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Turkish Journal of Biology Turk J Biol(2016) 40: 643-651© TÜBİTAKdoi:10.3906/biy-1505-71
Synthesis and characterization of silver nanoparticles integrated in polyvinyl alcohol nanofibers for bionanotechnological applications
Gamze TAN1, Semran SAĞLAM2, Ezgi EMÜL3, Demet ERDÖNMEZ1, Necdet SAĞLAM3,*1Department of Biology, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey
2Department of Physics, Faculty of Science, Section of Atom and Molecule Physics, Gazi University, Ankara, Turkey3Graduate School of Science and Engineering, Division of Nanotechnology and Nanomedicine, Hacettepe University, Ankara, Turkey
* Correspondence: [email protected]
1. IntroductionSeveral processing techniques such as phase separation, template synthesis, and self-assembly have been used to prepare polymeric nanofibers (NFs). In electrospinning, high voltages are applied to a polymer solution to produce NFs (Bhardwaj and Kundu, 2010). Compared with other techniques, electrospinning is a cheap and simple method of nanofabrication. NFs produced by this method are cost effective, porous, long, and stable (Ramakrishna et al., 2005).
The electrospinning system generally consists of four main components: (i) a syringe pump, (ii) voltage DC power supply, (iii) needle, and (iv) collector. The polymer, which is dissolved in the solvent, is transferred to the syringe. When the syringe is placed upright, polymer solution is directed to the collector under the influence of gravity. In some cases, to control the flow the needle can be placed at a certain angle, and if the syringe is positioned horizontally the syringe pump is needed to start the flow. The power supply is used to create a high voltage difference between the polymer solution, which
is in the tip of the needle, and the metal collector. As the electric field intensity increases, the semispherical surface of the liquid at the end of the capillary tube elongates into a cone, called a Taylor cone. When the electric field reaches a critical value, the repelling electrical forces exceed the surface tension, and the charged polymer solution escapes from the end of the Taylor cone. The solvent evaporates, and charged polymer fibers are deposited onto the collector. The major parameters affecting polymeric fiber morphology are the molecular weight (Mw) and concentration of the polymer, feed rate, applied voltage, viscosity of the solution, and distance between the tip and the collector. These parameters can be tuned to obtain the desired fiber properties (Sill and von Recum, 2008).
NFs produced by electrospinning have been used in many different applications, including wound healing, biological tissue scaffold, optical and chemical sensors, electrode materials, enzyme immobilization, affinity membranes, and filtration (Frenot and Chronakis, 2003; Cui et al., 2010; Aktürk and Keskin, 2015). Silver nanoparticles (AgNPs) are attractive candidates for
Abstract: Characterization and microbial growth activity of polyvinyl alcohol (PVA) nanofibers (NFs) produced by an electrospinning technique containing different amounts of silver nanoparticles (AgNPs) was investigated. AgNPs were synthesized by chemical reduction of silver nitrate (AgNO3) (in different weight ratios) in PVA solution followed by electrospinning to produce PVA/AgNPs NFs. The properties of PVA/AgNPs NFs were studied as a function of the silver content. The morphology and distribution of NFs and AgNPs were characterized by transmission and scanning electron microscopy. The size of NFs was 135–160 nm, and the size of AgNPs was 15–27 nm. TEM images clearly showed that spherical AgNPs were homogeneously integrated in PVA NFs. The microbial activity of NFs containing different amounts of AgNPs was examined against both Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Bacillus megaterium and Staphylococcus aureus) bacteria and against Candida albicans. PVA NFs containing AgNPs exhibited increasing inhibitory effects against microbial pathogenic strains with increasing AgNPs amounts. The results suggest that PVA/AgNPs NFs could be used for wound dressing components, protective coatings, biomedical devices, and water purification.
Key words: Electrospinning, polyvinyl alcohol, silver nanoparticles, antimicrobial activity, nanofibers
Received: 24.05.2015 Accepted/Published Online: 19.08.2015 Final Version: 18.05.2016
Research Article
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biomedical applications due to their unique optical, electronic, thermal, and antibacterial properties. For instance, because of their size-tunable light scattering properties and plasmon resonance characteristics, AgNPs are attractive tools for signal enhancers and biotags in biosensing and bioimaging applications (Biju et al., 2008; Arvizo et al., 2012). AgNPs are also known for their antibacterial characteristics against gram-positive and gram-negative bacteria (Balazs et al., 2004; Jafari et al., 2015). At the nanoscale, AgNPs with their large surface area act as effective weapons against pathogenic bacterial strains. Feng et al. (2000) synthesized nanoscale silver particles with a size of 1–20 nm. These attached to the surface of Escherichia coli and damaged the cells by penetration and by lowering the lipopolysaccharide (LPS) level. Today, nanocomposites containing AgNPs are widely used as coatings on medical devices and are also used in tissue engineering and consumer products including toothpaste, cutting boards, and air sanitizer sprays, etc.
The preparation methods for electrospun NFs are convenient for designing functional composite NFs by direct inclusion of nanomaterials (Celebioglu et al., 2014). Lee et al. (2005) prepared polyurethane (PU), poly (N-vinylpyrrolidone) (PVP), and polyacrylonitrile (PAN) electrospun NFs containing AgNPs but showed that these polymers were not suitable for biomedical applications. In our study, we used polyvinyl alcohol (PVA) as a biodegradable and water-soluble polymer. It has been used in practical applications (e.g., in the medical, cosmetic, food, pharmaceutical, and packing industries) due to its good fiber formation ability, physical properties, and high chemical resistance (Kim H and Kim J, 2011; Mahanta and Valiyaveettil, 2012).
There have been several studies of AgNPs; however, none provide detailed information regarding AgNPs integrated in PVA NFs and their application against different types of bacteria. In the present study, we used the electrospinning method to prepare PVA/AgNPs NFs and investigated their antimicrobial effects for nanomedical and nanotechnological applications.
2. Materials and methods2.1. MaterialsElectrospinning experiments were performed using 87%–90% hydrolyzed granules of PVA (Mw 30,000–70,000) and silver nitrate (AgNO3, 99.998%), which were purchased from Sigma-Aldrich and used as received. All aqueous solutions were prepared using double-distilled water. 2.2. Preparation of PVA/water solutions containing AgNPsAll glassware was washed with deionized (DI) water before use. A 12 wt% stock solution of PVA was prepared in DI water by heating and stirring for 4 h and 85 °C until
the polymer dissolved completely and the solution was homogeneous.
The amount of PVA required to obtain the desired weight percentage (wt%) of polymer solution was calculated using the following equation:
WP (%) = Mp/(Mp + Ms) (1)WP%: weight percent,Mp: mass of polymer (g),Ms: mass of solvent (g).When the PVA solution was cooled to room temperature
before electrospinning, different concentrations (1 wt%, 5 wt%, and 10 wt%) of AgNO3 were added to the solution in glass vials. To avoid photoreduction, after the addition of AgNO3 all mixtures were kept in the dark. To reduce the Ag+ ions to AgNPs, the mixtures were refluxed at 100 °C with gentle stirring for 30, 60, or 90 min, resulting in viscous PVA/AgNP solutions.2.3. ElectrospinningPVA/AgNPs NFs were prepared by electrospinning the 12 wt% PVA solution containing AgNPs. Electrospinning was performed with PVA and PVA/AgNP solutions (12/0 wt%, 12/1 wt%, 12/5 wt%, and 12/10 wt%). The solutions were loaded into a syringe with a 5-mL capacity and 0.8-mm needle diameter and injected into a metal collector covered with aluminum foil under high electric field strength (15 kV/10 cm) at an injection rate of 10 µL/min. The electrospun samples were collected on aluminum foil and prepared for characterization and research into their effects on microbial activity. 2.4. CharacterizationThe PVA/AgNPs NF solutions were characterized by UV-vis spectroscopy (UV-Visible, Agilent 8453) and Raman spectroscopy. Raman spectroscopy was performed on a Delta Nu Examiner Raman microscopy system (Deltanu Inc., Laramie, WY, USA). This system consists of a 532-nm laser source, motorized moving table, and CCD sensor. For each sample, automatic baseline correction was applied. With low laser power, each sample was measured for 5 s. Raman measurements were performed in a Raman cuvette, and the sample volume was 200 µL at room temperature.
PVA/AgNPs NFs and AgNPs were characterized with both SEM (FEI Quanta FEG 250, USA) and TEM (FEI Tecnai G2 Spirit BioTwin, USA). SEM was used to determine the morphologies of PVA/AgNPs NFs. The mean fiber diameter was determined by measuring the diameters of 30 randomly selected fibers from an SEM image using image analysis software (NIH Image J version 1.48, USA). The size distribution of PVA/AgNPs NFs was determined by image analysis software from TEM images, and a histogram of fiber diameter distribution was also plotted.
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2.5. Determination of microbial activity by the disc dif-fusion methodThe effects of antimicrobial activity of NFs were evaluated by the disc diffusion method. Therefore, antimicrobial activity of NFs against Candida albicans ATCC 10231, gram-positive (Bacillus megaterium and Staphylococcus aureus ATCC 25923), and gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922) was evaluated. For bacterial growth, Luria–Bertani broth (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl; pH 7) was used. For fungal growth, Mueller–Hinton (MH) agar was used. Each microorganism was maintained by subculturing regularly on the same medium and storing at 4 °C before use in experiments. For the disc diffusion method, a loop of the bacteria and yeast was inoculated into the Mueller–Hinton broth medium (MHB) and incubated for 24 h at 30–37 °C. The turbidity of the suspensions was compared with 0.5 McFarland standard. Then 100 µL of microorganism suspension was inoculated onto a Mueller–Hinton (MH) agar plate before the antimicrobial assay discs were placed on the plate. PVA NFs with different amounts of AgNPs (0 wt%, 1 wt%, 5 wt%, and 10 wt%) on filter discs (5 mm in diameter) were embedded onto inoculated petri plates (10 × 90 mm) and incubated at 30–37 °C for 24 h. If PVA/AgNPs NFs were effective, they inhibited microbial growth and produced a clear zone, called the zone of inhibition, on the agar plate. At the end of incubation, the inhibition zone diameter was measured with a transparent ruler (in millimeters), and the mean values were calculated. These tests were performed in triplicate.
3. Results and discussionNFs formed from PVA only were white, as expected. Increasing concentrations of AgNO3 resulted in colored solution, from yellow to brownish yellow, with increasing Ag levels. When the heating duration was increased, the color of the NFs changed from brownish yellow to brown. The color changes may have been due to the degradation of the polymer and the growth of AgNPs. 3.1. UV-vis characterizationThe dispersion of discrete AgNPs leads to intense colors resulting from plasmonic characteristics. Metals have free electrons forming a cloud in the conduction band around nuclei. The interaction of NPs and incident light with wavelengths greater than the NP size induces electron vibration and collective oscillations along with a conduction band. This optical phenomenon typically occurs at the metal surface and is called localized surface plasmon resonance. The plasmon oscillation of NPs with light at the resonant wavelength depends on the composition, size, shape, surface, aggregation, and dielectric environment of NPs. Thus, metal NPs exhibit
different absorption spectrum bands in the visible and near-infrared region (Kulkarni, 2015).
Figure 1 shows the optical absorption spectra of pure PVA and PVA/AgNO3 samples. Pure PVA samples exhibit almost zero absorption in the wavelength range of 300–600 nm. A transparent PVA solution shows only a single band. The surface plasmon band exhibits a blue shift, where the maximum absorption peak shifts from 422 to 400 nm. This observation indicates the formation of AgNPs within the PVA matrix.
The PVA/AgNPs nanocomposite displays a broad surface plasmon absorption band (Figure 1). This spectrum is similar to the optical absorption spectra of AgNPs integrated in other polymers such as polyacrylonitrile (PAN). After the integration of AgNPs into PVA, the band expands and shifts to a longer wavelength. This change may reflect AgNP agglomeration or change in dielectric properties of the surrounding environment.3.2. Raman spectroscopic characterizationRaman spectroscopy is suitable for the detection of chemical species formed on silver substrates. Martina et al. (2012) analyzed silver compounds at three laser wavelengths in order to decide which laser would be the most suitable. They found that the most suitable instrumental parameters for analyses of silver compounds were the Nd:YAG laser at 532 nm. We used these parameters for Raman spectroscopy measurements in the current study.
Ag–O and Ag–N vibrational bands occupy the range between 230 and 248 cm−1 (Kai et al., 1989; Michota et al., 2002; Panicker et al., 2006). Due to the AgNPs embedded in PVA, the Ag–N vibrational band at 245 cm−1 was reduced. The stretching vibration band of the nitrate ion appeared at 1045 cm−1 (Oliver and Janz, 1970).
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Figure 1. UV-vis spectra of PVA/AgNO3 nanocomposites at different concentrations. a) Black line, pure PVA; b) red line, 1% Ag; c) blue line, 5% Ag; d) pink line, 10% Ag, e) green line, AgNPs.
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With increasing percentages of AgNO3 in PVA NFs, the intensity increased. The highest intensity peak was obtained with 10% AgNO3, indicating that increasing the amount of AgNO3 increases Raman intensity (Figures 2 and 3). However, there was no band shift for PVA NFs compared with the corresponding bands in the Raman spectrum of PVA/AgNPs.3.3. Morphology and diameter distribution of NFs and AgNPsSEM images, diagrams, and diameter distribution of NFs are shown in Figure 4.
Figure 4a shows SEM images of PVA electrospun into NFs. Electrospinning generated NFs with relatively uniform diameters. The average diameters of electrospun NFs with 0, 1, 5, and 10 wt% of AgNO3 were 160, 141, 136, and 146 nm, respectively. The average diameters decreased with increasing amounts of AgNO3 and were dependent on the conductivity of the PVA solutions. Table 1 summarizes the size distribution of NFs.
Zhang et al. (2005) studied the electrospinning of PVA NFs and reported that with increasing polymer concentrations, the morphology changed from beaded fibers to uniform fibers, and the diameter also increased.
The concentration also had a significant effect on the morphology of the fibers.
The size distribution of AgNPs in PVA NFs was uniform at 17, 20, and 15 nm (bottom row, Figure 5). AgNPs appeared to be distributed mostly within the fibers, as seen from the SEM images (Figure 4), with just a few Ag clusters on the surface of the fibers.3.4. Antimicrobial activityThe aim of this study was to evaluate the antimicrobial activity of pure PVA and PVA/AgNPs NFs and to determine whether increasing the amount of AgNPs in PVA NFs affects the inhibition of bacterial growth.
The results are summarized in Table 2 and shown in Figures 6–8. The scatter plot of inhibition zones versus various bacteria is presented in Figure 9. In the disc diffusion assays, each of the tested PVA/AgNPs NFs inhibited the growth of microorganisms. However, AgNP-free PVA NFs (negative controls) did not show inhibition zones (Figures 6 and 9). Different amounts of PVA/AgNPs NFs showed inhibition zones from 6 to 12 mm against all fungi and bacteria tested. The antifungal and antibacterial activity increased with silver precursor content, whereas PVA NFs had no effect. Better results were observed for C. albicans and the gram-positive bacterium B. megaterium. AgNP has size-dependent antibacterial properties (Morones et al., 2005). PVA/AgNPs (10 wt%) showed higher antibacterial activity because these AgNPs were the smallest in this sample (Figure 5c). The explanation may be that smaller particle sizes present a higher surface-to-volume ratio, enhancing the antibacterial activity of AgNPs (Martinez-Castanon et al., 2008; Mollahosseini et al., 2012).
In general, the activity of AgNPs against gram-positive bacteria was higher than against gram-negative bacteria. This finding has been previously reported and may be explained by the different cell wall structures of
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Figure 2. Raman spectrum of PVA NFs and PVA/AgNPs.
Figure 3. Raman spectrum of PVA NFs and PVA/AgNPs.
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Figure 4. Diameter distributions from SEM images of PVA NFs (a), 1 wt% AgNPs integrated in PVA NFs (b), 5 wt% AgNPs integrated in PVA NFs (c), 10 wt% AgNPs integrated in PVA NFs (d).
Table 1. Summary of the size distribution of NFs.
Nanofiber types Mean diameter (nm) Std. err. (nm)PVA NFs (12 wt%) 159.95 11.95PVA/AgNPs (12/1 wt%) 140.72 7.90PVA/AgNPs (12/5 wt%) 135.81 6.19PVA/AgNPs (12/10 wt%) 146.0364 6.598
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Figure 5. TEM images (top row) and size distribution of AgNP (bottom row, under TEM images) of prepared PVA/AgNPs (1 wt%) (a), PVA/AgNPs (5 wt%) (b), and PVA/AgNPs (10 wt%) (c) NFs.
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Table 2. Antimicrobial activity of PVA NFs containing different amounts of AgNP (wt%). A- PVA NFs (not containing a silver precursor), B- 1 wt%, C- 5 wt%, D- 10 wt%.
SamplesAverage of formed inhibition zones (mm)C. albicans E. coli P. aeruginosa S. aureus B. megaterium
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Figure 6. Microbial activity of PVA/AgNPs NFs against C. albicans and the effects of silver loading on the diameter of the inhibition zone for the fungus.
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Figure 7. Microbial activity of PVA/AgNPs NFs against gram-negative bacteria (P. aeruginosa and E. coli) and the effects of silver loading on the diameter of the inhibition zone.
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these bacteria (Thiel et al., 2007). Gram-negative bacteria possess an outer phospholipid membrane with structural LPS components; this membrane is not found in gram-positive bacteria. AgNPs attach to the surface of the cell and damage the cell, leading to DNA damage and, finally, death (Feng et al., 2000). Marambio-Jones and Hoek (2010) summarized the possible mechanisms of toxicity behind the activity of nano-scaled silver on bacteria as follows: (1)
disruption of ATP production and DNA replication upon uptake of free Ag ions; (2) generation of reactive oxygen species (ROS) that may affect DNA, cell membrane, and membrane proteins; and (3) direct damage to cell membranes by AgNPs.
In this study, we used electrospinning to prepare PVA/AgNPs NFs and investigated their antifungal and antibacterial effects. Antibacterial activity of NPs is a very
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Figure 8. Antibacterial activity of PVA/AgNPs NFs against gram-positive bacteria (S. aureus and B. megaterium) and the effects of silver loading on the diameter of the inhibition zone.
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Figure 9. Scatter plot of inhibition zones versus various bacteria at different concentrations of AgNPs: 0% (A); 1 wt% (B); 5 wt% (C); 10 wt% (D).
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complicated issue and depends on several factors (e.g., NP size, surface charge, shape, concentration, synthesis methods, and bacterial species). PVA has great application potential in biology because it is a water-soluble and biodegradable polymer (Hong et al., 2006). The results of SEM and TEM analysis of PVA NFs containing different amounts of silver precursors showed that NFs exhibited a well-distributed nanofibrous structure, and AgNPs occurred on the surfaces of NFs. Silver salts played an important role in the formation of NFs in PVA.
There are several studies of AgNPs; however, none provide detailed information regarding AgNPs integrated in PVA NFs and their application against several types of bacteria that cause serious diseases. Wounds heal faster if covered by a thin web of silver containing NFs, especially those from a biodegradable polymer (Hong, 2007). Our results showed that PVA/AgNPs NFs could be used for wound dressing components, protective coatings, biomedical devices, and water purification purposes.
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