, H.Y. Kim, Colloids Surf. Physicochem. Eng. Aspects, 395 (2012) 94-99.
ip t
[21] J.H. Sohn, L.Q. Pham, H.S. Kang, J.H. Park, B.C. Lee, Y.S. Kang, Radiat. Phys. Chem., 79 (2010) 1149-1153.
cr
[22] S.-E. Kim, J. Hyun Park, B.c. Lee, J.-C. Lee, Y. Ku Kwon, Radiat. Phys. Chem., 81 (2012)
us
978-981.
[23] Y. Li, Y.N. Kim, E.J. Lee, W.P. Cai, S.O. Cho, Nuclear Instruments and Methods in Physics
an
Research Section B: Beam Interactions with Materials and Atoms, 251 (2006) 425-428.
Chem. Soc., 34 (2013) 3899-3902.
M
[24] H.S. Kang, B. Kim, J.H. Park, H.W. Kim, Y.H. Koo, H.B. Bae, C. Park, B.C. Lee, Bull. Korean
8 (2013) 4131-4145.
ed
[25] C. Li, R. Fu, C. Yu, Z. Li, H. Guan, D. Hu, D. Zhao, L. Lu, International journal of nanomedicine,
ce pt
[26] S. Akmaz, E.D. Adiguzel, M. Yasar, O. Erguven, Advances in Materials Science and Engineering, 2013 (2013) 1-6.
Ac
[27] Y. Liu, M. Park, H.K. Shin, B. Pant, J. Choi, Y.W. Park, J.Y. Lee, S.-J. Park, H.-Y. Kim, Journal of Industrial and Engineering Chemistry, 20 (2014) 4415-4420. [28] T.T.T. Nguyen, B. Tae, J.S. Park, J Mater Sci, 46 (2011) 6528-6537. [29] E. Hadipour-Goudarzi, M. Montazer, M. Latifi, A.A. Aghaji, Carbohydr. Polym., 113 (2014) 231-239. [30] R. Kumar, H. Munstedt, Biomaterials, 26 (2005) 2081-2088. [31] Q. Shi, N. Vitchuli, J. Nowak, J. Noar, J.M. Caldwell, F. Breidt, M. Bourham, M. McCord, X.W.
Page 20 of 30
Zhang, J. Mater. Chem., 21 (2011) 10330-10335. [32] Z.M. Xiu, Q.B. Zhang, H.L. Puppala, V.L. Colvin, P.J. Alvarez, Nano Lett., 12 (2012) 4271-4275. [33] R.C. Goy, D. Britto, O.B.G. Assis, Polímeros: Ciência e Tecnologia, 19 (2009) 241-247.
[35] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Bull. Mater. Sci., 34 (2011) 29-35.
ip t
[34] M.M. Fouda, M.R. El-Aassar, S.S. Al-Deyab, Carbohydr. Polym., 92 (2013) 1012-1017.
Ac
ce pt
ed
M
an
us
cr
[36] F. Xu, C. Piett, S. Farkas, M. Qazzaz, N.I. Syed, Molecular brain, 6:29 (2013) 1-15.
Page 21 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 22 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 23 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 24 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 27 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 29 of 30
Highlights (for review)
Highlights ►
Eco-friendly CS/AgNPs hybrid nanofibers were successfully electrospun.
►8
wt % CS/AgNO3 paste in 1% acetic acid became transparent solution after EBI.
CS hybrid NFs provided a steady and prolonged silver ion release over 16 days.
►
CS NFs with AgNPs (0.5 wt %) showed best antibacterial activity toward E. coli
ip t
►
Ac
ce pt
ed
M
an
us
cr
and S. aureus bacterias.
Page 30 of 30
S0141-8130(15)00399-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.05.058 BIOMAC 5142
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-2-2015 26-5-2015 30-5-2015
Please cite this article as: Y. Liu, Y. Liu, N. Liao, F. Cui, M. Park, H.-Y. Kim, Fabrication and durable antibacterial properties of electrospun chitosan nanofibers with silver nanoparticles, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.05.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Manuscript Click here to view linked References
Fabrication and durable antibacterial properties of electrospun chitosan nanofibers with silver nanoparticles
Department of BIN fusion technology, Chonbuk National University, Jeonju 561-756,
cr
a
ip t
Yanan Liua, Yang Liua, Nina Liaob, Fuhai Cuia, Mira Parkc, *, Hak-Yong Kima,* *
b
us
Republic of Korea
Department of Bionanosyestem Engineering, Chonbuk National University, Jeonju
Department of Organic Materials and Fiber Engineering, Chonbuk National
M
c
an
561-756, Republic of Korea
Ac
ce pt
ed
University, Jeonju 561-756, Republic of Korea
* Corresponding author: Tel: +82-63-270-2351, Fax: +82-63-270-4249. E-mail address: [email protected] (M. Park) ** Corresponding author: Tel: +82-63-270-2351, Fax: +82-63-270-4249. E-mail address: [email protected] (H. Y. Kim)
Page 1 of 30
Abstract Non-precipitation Chitosan/silver nanoparticles (AgNPs) in 1% acetic acid aqueous solution was prepared from chitosan colloidal gel with various contents of silver
ip t
nitrate via electron beam irradiation (EBI). Electrospun chitosan-based nanofibers decorated with AgNPs were successfully performed by blending poly(vinyl alcohol).
cr
The morphology of as-prepared nanofibers and the size of AgNPs in the nanofibers
us
were investigated by field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). The presence of AgNPs in as-obtained
an
nanofibers was also confirmed by ultraviolet-visible spectroscopy (UV), Fourier
M
transform infrared (FT-IR) spectroscopy, EDX spectrum and metal mapping. Silver ion release behavior indicated that these hybrid nanofibers continually release
ed
adequate silver to exhibit antibacterial activity over 16 days. These biocomposite nanofibers showed pronounced antibacterial activity against Staphylococcus aureus (S.
ce pt
aureus) and Escherichia coli (E. coli).
1.
Ac
Keywords: chitosan, silver nanoparticles, electrospinning, antibacterial nanofibers
Introduction
Chitosan (CS), as the second abundance of natural polysaccharide, has been
widely used in numerous applications such as medicine, food and chemical engineering, pharmaceuticals, nutrition, and agriculture [1]. CS-based nanofibers (NFs) have been identified as an excellent biomaterial, especially for tissue scaffold and wound healing because CS possesses renewability, nontoxicity, absorption of
Page 2 of 30
exudates, and antibacterial activity [2, 3], simultaneously nanofibrous architectural morphology is similar to natural extracellular matrix which can promote cell’s attachment, proliferation and differentiation [4]. However, electrospun pristine CS
ip t
NFs is considered extremely difficult due to its high molecular weight, its low stability and its low solubility in common organic solvent. It is essential to manage
cr
molecular weight of CS via depolymerization, chemical or radiation-treatment to
us
improve its solubility [5-8]. CS-based nanofibrous mats are supposed to be fabricated successfully via electrospinning using appropriate chemical structure of CS.
an
Electrospinning is recognized as an efficient technology to prepare micro or nano
M
sized fibers for various applications spreading in different fields, such as environment, energy, electronic and biomaterials [9-11]. The distinct features of electrospun
ed
nanofibers emerge to be high porosity, large surface area, and simple fabrication process. In addition, nanofibers possess high capacity for incorporating of various
ce pt
biological materials or active substances, for instance, drugs, natural remedies, or metal nanoparticles [12, 13]. Moreover, the combination of nanofibers and
Ac
antibacterial substances as a fascinating wound healing material has demonstrated antibacterial efficacy to prevent wound contamination by some bacteria. Owing to AgNPs possess outstanding antibacterial activity toward germs on
contact without the release of toxic biocides [14, 15], utilization of AgNPs to integrate into electrospun NFs has been drawn considerable attention for wound healing, tissue engineering, biological protective materials, and medical devices applications [16-18]. In order to attain hybrid NFs containing AgNPs, reduction of Ag ion to AgNPs has to
Page 3 of 30
be carried out before or after elelctrospinning [19, 20], which can be accomplished by irradiation, thermal or chemical reduction process [21]. Among these treatment method, electron beam irradiation (EBI) is considered as an efficient method for
ip t
large-scale synthesis of AgNPs [22], and has more advantages over others, including easy operation, inexpensive, short irradiation time, and absence of chemical residues
cr
after irradiated. Furthermore, EBI treatment is possible to control the size and
us
suppress the agglomeration of as-obtained AgNPs, which affect the property of nanoparticles [21, 23]. Generally, radiation-induced reduction performs as follows
an
[24],
M
1) Water radiolysis:
(1)
ed
2) Reduction by hydrated electron and hydrogen atoms: (2) (3)
ce pt
Therefore, EBI is a facile, feasible, and highly desirable approach to attain AgNPs-loaded polymer fibers.
Ac
CS/PVA NFs were successfully prepared from biocompatible polymer-solution via EBI and electrospinning technique in our previous work [8]. The advantages of this strategy were non-toxic and environmental-friendly throughout the overall system with no chemical residues for NFs. In spite of the antibacterial property showed from CS, the obtained NFs still need to improve their durably antibacterial activity. Thus, we develop advanced CS-based NFs by adding AgNPs as a reinforced antibacterial agent. It is expected to associate the superiorities of CS/PVA composite with
Page 4 of 30
antibacterial activity of AgNPs. In order to reduce process steps, EBI treatment is managed to diminish molecular weight of CS and fabricate AgNPs from Ag ions synchronously. It is a facile fabrication process to accomplish CS/AgNPs solution.
ip t
Then CS/PVA/AgNPs biocomposite NFs are prepared by blending non-toxic PVA solution to enhance the spinnability of CS/AgNPs solution [4]. AgNPs effect on the
Experimental
us
2.
cr
morphology, diameter and antibacterial properties of NFs is further evaluated.
2.1 Materials (200,000cps,
75-85%
deacetylated),
an
Chitosan
poly(vinyl
alcohol)
M
(Mw=85,000-124,000, 87-89% hydrolyzed) and glyoxal solution (40 wt % in H2O) were purchased from Aldrich Co., USA. Silver nitrate (Showa, Japan) was used as
ed
metal precursor. Acetic acid and phosphoric acid were obtained from Samchun Pure
purification.
ce pt
Chemical Co., Ltd, South Korea. All the chemicals were used without further
2.2 Preparation of CS/AgNPs homogenous solution
Ac
8 g CS powder was dispersed in 92 ml deionized water at 90 °C for 24 h, then Silver nitrate (0, 0.5, 1 and 2 wt % with weight of CS) was added with stirring for 8 h at 25 °C, respectively. CS/AgNO3 gelatinous solid was obtained by pouring 1 mL acetic acid. All the gelatinous CS/AgNPs samples were irradiated with an electron-beam at a dose of 150 kGy to get non-precipitate solutions. 2.3 Fabrication of CS/PVA/AgNPs NFs via electrospinning 10 wt % PVA solution was prepared by dissolving PVA powder in deionized
Page 5 of 30
water at 90 °C for 24 h. The homogeneous solution was obtained by mixing 4g PVA solution and 5g CS/AgNPs solution (AgNPs contents = 0, 0.5, 1, and 2 wt % of CS) under vigorously magnetic stirrer for 4h, respectively. And then 0.06 ml glyoxal
ip t
solution was added as cross-linking agent when the pH values of these system were adjusted to 2~3 by phosphoric acid. In a typical electrospinning experiment, CS/PVA
cr
with various contents of AgNPs (0, 0.25, 0.5 and 1 wt %) NFs were successfully
us
electrospun at 15 kV using a high-voltage DC power supply with a 14 cm tip-to-collector distance and 35% relative humidity, respectively. As-spun
an
CS/PVA/AgNPs nanofibers were collected by Teflon papers. In order to make
M
resulting fibrous mats cross-linking effectively, put them in the heat oven under alcohol vapor at 50 °C for 24 h, subsequently cured at 120 °C for 10 min. A schematic
ce pt
2.4 Characterizations
ed
process of CS/PVA/AgNPs NFs is shown in Fig. 1.
CS/AgNPs solution was performed from CS/AgNO3 gelatinous paste via electron beam accelerator (EBTECH Co., Ltd., Korea, beam energy of 2.5 MeV,
Ac
beam current of 8.5 mA, and dose rate of 6.67 kGy/sec) at 25 °C in an air atmosphere. The chemical structure of CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs was confirmed by Fourier transform infrared spectroscopy (FT-IR, Varian 1000 Scimitar series). Ultraviolet–visible (UV–vis) absorption spectra of as-obtained NFs was measured by UV-2600 UV-Vis Spectrophotometer (Shimadzu, Japan). The surface morphologies and diameters were determined by Field emission scanning electron microscopy (FESEM, S-7400, Hitachi, Japan). The size, shape, and manner of
Page 6 of 30
deposition of AgNPs on the fiber surface were investigated via biological transmission electron microscopy (Bio-TEM, JEM-2010, JEOL, Japan). The metal mapping and EDX spectrum of hybrid NFs mat were recorded with the scanning
ip t
electron microscopy instrument (SEM, JSM-5900JEOL Co.). Mechanical properties were measured with a universal testing machine (AG-5000G, Shimadzu, Japan),
cr
under a crosshead speed of 5 mm/min at room temperature. The samples were
us
prepared in the form of standard dumbbell shapes according to ASTM Standard D 638 via die cutting from the electrospun mats and tested in the machine direction. Each
an
sample was performed at five times.
M
2.5 Silver ion release
Silver ion release behavior of the NFs was determined as followed, 50 mg hybrid
ed
NFs was placed in a conical flask and 50 ml deionized water was added into it as the release medium. The flask was shaken to make sure the NFs was completely
ce pt
immersed, then sealed and incubated at room temperature. The deionized water was collected every 12, 24, 36, 72 or 96 h. Silver ion concentration in the solution was
Ac
measured using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (ICPS-7500, SHIMADZU, Japan). 2.6 Antibacterial Properties Characterizations The antibacterial activities of CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs membranes were investigated by a zone inhibition method. Gram-negative Escherichia coli (E. coli) and gram-positive Staphylococcus aureus (S. aureus) cells were used as the model microorganisms. All CS/PVA/AgNPs NFs with different
Page 7 of 30
contents of AgNPs were cut into 6 mm diameter discs and sterilized by UV light prior to bacterial viability test. Then, NFs discs were transferred to E. coli or S. aureus bacterium suspension containing around 106 colony forming units (CFUs)/mL and
diameter of inhibition area around each disc. 3.
Results and Discussions
us
3.1 Preparation of CS/AgNPs solution and electrospinning
cr
ip t
then incubated for 24 h at 37 °C. The zone of inhibition was measured by testing the
One role of EBI treatment was to decrease the molecular weight of CS for
an
dissolving in 1% acetic acid aqueous solution [8], the other role was large scale
M
production of AgNPs according to the procedure described by Kang et al [24]. Interestingly, the AgNPs grow in size with an increase of the absorbed dose. Therefore,
ed
an EBI dose at 150 KGy has effectively transformed CS/Ag+ gelatinous paste to non-precipitation solution. In addition, biocompatible and biodegradable PVA was
ce pt
chosen as a composition to improve the viscosity and spinability of CS/AgNPs solution owing to its easy-to-obtain, its good fiber-forming ability, its non-toxic and
Ac
water-solubility [4]. Finally, the weight ratio of PVA and CS/AgNPs was permanent at 1:1 in order to make NFs contain the high content of antibacterial component on the premise of forming good fiber morphology. To detect the formation of AgNPs in the CS/AgNPs solution, UV spectroscopy was performed to test the characteristic ultraviolet peak of AgNPs. The spectra of as-prepared CS/AgNPs solutions are shown in Fig. 2 (e). For pristine CS solution, one peak appeared at 300 nm, likely due to the absorption peak of chitosan
Page 8 of 30
oligosaccharide [25].The maximum absorption peak was observed at approximately 410 nm, which is characteristic surface Plasmon absorption for AgNPs. UV absorption peak of as-prepared AgNPs was consistent with other researchers recorded
ip t
in the range 410–420 nm [16, 26]. In addition, the intensity of AgNPs absorption peak increased when increasing the concentration of AgNO3 in precursor solution, implying
cr
that the maximum efficiency of large scale producing AgNPs was realized via EBI
us
treatment. 3.2 Morphology of electrospun NFs
an
FESEM images in Fig. 2 (a~d) show the morphology of as-obtained hybrid
M
nanofibrous mats with various contents of AgNPs (0, 0.25, 0.5 and 1 wt %). All electrospun NFs possessed the uniform, smooth, continuously and bead-free
ed
morphology. An average diameter of pristine CS/PVA NFs was 155 nm, the average diameter of hybrid NFs decreased by increasing AgNPs content, 148, 144 and 139 nm,
ce pt
respectively, which were determined by averaging the diameter of 50 random fibers. But this was not significantly difference, because the AgNPs content is very low and
Ac
all the CS/AgNPs solution possessed the similar viscosity (about 25 cps) and conductivity (about 0.7 ms/m). To quantify the average size and distribution of AgNPs and assembling structure of AgNPs on the NFs, TEM images were used to measure the NPs size and distribution of NFs. Fig. 2 (f~h) exhibited that many individual AgNPs with oval and spherical shape distributing on the surface of CS/PVA NFs were observed. The particle size was on the range of 1-30 nm and average size of particles increased with increasing AgNPs content, 14.0, 15.4 and 18.6
Page 9 of 30
nm, respectively. These results were ascribed to the aggregation of AgNPs, which may be caused by constantly attack of the Ag atoms by hydrated electrons and hydrogen atoms generated during the e-beam process at relatively high dose and high
ip t
concentration [21, 24]. To further explore the distribution of AgNPs on the NFs, metal mapping image and EDX of hybrid NFs was taken (Fig. 3), which also confirmed the
cr
deposition of AgNPs on the surface of CS/PVA NFs. In addition, it was obvious that
us
the particles dispersed uniformly on the NFs. The content of AgNPs was 0.02, 0.06, 0.09 %, respectively, which was found to be similar with theoretical calculated value.
an
All of these results implied nanoparticles to the utmost extent to be utilized without
3.3 FT-IR spectra of electrospun NFs
M
loss.
ed
The FT-IR spectra of electrospun CS/PVA/AgNPs NFs are shown in Fig. 4. The frequencies and assignments for the pristine CS/PVA were indicated as follows: C–H
ce pt
asymmetric vibration (2933 cm−1), –OH and –NH2 groups (3700-3000 cm−1), C=O bond (1733 cm−1), C=O bond (1640 cm-1) in NHCO–, and C–C stretching vibration
Ac
(851cm−1) [25, 27]. The presence of AgNPs in the NFs was evidenced by the vibration band of N–C=O group taken place the blue shift from 1534 cm-1 to 1552cm-1 with increasing AgNPs content [28], which indicated that N-H vibration was affected by attachment of the AgNPs. In short, these results implied the existence of AgNPs in the NFs and the interaction force between NPs and –NH2 groups of CS via FT-IR spectra. 3.4 Mechanical properties Typical stress−strain curves for hybrid NFs with varying AgNPs content are
Page 10 of 30
illustrated in Fig. 5. It was observed that the tensile strengths of as-prepared NFs were significantly increased with more AgNPs loaded, from 3.65 MPa of PVA/CS NFs to 3.78 and 4.52 MPa with 0.25 and 0.5 wt % NPs loading, respectively. The possible
ip t
interactions between CS/PVA with AgNPs enhanced by intermolecular interactions (-OH of PVA, Ag, -CH2OH of CS) [29]. When 1 wt % AgNPs loaded to NFs, the
cr
tensile strength of hybrid NFs decreased appreciably to 3.74 MPa probably due to
us
regularity and compactness of NFs affected by the aggregation of AgNPs. Thus, CS/PVA/AgNPs (0.5 wt %) NFs showed the highest tensile strength, this implied that
an
the tensile strengths of NFs membranes were sufficient for practical handling during
M
the wound healing process. 3.5 Silver ion release
ed
In order to evaluate Ag+ release rate for these CS/AgNPs-based antibacterial materials, ICP-AES spectra was used to test Ag+ release behavior. For purpose of
ce pt
providing effective antibacterial properties, it is necessary to keep a steady and prolonged Ag+ release rate at a concentration level at least 0.1 ppb [13, 30]. When the
Ac
hybrid NFs was incubated into aqueous solution, the AgNPs gradually release Ag+ into the water. The release rate is mainly determined by the concentration of NPs and the diffusion distance from inside the fiber to the surface [31]. Ag+ release profiles of hybrid NFs are presented in Fig. 6. It is found that the release rate is relatively high in the first few days and then continuously release over time. For the electrospun hybrid NFs loaded with 0.25, 0.5 and 1 wt % AgNPs, the accumulated released amount of Ag after 16 days were approximately 40, 56 and 36 %, respectively. After 9 days,
Page 11 of 30
AgNPs-loaded hybrid NFs with (0.5 and 1 wt %) still gave a steady at 0.13–0.14 ppm per day, and release rate of NFs with 0.25 % AgNPs went to level off at 0.005 ppm. The results indicated that CS/PVA/AgNPs NFs prepared by EBI and electrospinning
ip t
can release sufficient amounts of silver and have a long lifetime to exhibit sustained antibacterial activity.
cr
3.6 Zone of inhibition testing of antibacterial efficacy (E. coli and S. aureus)
us
AgNPs leads to bacterial cell death by interacting with sulfur-containing proteins in germ membrane and releasing Ag+ to attack the respiratory chain, cell division [32].
an
Furthermore, an extremely large surface area of AgNPs can provide larger action sites
M
for microorganisms to promote bactericidal activity. The antibacterial property of pristine CS/PVA and CS/PVA/AgNPs (0, 0.25, 0.5 and 1 wt %) NFs against E. coli
ed
and S. aureus were performed by the disc diffusion susceptibility test after 24 h incubation. As presented in Fig. 7, both of E. coli and S. aureus clearly showed a zone
ce pt
of inhibited growth of the bacteria around the CS/PVA and CS/PVA/AgNPs NFs. The diameter of inhibition ring for each sample was shown in the schematic diagram.
Ac
Pristine CS/PVA possessed inhibition ring diameter at 7.0 mm owing to the cationic nature of CS as antimicrobial agents [33]. The inhibition zones of CS/PVA/AgNPs (0.25, 0.5 and 1 wt %) NFs against E. coli were 7.7, 13.3 and 9.0 mm, respectively. Furthermore, CS/PVA/AgNPs (0.25 and 0.5 wt %) NFs were exhibited to inhibit the growth of bacteria with slightly higher effectiveness against S. aureus compared with E. coli, the zones of inhibition increased to 9.1 and 14.3 mm, which was in accordance with CS-Ag composite [34, 35]. It was mentioned that the most
Page 12 of 30
antibacterial effected against for both two microorganisms when AgNPs (0.5 wt %) loaded to hybrid NFs. After adding more AgNPs, the antibacterial property of hybrid NFs weakened mainly owing to the aggregation of AgNPs at relative high
ip t
concentration and the potentially devastating effects of AgNPs [15, 36]. Therefore, this work indicated the wonderful antibacterial performance of CS/PVA/AgNPs (0.5
Conclusions
us
4.
cr
wt %) which can be used as excellent materials in biomedical application.
In this study, continuous and uniform CS/PVA/AgNPs NFs were successfully
an
electrospun using eco-friendly solvent. It is a wonderful approach to fabricate
M
CS/AgNPs-based biocomposite NFs by combine EBI and electrospinning technique. The green EBI process was achievable to gain CS solution from CS gel and
ed
large-production of AgNPs from Ag+. It was observed that AgNPs dispersed uniformly in the CS/PVA NFs, which remained utilization of nanoparticles to the
ce pt
utmost extent. Furthermore, the properties of PVA/CS NFs were improved greatly by AgNPs loaded. These hybrid NFs mats showed good mechanical property and
Ac
antibacterial activity toward E. bacteria and S. aureus, and latter bacteria was the more sensitive microbe against antimicrobial disk when the concentration of AgNPs was only 0.5 wt %. The resultant hybrid NFs provide a steady and prolonged silver ion release. This work provides a new direction for efficient CS/AgNPs-based NFs as antibacterial biomedical applications.
Page 13 of 30
Acknowledgements Following are results of a study on the “Basic Research Program through the National Research Foundation of Korea (NRF)” project, funded by the Ministry of Education
ip t
(No.NRF-2014R1A1A2008489). This research was supported by the National Research Foundation of Korea (NRF) Grant funded by Korea Government (MISP)
Figure Captions
an
Fig. 1. A schematic process of CS/PVA/AgNPs NFs.
us
cr
(NO. 2014R1A4A1008140).
M
Fig. 2. FESEM images of CS/PVA NFs (a), FESEM and TEM images of hybrid NFs with contents of AgNPs, 0.25 wt % (b and e), 0.5 wt % (c and f) and 1 wt % (d and g), respectively. Vis-UV spectra of CS solutions with various contents of AgNPs (e).
ed
Fig. 3. SEM metal mapping images and EDX of hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively (a~c).
ce pt
Fig. 4. FTIR spectra of electrospun fibrous mats: (a) CS/PVA, (b~d) hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively. Fig. 5. Stress−strain curves of hybrid NFs with different contents of AgNPs, 0, 0.25,
Ac
0.5 and 1 wt %, respectively (a~d).
Fig. 6. Silver release profiles of hybrid NFs with different contents of AgNPs, 0.25, 0.5 and 1 wt %, respectively (a~c). Fig. 7. Zone of inhibition antibacterial testing against E. coli (left) and S. aureus (right). (a~d) CS/PVA /AgNPs NFs with different contents of AgNPs, 0, 0.25, 0.5 and 1 wt %, respectively. Schematic diagram is shown that measured dimension of inhibited bacteria.
Page 14 of 30
ip t
Fig. 1.
Fig. 3.
Ac
ce pt
ed
M
an
us
cr
Fig. 2.
Page 15 of 30
ip t cr us an M Ac
ce pt
ed
Fig. 4.
Page 16 of 30
Fig. 6.
Ac
ce pt
ed
M
an
us
cr
ip t
Fig. 5.
Page 17 of 30
us
cr
ip t
Fig. 7.
Ac
ce pt
ed
M
an
GRAPHICAL ABSTRACT
References
[1] A. Domard, Carbohydr. Polym., 84 (2011) 696-703. [2] D. Archana, B.K. Singh, J. Dutta, P.K. Dutta, Int. J. Biol. Macromol., 73 (2015) 49-57. [3] A.S. Mehta, B.K. Singh, P.K. Dutta, Asian Chitin Journal, 10 (2014) 25-28. [4] M.Z. Elsabee, H.F. Naguib, R.E. Morsi, Mat Sci Eng C-Mater, 32 (2012) 1711-1726.
Page 18 of 30
[5] I. Prasertsung, S. Damrongsakkul, C. Terashima, N. Saito, O. Takai, Carbohydr. Polym., 87 (2012) 2745-2749. [6] C.T. Tsao, C.H. Chang, Y.Y. Lin, M.F. Wu, J.L. Han, K.H. Hsieh, Carbohydr. Res., 346 (2011)
ip t
94-102. [7] H.A. Abd El-Rehim, N.M. El-Sawy, S.A. Hegazy el, S.A. Soliman el, A.M. Elbarbary, Int. J. Biol.
cr
Macromol., 50 (2012) 403-413.
[9] R.J. Wade, J.A. Burdick, Nano Today, 9 (2014) 722-742.
us
[8] Y. Liu, M. Park, H.K. Shin, B. Pant, S.-J. Park, H.-Y. Kim, Mater. Lett., 132 (2014) 23-26.
an
[10] X. Wang, B. Ding, J. Yu, M. Wang, Nano Today, 6 (2011) 510-530.
M
[11] V.J. Babu, S. Vempati, S. Sundarrajan, M. Sireesha, S. Ramakrishna, Solar Energy, 106 (2014) 1-22.
ed
[12] A.J. Meinel, O. Germershaus, T. Luhmann, H.P. Merkle, L. Meinel, Eur. J. Pharm. Biopharm., 81 (2012) 1-13.
ce pt
[13] Q. Shi, N. Vitchuli, J. Nowak, J.M. Caldwell, F. Breidt, M. Bourham, X. Zhang, M. McCord, Eur. Polym. J., 47 (2011) 1402-1409.
Ac
[14] M. Rai, A. Yadav, A. Gade, Biotechnol. Adv., 27 (2009) 76-83. [15] K.-H. Cho, J.-E. Park, T. Osaka, S.-G. Park, Electrochim. Acta, 51 (2005) 956-960. [16] A.M. Abdelgawad, S.M. Hudson, O.J. Rojas, Carbohydr. Polym., 100 (2014) 166-178. [17] Z.C. Xing, W.P. Chae, J.Y. Baek, M.J. Choi, Y. Jung, I.K. Kang, Biomacromolecules, 11 (2010) 1248-1253. [18] H. Deka, N. Karak, R.D. Kalita, A.K. Buragohain, Polym. Degrad. Stab., 95 (2010) 1509-1517. [19] S.-W. Park, H.-S. Bae, Z.-C. Xing, O.H. Kwon, M.-W. Huh, I.-K. Kang, J. Appl. Polym. Sci., 112
Page 19 of 30
(2009) 2320-2326. [20] B. Pant, H.R. Pant, D.R. Pandeya, G. Panthi, K.T. Nam, S.T. Hong, C.S. Kim, H.Y. Kim, Colloids Surf. Physicochem. Eng. Aspects, 395 (2012) 94-99.
ip t
[21] J.H. Sohn, L.Q. Pham, H.S. Kang, J.H. Park, B.C. Lee, Y.S. Kang, Radiat. Phys. Chem., 79 (2010) 1149-1153.
cr
[22] S.-E. Kim, J. Hyun Park, B.c. Lee, J.-C. Lee, Y. Ku Kwon, Radiat. Phys. Chem., 81 (2012)
us
978-981.
[23] Y. Li, Y.N. Kim, E.J. Lee, W.P. Cai, S.O. Cho, Nuclear Instruments and Methods in Physics
an
Research Section B: Beam Interactions with Materials and Atoms, 251 (2006) 425-428.
Chem. Soc., 34 (2013) 3899-3902.
M
[24] H.S. Kang, B. Kim, J.H. Park, H.W. Kim, Y.H. Koo, H.B. Bae, C. Park, B.C. Lee, Bull. Korean
8 (2013) 4131-4145.
ed
[25] C. Li, R. Fu, C. Yu, Z. Li, H. Guan, D. Hu, D. Zhao, L. Lu, International journal of nanomedicine,
ce pt
[26] S. Akmaz, E.D. Adiguzel, M. Yasar, O. Erguven, Advances in Materials Science and Engineering, 2013 (2013) 1-6.
Ac
[27] Y. Liu, M. Park, H.K. Shin, B. Pant, J. Choi, Y.W. Park, J.Y. Lee, S.-J. Park, H.-Y. Kim, Journal of Industrial and Engineering Chemistry, 20 (2014) 4415-4420. [28] T.T.T. Nguyen, B. Tae, J.S. Park, J Mater Sci, 46 (2011) 6528-6537. [29] E. Hadipour-Goudarzi, M. Montazer, M. Latifi, A.A. Aghaji, Carbohydr. Polym., 113 (2014) 231-239. [30] R. Kumar, H. Munstedt, Biomaterials, 26 (2005) 2081-2088. [31] Q. Shi, N. Vitchuli, J. Nowak, J. Noar, J.M. Caldwell, F. Breidt, M. Bourham, M. McCord, X.W.
Page 20 of 30
Zhang, J. Mater. Chem., 21 (2011) 10330-10335. [32] Z.M. Xiu, Q.B. Zhang, H.L. Puppala, V.L. Colvin, P.J. Alvarez, Nano Lett., 12 (2012) 4271-4275. [33] R.C. Goy, D. Britto, O.B.G. Assis, Polímeros: Ciência e Tecnologia, 19 (2009) 241-247.
[35] S. Tripathi, G.K. Mehrotra, P.K. Dutta, Bull. Mater. Sci., 34 (2011) 29-35.
ip t
[34] M.M. Fouda, M.R. El-Aassar, S.S. Al-Deyab, Carbohydr. Polym., 92 (2013) 1012-1017.
Ac
ce pt
ed
M
an
us
cr
[36] F. Xu, C. Piett, S. Farkas, M. Qazzaz, N.I. Syed, Molecular brain, 6:29 (2013) 1-15.
Page 21 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 22 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 23 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 24 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 27 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s) Click here to download high resolution image
Page 29 of 30
Highlights (for review)
Highlights ►
Eco-friendly CS/AgNPs hybrid nanofibers were successfully electrospun.
►8
wt % CS/AgNO3 paste in 1% acetic acid became transparent solution after EBI.
CS hybrid NFs provided a steady and prolonged silver ion release over 16 days.
►
CS NFs with AgNPs (0.5 wt %) showed best antibacterial activity toward E. coli
ip t
►
Ac
ce pt
ed
M
an
us
cr
and S. aureus bacterias.
Page 30 of 30
