Materials Science and Engineering C 54 (2015) 8–13
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Antibacterial and hemolysis activity of polypyrrole nanotubes decorated with silver nanoparticles by an in-situ reduction process J. Upadhyay a, A. Kumar a,⁎, B. Gogoi b, A.K. Buragohain b,c a b c
Department of Physics, Tezpur University, Napaam, Tezpur 784028, Assam, India Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Assam, India Dibrugarh University, Dibrugarh 786004, Assam, India
a r t i c l e
i n f o
Available online 22 April 2015 Keywords: Polypyrrole nanotubes Silver nanoparticles Nanocomposites Antibacterial activity Hemolysis activity
a b s t r a c t Polypyrrole nanotube–silver nanoparticle nanocomposites (PPy-NTs:Ag-NPs) have been synthesized by in-situ reduction of silver nitrate (AgNO3) to suppress the agglomeration of Ag-NPs. The morphology and chemical structure of the nanocomposites have been studied by HRTEM, SEM, XRD, FTIR and UV–vis spectroscopy. The average diameter of the polypyrrole nanotubes (PPy-NTs) is measured to be 130.59 ± 5.5 nm with their length in the micrometer range, while the silver nanoparticles (Ag-NPs) exhibit spherical shape with an average diameter of 23.12 ± 3.23 nm. In-vitro blood compatibility of the nanocomposites has been carried out via hemolysis assay. Antimicrobial activity of the nanocomposites has been investigated with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria. The results depict that the hemolysis and antimicrobial activities of the nanocomposites increase with increasing Ag-NP concentration that can be controlled by the AgNO3 precursor concentration in the in-situ process. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Combinations of electrical, optical, and biological properties of multifunctional nanomaterials have shown immense potential for biosensing, optoelectronics, energy conversion and storage, and biocidal applications [1–3]. Successful application of nano-materials into functional nanodevices requires controlled design at nano level. The characterization of these nanostructures is also a vital issue in fundamental research as well as for technological applications. Conducting polymers are emerging as a promising material for their synthesis as nanostructured form as they exhibit electrical, magnetic, electronic, and optical properties analogous to that of metals or semiconductors while retaining polymer like properties such as their flexibility, ease of processing, low toxicity and modifiable electrical conductivity [4]. The nanostructures of electrically conducting polymer can be synthesized by chemical and electrochemical techniques. Fine metal nano particles are of particular interest because of their extraordinary size dependent optical, electrical, catalytic and biological properties. The current progress in research on metal nanoparticles has revived the use of silver nanoparticles for antibacterial application. The use of excellent antibacterial activity of nano-silver is reasonably new in the field of biotechnology [5]. Silver-based antibacterial materials captured much attention because of their being a long lasting biocide with high temperature stability and low volatility [6]. The main problem associated with nano⁎ Corresponding author. E-mail address: [email protected] (A. Kumar).
http://dx.doi.org/10.1016/j.msec.2015.04.027 0928-4931/© 2015 Elsevier B.V. All rights reserved.
sized silver particles is that they form clusters and agglomerate due to high surface energy which reduces the possibility of commercialization of Ag-NPs in terms of reusability [7]. The antibacterial activity of Ag-NPs is size dependent and once they aggregate, their activity decreases noticeably. To overcome this difficulty several techniques such as dispersion of the nanoparticles in different matrices or stabilizing metal nanoparticles by ligands have been considered [8]. The nanostructures of conducting polymers such as polyaniline (PAni) or polypyrrole (PPy) have proven to be good matrices for loading metal nanoparticles to form conducting polymer–metal nanocomposites. The entrapment of metal nanoparticles within the conducting polymer matrix is an alluring aspect as it increases the processability of the nanoparticles due to high thermal stability of conducting polymers. It has been reported that conducting polymers such as PAni and PPy also possess antibacterial activity [9,10]. Therefore, the synergy between 1D conducting polymers and metal nano-particles provides resultant nanocomposites with additional functionalities over individual materials with the possibility of designing device functionality. Several new approaches have been considered in synthesis, characterization and applications of nanocomposites with diverse combinations of properties of conducting polymers (PAni, PPy) and metallic nanoparticles. S. Fujii et al. have developed polypyrrole-coated silver nanocomposites with core shell morphology by aqueous chemical oxidative dispersion polymerization of PPy with silver nitrate as oxidant [11]. The synthesis of polypyrrole silver nanocomposites has been reported by P. Dallas et al. by interfacial polymerization method at water chloroform interface using sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium
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Fig. 1. SEM images of (a) PPy-NTs and PPy-NT:Ag-NP nanocomposites with (b) 6 wt.% and (c) 15 wt.% of Ag-NPs.
bromide (DTAB) as surfactants [12]. J. Zhang et al. [13] reported the synthesis of PPy-NTs and metal nanoparticle nanocomposites via MnO2 nanowires as a reactive template. MnO2 nanowires induce the 1D polymerization of pyrrole and the simultaneous dissolution of the templates gives the hollow tube-like structure. In spite of the antibacterial activity of Ag-NPs, safety of their use needs to be carefully investigated since the expanding applications of nano-silver may also increase the risk of overexposure to human cells [14]. Quantification of hemolysis activity of a material is considered as one of the fundamental tests in determining the safety of a blood contacting biomaterial. If hemolysis occurs to a significant extent it might lead to dangerous pathological conditions. The side effects of Ag-NPs or silver based nanocomposites on mammalian cells have been reported by various groups [15]. J. Choi et al. compared the hemolysis activity of silver micro and nano particles and reported that silver nanoparticles are more hemolytic as compared to micron-size particles due to their high activity owing to higher surface to volume ratio. According to their report the nano particles exceed the limit of 5% hemolysis at a concentration of 70 μg/ml [16]. In other report, Ag-NPs of average diameter of 30 nm at a concentration of 200 μg/ml cause 14.2% hemolysis [17]. In the present study, PPy-NT:Ag-NP nanocomposites have been synthesized by in-situ reduction of AgNO3 and their antibacterial and hemolysis activities have been investigated as a function of varying silver nanoparticle concentrations. Antibacterial activity of the nanocomposites has been investigated as a function of silver content by the diffusion plate method. In-vitro hemolysis activity measurements of the nanocomposites have been carried out against mammalian red blood cells (RBC). 2. Experimental 2.1. Materials and methodology Pyrrole (Sigma Aldrich) was vacuum dried prior to use. Ferric chloride (FeCl3), AgNO3, methyl orange (MO) and NaBH4 from Sisco Research Laboratory were used as received.
2.2. Preparation of polypyrrole nanotubes PPy-NTs were synthesized using the method by T. Dai et al. [18]. 0.243 g of FeCl3 was added to 30 ml of 5 mM MO solution. After the formation of flocculent precipitation 105 μl of pyrrole were added to the mixture and stirred for 24 h. The black precipitate formed was thoroughly rinsed three times with DD water and ethanol, and separated by centrifugation. Subsequently, the PPy-NTs were dried in a desiccator overnight. 2.3. Preparation of polypyrrole nanotube–silver nanoparticle nanocomposites 70 mg of PPy-NTs were dispersed in 25 ml DD water and ultrasonicated for 20 min. A calculated amount of AgNO3 was mixed with PPy-NT solution by stirring up to 2 h. Sodium borohydride solution was prepared separately by dissolving in 75 ml of DD water. The silver nitrate solution was mixed with sodium borohydride solution dropwise and magnetically stirred for 24 h at room temperature. The molar ratio of silver nitrate to sodium borohydride was maintained at 1:2 during the synthesis process. Finally the filtrate was thoroughly washed in ethanol and DD water, and dried. Four different compositions with different Ag-NP concentration were prepared by varying AgNO3 concentration as 6, 9, 12 and 15 wt.% with respect to PPy-NTs. 2.4. Characterization techniques The morphology of the nanocomposites was visualized using scanning electron microscopy (SEM, Jeol 6390 LV) and high resolution transmission electron microscopy (HRTEM, Jeol JEM 2100, 200 kV). For HRTEM imaging, a small drop of the prepared sample was placed on a copper grid following solvent evaporation in ambient air at room temperature. The crystalline nature of the nanocomposites was investigated using Rigaku miniflex X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å). The scan rate, accelerating voltage and current were kept at 5°/min, 30 kV and 15 mA, respectively during the XRD recording process. Optical absorption in the range of 200–800 nm was measured
Fig. 2. HRTEM images of (a) PPy-NTs and (b) PPy-NT:Ag-NP nanocomposites with 15 wt.% of Ag-NPs. (c) Gaussian distribution of diameters of PPy-NTs.
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employing a Shimadzu 1700 UV–visible spectrophotometer using quartz cuvettes of 1 cm path length. The FTIR spectra of PPy-NTs and PPy-NT:Ag-NP nanocomposite pellets pressed with KBR powder were recorded by using Perkin Elmer FTIR spectrometer model spectrum 100. 2.5. Measurement of antimicrobial activity The antibacterial activity of the nanocomposites was investigated by modified Kirby–Bauer diffusion plate method. E. coli and S. aureus were cultivated in sterilized Luria Bertani (LB) broth and incubated overnight at 37 °C in a shaking incubator. The overnight grown bacteria were spread in Luria Bertani (LB) agar plates by spread plate method. An equal quantity of the nanocomposites having different silver loadings was formed into pellets of diameter about 13 mm and thickness of 1–2 mm using hydraulic press and placed on each lawn of microorganisms in agar plates. Zone of inhibition was measured after 24 h incubation at 37 °C. To determine the value of MIC (minimum inhibitory concentration) of PPyNT:Ag-NP nanocomposites against E. coli and S. aureus, the broth dilution method described by M. T. Yilmaz [19] was followed. Fig. 3. X-ray diffractogram of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
2.6. Hemolysis assay Hemolysis is a phenomenon by which the breakdown or destruction of red blood cells (RBC) takes place so that the contained hemoglobin is freed into the surrounding medium. The in-vitro hemolysis activity test of PPy-NT:Ag-NP nanocomposites has been investigated by following the procedure of Zhu et al. [20]. Mammalian blood was collected into a heparinized tube having 4% sodium citrate followed by centrifugation at 3000 rpm at 4 °C for 20 min. Erythrocytes were then washed repeatedly with phosphate saline buffer (PBS, pH 7.4). 5% of the erythrocytes were gently resuspended with PBS to prepare the hematocrit. PPyNT:Ag-NP nanocomposites with different concentrations of 1.25 mg/ ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml were dispersed in PBS followed by ultrasonication. As positive control Triton X-100 was used which is capable of damaging the red blood cells and PBS is used as negative control. 100 μl of the dissolved samples of different concentrations were mixed with 1900 μl of hematocrit in different microfuge tubes and incubated at 37 °C for 1 h. After incubation the samples were placed in an ice bath for 60 s followed by centrifuging at 3000 rpm for 5 min. Supernatants were carefully used for determining the free hemoglobin concentration as a measure of hemolysis by taking absorbance at 540 nm [18]. The percentage of hemolysis was calculated as follows: A −AN 100 Hemolysis percentage ¼ S AP −AN
ð1Þ
decrease their surface energy, which is suppressed by PPy-NTs by capping them and enhanced their stability. The Gaussian distribution of outer diameters of PPy-NTs as determined from the HRTEM micrographs in Fig. 2(a) and (b) is shown in Fig. 2(c). The average diameters of PPy-NTs and Ag-NPs are measured to be 130.59 ± 5.5 and 23.12 ± 3.23 nm, respectively. 3.2. XRD analysis We further pursued X-ray diffraction measurement to confirm the presence of Ag-NPs in the nanocomposites. The XRD pattern (Fig. 3) shows the peaks at 2θ values of 38.3°, 44.6° and 64.4° corresponding to the (111), (200) and (220) planes, respectively of face-centered cubic silver [21]. A suppressed hump of PPy-NTs is observed in the 2θ range of 20°–30°, which can be attributed to the semi-crystalline nature of the polypyrrole which arises due to π–π interaction of polypyrrole chain. 3.3. FTIR spectra analysis The FTIR spectra of PPy-NTs and the nanocomposites with different Ag-NP loading are depicted in Fig. 4. Polypyrrole exhibits its characteristic bands at 1547 and 1470 cm−1, which are assigned to C_C
where AS, AP and AN are the absorbance of the sample, positive control and negative control, respectively. 3. Results and discussion 3.1. Morphological analysis The morphology of the nanocomposites was analyzed by means of SEM (Fig. 1) and HRTEM (Fig. 2). Both the electron microscopy techniques confirm the nanotubular morphology of PPy and nanoparticle nature of Ag. SEM micrographs of PPy-NTs and PPy-NT:Ag-NP nanocomposites with 6 and 15 wt.% of AgNO3 concentrations are depicted in Fig. 1 (a), (b) and (c), respectively. In Fig. 1 (b) and (c), the Ag-NPs are displayed as white dots and an increase in concentration of AgNPs is observed as the concentration of AgNO3 increases from 6 to 15 wt.%. However, the AgNO3 concentration does not have any effect on PPy-NTs as Ag-NPs are grown within the pre-synthesized PPy-NT matrix by in-situ reduction of AgNO3 by NaBH4. From the SEM micrographs it is observed that the Ag-NPs are distributed uniformly within the polymeric nanotubular matrix. Ag-NPs tend to agglomerate to
Fig. 4. FTIR spectra of (a) PPy-NTs and PPy-NT:Ag-NP nanocomposites with (b) 6 wt.%, (c) 9 wt.%, (d) 12 wt.% and (e) 15 wt.% of Ag-NPs.
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suppressed by the absorption band of PPy-NTs. As the amount of silver in the nanocomposites is increased up to 15 wt.%, blue shifting of the band to 460 nm is observed which is attributed to the increased absorption of Ag-NPs as a result of which the band is shifted towards the absorption of Ag-NPs. 3.5. Antimicrobial activity of the nanocomposite
Fig. 5. UV–vis absorption spectra of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
stretching and C\\N stretching, respectively [22]. The C\\H in-plane vibration is observed at 1037 cm−1 [23]. Strong peaks near 1176 and 910 cm−1 represent the doped state of polypyrrole, which shifts towards higher wave number side in the case of nanocomposites [24, 25]. The C\\H out-of-plane ring deformation vibration appears around 800–770 cm− 1 for both PPy-NTs and nanocomposites. Compared to the pure polypyrrole, the characteristic bands at 1547 and 1470 cm−1 shift to 1556 and 1481 cm−1, respectively in the case of the nanocomposites but the C\\H in-plane and out-of-plane band positions remain unchanged. This is ascribed to the difference in electron density as Ag makes the electron cloud of PPy more delocalized, which indicates that Ag-NPs segregate near the C_C and C\\N in the nanocomposites [26]. 3.4. Optical property analysis The UV–vis absorption spectrum of PPy-NT:Ag-NP nanocomposites is depicted in Fig. 5. PPy-NTs exhibit π–π* absorption band at 485 nm while, Ag-NPs show the absorbance in the range of 410–440 nm due to surface plasmon resonance depending on the particles size [27,28]. Therefore, the single broad absorbance band of PPy-NTs:Ag-NPs nanocomposites in the range of 310–550 nm is attributed to the overlapping of the π-π* transition of polypyrrole and surface plasmon resonance absorption of Ag-NPs. Due to the scanty intensity of the absorbance band of silver nanoparticles it became difficult to detect separately and was
The antibacterial activity of PPy-NT:Ag-NP nanocomposites against the gram positive (S. aureus) and gram negative (E. coli) bacteria has been estimated and presented in Fig. 6. Zones of inhibition (clear areas where no bacteria grows) are observed against both E. coli and S. aureus when incubated for 24 h with the nanocomposites on bacteria-inoculated agar plates. The generation of these zones of inhibition is ascribed to antibacterial activity of the nanocomposites. It is observed that the bactericidal performance of the nanocomposites increases with the concentration of Ag-NPs in the nanocomposites. Maximum zone of inhibition is measured around 23 mm with 15 wt.% of silver against both the organisms. In contrast the antibacterial activity of pristine PPy-NTs cannot be estimated by the diffusion plate method as it does not produce any zone of inhibition when in contact with E. coli and S. aureus bacteria. Therefore the antibacterial activity of the nanocomposites is mainly influenced by the existence of Ag-NPs which are active biocidal agents as compared to the PPy-NTs. The mechanism of the bactericidal effect of silver nanoparticles against the bacteria is not very well-understood. Ag-NPs may attach to the bacterial cell wall and form ‘pits’ thereby causing structural changes in the cell membrane, such as increasing the permeability of the cell membrane, resulting in cell death [29]. This mainly depends on the free surface area of the Ag-NPs. The Ag-NPs are dispersed and encapsulated within the PPy-NT matrix, which prevents their agglomeration. As a result of which the larger surface area of the nanoparticles is exposed, giving rise to the increased bacterial cell attachment resulting in improved antimicrobial action. It is also possible that Ag+ ions originating from AgNPs can coordinate with electron donating groups for instance thiols, carboxylates, amides, indoles, etc. damaging and deactivating the DNA and cellular enzymes [30,31]. Additionally, Ag+ ions may produce excess OH• radical by interacting with intercellular Fe–S cluster resulting in cell death [32]. On the other hand, during the oxidative polymerization of pyrrole, positive charges are produced along the backbone chains of polypyrrole. The electrostatic adherence between the positively charged polypyrrole molecule and negatively charged bacteria cell membrane may affect the membrane permeability, disturb internal osmotic imbalances, and lead to microbial growth inhibition. Furthermore, the nanotubular structure of polypyrrole can penetrate the cell wall and release the active silver through the nanopores enhancing the antibacterial activity of the nanocomposites [33].
Fig. 6. Photograph image of the zone of inhibition of PPy-NT:Ag-NP nanocomposites with different Ag-NP concentrations by modified Kirby–Bauer test against (a) E. coli and (b) S. aureus.
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Table 1 MIC values of PPy-NT:Ag-NP nanocomposites against E. coli and S. aureus with different loading concentrations of Ag-NPs. Sample
6 wt.% Ag-NPs 9 wt.% Ag-NPs 12 wt.% Ag-NPs 15 wt.% Ag-NPs
MIC value (mg/ml) E. coli
S. aureus
0.625 0.3125 0.156 0.078
0.625 0.625 0.3125 0.156
The MIC value which is defined as the minimum concentration of an antimicrobial that is able to restrict the growth of a microbe after whole night incubation has been investigated by broth dilution method and presented in Table 1 [34]. Concentration of PPy-NT:Ag-NP nanocomposites below the MIC value fail to inhibit the growth of bacteria. Different concentrations of the nanocomposites with varying Ag content (10 mg/ ml–0.0195 mg/ml) have been subjected to E. coli and S. aureus to investigate the value of MIC. After 16 h of incubation in Luria Bertani media in 96 well microtiter plate, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to the solution followed by 45 minute incubation in the dark. Live bacterial cells are capable of producing succinate dehydrogenase (enzyme) which reacts with MTT and forms formazan complexes with purple appearance. So the well having minimum concentration of sample where the complex does not form i.e. which does not appear purple is considered as the MIC of the compound. It is observed that the MIC values for both E. coli and S. aureus decrease with increase in the concentration of Ag-NPs in the
nanocomposites as expected. The lowest MIC value is calculated to be 0.078 mg/ml and 0.15625 mg/ml for the nanocomposites having 15 wt.% of Ag-NPs against E. coli and S. aureus, respectively. The lower MIC value against E. coli is attributed to the thinner cell wall of the gram negative bacteria as compared to that of S. aureus. Therefore, the penetration of the cell wall becomes easier which increases the antimicrobial efficiency of the nanocomposites against E. coli. 3.6. Hemolysis activity study The fundamental requirement of a biomaterial is that it must be biocompatible and not generate any toxic effects. The biocompatibility is generally a surface property, and can be determined based on the adverse host response intensity. In the present work we have investigated the biocompatibility of PPy-NT:Ag-NP nanocomposites via hemolysis assay. The percentage of hemolysis caused by the nanocomposites with different silver loading is depicted in Fig. 7. It is observed that the percent hemolysis increases with increase in sample concentration as well as the amount of Ag-NPs in the nanocomposites. Thus the concentration and surface to volume ratio of silver nanoparticles greatly influence the hemolysis activity of the nanocomposites. It has been reported that the hemolysis activity of Ag-NPs is not due to leaching of Ag+ ions from nanoparticles but due to the toxicity of the nanoparticles. Ag-NPs are capable of forming depressions and pits on the RBC membrane that could ultimately result in hemolysis by forming pores on the membrane and osmotic lysis [35,36]. All the nanocomposites exhibit hemolysis below 5% up to a concentration of 2.5 mg/ml which is permissible for biomaterials. Therefore the enhanced hemo-compatibility of the PPy-
Fig. 7. Hemolysis activity of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
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NT:Ag-NP nanocomposites is attributed to the biocompatible nature of PPy-NTs as polypyrrole is less toxic compared to silver. 4. Conclusions In summary, we synthesized nanocomposites of PPy-NTs and AgNPs by in-situ reduction of AgNO3 in the existence of PPy-NTs. Morphological studies demonstrate that PPy-NTs act as capping agent and prevent the agglomeration of Ag-NPs. It is found that the loading of Ag-NPs can be controlled by changing the amount of metal precursor silver nitrate during the in-situ reduction process. XRD results confirm the formation of the nanocomposites as they exhibit the characteristic peaks of both PPy and Ag. The shifting of C_C and C\\N peaks of the nanocomposites in the FTIR spectra indicates that Ag-NPs interact and segregate near these bonds in the nanocomposites. The single broad UV–vis absorbance band of PPy-NT:Ag-NP nanocomposites in the range of 310–550 nm is attributed to the overlapping of the π–π* transition of polypyrrole and surface plasmon resonance absorption of Ag-NPs. The absence of a separate absorbance band of silver is attributed to the suppression of scanty absorption of Ag-NPs by the absorbance of PPy-NTs. It is observed that the antibacterial activity of the nanocomposites against E. coli and S. aureus bacteria can be facilely tuned by controlling the silver concentration in the nanocomposites. The antibacterial activity of the PPy-NT:Ag-NP nanocomposites may be attributed to the interaction of Ag-NPs and/or Ag+ ions originating at Ag-NPs with the bacteria. The limited antibacterial properties of the PPy-NTs may be ascribed to their tubular structure and electrostatic interaction between the positively charged polypyrrole molecules and negatively charged bacterial cell membrane. The lowest MIC values are calculated as 0.078 mg/ml and 0.15625 mg/ml for the nanocomposites having 15 wt.% of silver against E. coli and S. aureus, respectively. It is observed that the hemolysis activity of the nanocomposites increases with the concentration of Ag-NPs. Acknowledgment The authors gratefully acknowledge the help extended for TEM measurements by the Electron Microscopy Division, SAIF, Shillong. References [1] J. Xu, J. Hu, B. Quan, Z. Wei, Decorating polypyrrole nanotubes with Au nanoparticles by an in situ reduction process, Macromol. Rapid Commun. 30 (2009) 936–940. [2] Y. Shen, Y. Lin, C.-W. Nan, Interfacial effect on dielectric properties of polymer nanocomposites filled with core/shell-structured particles, Adv. Funct. Mater. 17 (2007) 2405–2410. [3] X. Lu, W. Zhang, C. Wang, T.-C. Wen, Y. Wei, One-dimensional conducting polymer nanocomposites: synthesis, properties and applications, Prog. Polym. Sci. 36 (2011) 671–712. [4] K. Ramanathan, M.A. Bangar, M. Yun, W. Chen, A. Mulchandani, N.V. Myung, Individually addressable conducting polymer nanowires array, Nano Lett. 4 (2004) 1237–1239. [5] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83. [6] R. Kumar, H. Münstedt, Silver ion release from antimicrobial polyamide/silver composites, Biomaterials 26 (2005) 2081–2088. [7] T. Yao, C. Wang, J. Wu, Q. Lin, H. Lv, K. Zhang, K. Yu, B. Yang, Preparation of raspberry-like polypyrrole composites with applications in catalysis, J. Colloid Interface Sci. 338 (2009) 573–577. [8] M.K. Corbierre, N.S. Cameron, M. Sutton, S.G. Mochrie, L.B. Lurio, A. Rühm, R.B. Lennox, Polymer-stabilized gold nanoparticles and their incorporation into polymer matrices, J. Am. Chem. Soc. 123 (2001) 10411–10412. [9] M.R. Gizdavic-Nikolaidis, J.R. Bennett, S. Swift, A.J. Easteal, M. Ambrose, Broad spectrum antimicrobial activity of functionalized polyanilines, Acta Biomater. 7 (2011) 4204–4209.
13
[10] E. Park, H. Kim, J. Song, H. Oh, H. Song, J. Jnag, Synthesis of silver nanoparticles decorated polypyrrole nanotubes for antimicrobial application, Macromol. Res. 20 (2012) 1096–1101. [11] S. Fujii, A. Aichi, K. Akamatsu, H. Nawafune, Y. Nakamura, One-step synthesis of polypyrrole-coated silver nanocomposite particles and their application as a coloured particulate emulsifier, J. Mater. Chem. 17 (2007) 3777–3779. [12] P. Dallas, D. Niarchos, D. Vrbanic, N. Boukos, S. Pejovnik, T. Christos, D. Petridis, Interfacial polymerization of pyrrole and in situ synthesis of polypyrrole/silver nanocomposites, Polymer 48 (2007) 2007–2013. [13] J. Zhang, X. Liu, L. Zhang, B. Cao, S. Wu, Reactive template synthesis of polypyrrole nanotubes for fabricating metal/conducting polymer nanocomposites, Macromol. Rapid Commun. 34 (2013) 528–532. [14] J.-J. Lin, W.-C. Lin, R.-X. Dong, S.-H. Hsu, The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers, Nanotechnology 23 (2012) 065102–065113. [15] J.-J. Lin, W.-C. Lin, S.-D. Li, C.-Y. Lin, S.-H. Hsu, Evaluation of the antibacterial activity and biocompatibility for silver nanoparticles immobilized on nano silicate platelets, Appl. Mater. Interfaces 5 (2012) 433–443. [16] J. Choi, V. Reipa, V.M. Hitchins, P.L. Goering, R.A. Malinauskas, Physicochemical characterization and in vitro hemolysis evaluation of silver nanoparticles, Toxicol. Sci. 123 (2011) 133–143. [17] M.J. Ki, S. Shin, Toxic effects of silver nanoparticles and nanowires on erythrocyte rheology, Food Chem. Toxicol. 67 (2014) 80–86. [18] T. Dai, X. Yang, Y. Lu, Controlled growth of polypyrrole nanotubule/wire in the presence of a cationic surfactant, Nanotechnology 17 (2006) 3028–3034. [19] M.T. Yilmaz, Minimum inhibitory and minimum bactericidal concentrations of boron compounds against several bacterial strains, Turk. J. Med. Sci. 12 (2012) 1423–1429. [20] Q.Y. Zhu, R.R. Holt, S.A. Lazarus, T.J. Orozco, C.L. Keen, Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte haemolysis, Exp. Biol. Med. 227 (2002) 321–329. [21] P. Boomi, H.G. Prabu, J. Mathiyarasu, Synthesis and characterization of polyaniline/Ag-Pt nanocomposite for improved antibacterial activity, Colloids Surf. B: Biointerfaces 103 (2013) 9–14. [22] P.-G. Su, L.-N. Huang, Humidity sensors based on TiO2 nanoparticles/polypyrrole composite thin films, Sens. Actuators B 123 (2007) 501–507. [23] D. Zhang, X. Zhang, Y. Chen, P. Yu, C. Wang, Y. Ma, Enhanced capacitance and rate capability of graphene/polypyrrole composite as electrode material for supercapacitors, J. Power Sources 196 (2011) 5990–5996. [24] J. Upadhyay, A. Kumar, Engineering polypyrrole nanotubes by 100 MeV Si9+ ion beam irradiation: enhancement of antioxidant activity, Mater. Sci. Eng. C 33 (2013) 4900–4904. [25] P. Xu, X. Han, C. Wang, B. Zhang, X. Wang, H.-L. Wang, Facile synthesis of polyaniline-polypyrrole nanofibers for application in chemical deposition of metal nanoparticles, Macromol. Rapid Commun. 29 (2008) 1392–1397. [26] H. Zhang, X. Zhong, J.-J. Xu, H.-Y. Chen, Fe3O4/polypyrrole/Au nanocomposites with core/shell/shell structure: synthesis, characterization, and their electrochemical properties, Langmuir 24 (2008) 13748–13752. [27] J. Upadhyay, A. Kumar, Investigation of structural, thermal and dielectric properties of polypyrrole nanotubes tailoring with silver nanoparticles, Compos. Sci. Technol. 97 (2014) 55–62. [28] S. Pal, Y.K. Tak, J.M. Song, Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli, Appl. Environ. Microbiol. 73 (2007) 1712–1720. [29] V. Sambhy, M.M. MacBride, B.R. Peterson, A. Sen, Silver bromide nanoparticle/polymer composites: dual action tunable antimicrobial materials, J. Am. Chem. Soc. 128 (2006) 9798–9808. [30] H. Kong, J. Jang, Antibacterial properties of novel poly(methyl methacrylate) nanofiber containing silver nanoparticles, Langmuir 24 (2008) 2051–2056. [31] M. Ignatova, D. Labaye, S. Lenoir, D. Strivay, R. Jerome, C. Jerome, Immobilization of silver in polypyrrole/polyanion composite coatings: preparation, characterization, and antibacterial activity, Langmuir 19 (2003) 8971–8979. [32] J.R. Morones-Ramirez, J.A. Winkler, C.S. Spina, J.J. Collins, Silver enhances antibiotic activity against Gram-negative bacteria, Sci. Transl. Med. 190 (2013) 190ra81. [33] A. Varesano, C. Vineis, A. Aluigi, F. Rombaldoni, C. Tonetti, G. Mazzuchetti, Antibacterial efficacy of polypyrrole in textile applications, Fibers Polym. 14 (2013) 36–42. [34] I. Wiegand, K. Hilpert, R.E. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [35] P.V. Asharani, S. Sethu, S. Vadukumpully, S. Zhong, C.T. Lim, M.P. Hande, S. Valiyaveettil, Investigations on the structural damage in human erythrocytes exposed to silver, gold, and platinum nanoparticles, Adv. Funct. Mater. 20 (2010) 1233–1242. [36] D. Drescher, T. Büchner, D. McNaughton, J. Kneipp, SERS reveals the specific interaction of silver and gold nanoparticles with hemoglobin and red blood cell components, Phys. Chem. Chem. Phys. 15 (2013) 5364–5373.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Antibacterial and hemolysis activity of polypyrrole nanotubes decorated with silver nanoparticles by an in-situ reduction process J. Upadhyay a, A. Kumar a,⁎, B. Gogoi b, A.K. Buragohain b,c a b c
Department of Physics, Tezpur University, Napaam, Tezpur 784028, Assam, India Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur 784028, Assam, India Dibrugarh University, Dibrugarh 786004, Assam, India
a r t i c l e
i n f o
Available online 22 April 2015 Keywords: Polypyrrole nanotubes Silver nanoparticles Nanocomposites Antibacterial activity Hemolysis activity
a b s t r a c t Polypyrrole nanotube–silver nanoparticle nanocomposites (PPy-NTs:Ag-NPs) have been synthesized by in-situ reduction of silver nitrate (AgNO3) to suppress the agglomeration of Ag-NPs. The morphology and chemical structure of the nanocomposites have been studied by HRTEM, SEM, XRD, FTIR and UV–vis spectroscopy. The average diameter of the polypyrrole nanotubes (PPy-NTs) is measured to be 130.59 ± 5.5 nm with their length in the micrometer range, while the silver nanoparticles (Ag-NPs) exhibit spherical shape with an average diameter of 23.12 ± 3.23 nm. In-vitro blood compatibility of the nanocomposites has been carried out via hemolysis assay. Antimicrobial activity of the nanocomposites has been investigated with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) bacteria. The results depict that the hemolysis and antimicrobial activities of the nanocomposites increase with increasing Ag-NP concentration that can be controlled by the AgNO3 precursor concentration in the in-situ process. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Combinations of electrical, optical, and biological properties of multifunctional nanomaterials have shown immense potential for biosensing, optoelectronics, energy conversion and storage, and biocidal applications [1–3]. Successful application of nano-materials into functional nanodevices requires controlled design at nano level. The characterization of these nanostructures is also a vital issue in fundamental research as well as for technological applications. Conducting polymers are emerging as a promising material for their synthesis as nanostructured form as they exhibit electrical, magnetic, electronic, and optical properties analogous to that of metals or semiconductors while retaining polymer like properties such as their flexibility, ease of processing, low toxicity and modifiable electrical conductivity [4]. The nanostructures of electrically conducting polymer can be synthesized by chemical and electrochemical techniques. Fine metal nano particles are of particular interest because of their extraordinary size dependent optical, electrical, catalytic and biological properties. The current progress in research on metal nanoparticles has revived the use of silver nanoparticles for antibacterial application. The use of excellent antibacterial activity of nano-silver is reasonably new in the field of biotechnology [5]. Silver-based antibacterial materials captured much attention because of their being a long lasting biocide with high temperature stability and low volatility [6]. The main problem associated with nano⁎ Corresponding author. E-mail address: [email protected] (A. Kumar).
http://dx.doi.org/10.1016/j.msec.2015.04.027 0928-4931/© 2015 Elsevier B.V. All rights reserved.
sized silver particles is that they form clusters and agglomerate due to high surface energy which reduces the possibility of commercialization of Ag-NPs in terms of reusability [7]. The antibacterial activity of Ag-NPs is size dependent and once they aggregate, their activity decreases noticeably. To overcome this difficulty several techniques such as dispersion of the nanoparticles in different matrices or stabilizing metal nanoparticles by ligands have been considered [8]. The nanostructures of conducting polymers such as polyaniline (PAni) or polypyrrole (PPy) have proven to be good matrices for loading metal nanoparticles to form conducting polymer–metal nanocomposites. The entrapment of metal nanoparticles within the conducting polymer matrix is an alluring aspect as it increases the processability of the nanoparticles due to high thermal stability of conducting polymers. It has been reported that conducting polymers such as PAni and PPy also possess antibacterial activity [9,10]. Therefore, the synergy between 1D conducting polymers and metal nano-particles provides resultant nanocomposites with additional functionalities over individual materials with the possibility of designing device functionality. Several new approaches have been considered in synthesis, characterization and applications of nanocomposites with diverse combinations of properties of conducting polymers (PAni, PPy) and metallic nanoparticles. S. Fujii et al. have developed polypyrrole-coated silver nanocomposites with core shell morphology by aqueous chemical oxidative dispersion polymerization of PPy with silver nitrate as oxidant [11]. The synthesis of polypyrrole silver nanocomposites has been reported by P. Dallas et al. by interfacial polymerization method at water chloroform interface using sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium
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Fig. 1. SEM images of (a) PPy-NTs and PPy-NT:Ag-NP nanocomposites with (b) 6 wt.% and (c) 15 wt.% of Ag-NPs.
bromide (DTAB) as surfactants [12]. J. Zhang et al. [13] reported the synthesis of PPy-NTs and metal nanoparticle nanocomposites via MnO2 nanowires as a reactive template. MnO2 nanowires induce the 1D polymerization of pyrrole and the simultaneous dissolution of the templates gives the hollow tube-like structure. In spite of the antibacterial activity of Ag-NPs, safety of their use needs to be carefully investigated since the expanding applications of nano-silver may also increase the risk of overexposure to human cells [14]. Quantification of hemolysis activity of a material is considered as one of the fundamental tests in determining the safety of a blood contacting biomaterial. If hemolysis occurs to a significant extent it might lead to dangerous pathological conditions. The side effects of Ag-NPs or silver based nanocomposites on mammalian cells have been reported by various groups [15]. J. Choi et al. compared the hemolysis activity of silver micro and nano particles and reported that silver nanoparticles are more hemolytic as compared to micron-size particles due to their high activity owing to higher surface to volume ratio. According to their report the nano particles exceed the limit of 5% hemolysis at a concentration of 70 μg/ml [16]. In other report, Ag-NPs of average diameter of 30 nm at a concentration of 200 μg/ml cause 14.2% hemolysis [17]. In the present study, PPy-NT:Ag-NP nanocomposites have been synthesized by in-situ reduction of AgNO3 and their antibacterial and hemolysis activities have been investigated as a function of varying silver nanoparticle concentrations. Antibacterial activity of the nanocomposites has been investigated as a function of silver content by the diffusion plate method. In-vitro hemolysis activity measurements of the nanocomposites have been carried out against mammalian red blood cells (RBC). 2. Experimental 2.1. Materials and methodology Pyrrole (Sigma Aldrich) was vacuum dried prior to use. Ferric chloride (FeCl3), AgNO3, methyl orange (MO) and NaBH4 from Sisco Research Laboratory were used as received.
2.2. Preparation of polypyrrole nanotubes PPy-NTs were synthesized using the method by T. Dai et al. [18]. 0.243 g of FeCl3 was added to 30 ml of 5 mM MO solution. After the formation of flocculent precipitation 105 μl of pyrrole were added to the mixture and stirred for 24 h. The black precipitate formed was thoroughly rinsed three times with DD water and ethanol, and separated by centrifugation. Subsequently, the PPy-NTs were dried in a desiccator overnight. 2.3. Preparation of polypyrrole nanotube–silver nanoparticle nanocomposites 70 mg of PPy-NTs were dispersed in 25 ml DD water and ultrasonicated for 20 min. A calculated amount of AgNO3 was mixed with PPy-NT solution by stirring up to 2 h. Sodium borohydride solution was prepared separately by dissolving in 75 ml of DD water. The silver nitrate solution was mixed with sodium borohydride solution dropwise and magnetically stirred for 24 h at room temperature. The molar ratio of silver nitrate to sodium borohydride was maintained at 1:2 during the synthesis process. Finally the filtrate was thoroughly washed in ethanol and DD water, and dried. Four different compositions with different Ag-NP concentration were prepared by varying AgNO3 concentration as 6, 9, 12 and 15 wt.% with respect to PPy-NTs. 2.4. Characterization techniques The morphology of the nanocomposites was visualized using scanning electron microscopy (SEM, Jeol 6390 LV) and high resolution transmission electron microscopy (HRTEM, Jeol JEM 2100, 200 kV). For HRTEM imaging, a small drop of the prepared sample was placed on a copper grid following solvent evaporation in ambient air at room temperature. The crystalline nature of the nanocomposites was investigated using Rigaku miniflex X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å). The scan rate, accelerating voltage and current were kept at 5°/min, 30 kV and 15 mA, respectively during the XRD recording process. Optical absorption in the range of 200–800 nm was measured
Fig. 2. HRTEM images of (a) PPy-NTs and (b) PPy-NT:Ag-NP nanocomposites with 15 wt.% of Ag-NPs. (c) Gaussian distribution of diameters of PPy-NTs.
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employing a Shimadzu 1700 UV–visible spectrophotometer using quartz cuvettes of 1 cm path length. The FTIR spectra of PPy-NTs and PPy-NT:Ag-NP nanocomposite pellets pressed with KBR powder were recorded by using Perkin Elmer FTIR spectrometer model spectrum 100. 2.5. Measurement of antimicrobial activity The antibacterial activity of the nanocomposites was investigated by modified Kirby–Bauer diffusion plate method. E. coli and S. aureus were cultivated in sterilized Luria Bertani (LB) broth and incubated overnight at 37 °C in a shaking incubator. The overnight grown bacteria were spread in Luria Bertani (LB) agar plates by spread plate method. An equal quantity of the nanocomposites having different silver loadings was formed into pellets of diameter about 13 mm and thickness of 1–2 mm using hydraulic press and placed on each lawn of microorganisms in agar plates. Zone of inhibition was measured after 24 h incubation at 37 °C. To determine the value of MIC (minimum inhibitory concentration) of PPyNT:Ag-NP nanocomposites against E. coli and S. aureus, the broth dilution method described by M. T. Yilmaz [19] was followed. Fig. 3. X-ray diffractogram of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
2.6. Hemolysis assay Hemolysis is a phenomenon by which the breakdown or destruction of red blood cells (RBC) takes place so that the contained hemoglobin is freed into the surrounding medium. The in-vitro hemolysis activity test of PPy-NT:Ag-NP nanocomposites has been investigated by following the procedure of Zhu et al. [20]. Mammalian blood was collected into a heparinized tube having 4% sodium citrate followed by centrifugation at 3000 rpm at 4 °C for 20 min. Erythrocytes were then washed repeatedly with phosphate saline buffer (PBS, pH 7.4). 5% of the erythrocytes were gently resuspended with PBS to prepare the hematocrit. PPyNT:Ag-NP nanocomposites with different concentrations of 1.25 mg/ ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml were dispersed in PBS followed by ultrasonication. As positive control Triton X-100 was used which is capable of damaging the red blood cells and PBS is used as negative control. 100 μl of the dissolved samples of different concentrations were mixed with 1900 μl of hematocrit in different microfuge tubes and incubated at 37 °C for 1 h. After incubation the samples were placed in an ice bath for 60 s followed by centrifuging at 3000 rpm for 5 min. Supernatants were carefully used for determining the free hemoglobin concentration as a measure of hemolysis by taking absorbance at 540 nm [18]. The percentage of hemolysis was calculated as follows: A −AN 100 Hemolysis percentage ¼ S AP −AN
ð1Þ
decrease their surface energy, which is suppressed by PPy-NTs by capping them and enhanced their stability. The Gaussian distribution of outer diameters of PPy-NTs as determined from the HRTEM micrographs in Fig. 2(a) and (b) is shown in Fig. 2(c). The average diameters of PPy-NTs and Ag-NPs are measured to be 130.59 ± 5.5 and 23.12 ± 3.23 nm, respectively. 3.2. XRD analysis We further pursued X-ray diffraction measurement to confirm the presence of Ag-NPs in the nanocomposites. The XRD pattern (Fig. 3) shows the peaks at 2θ values of 38.3°, 44.6° and 64.4° corresponding to the (111), (200) and (220) planes, respectively of face-centered cubic silver [21]. A suppressed hump of PPy-NTs is observed in the 2θ range of 20°–30°, which can be attributed to the semi-crystalline nature of the polypyrrole which arises due to π–π interaction of polypyrrole chain. 3.3. FTIR spectra analysis The FTIR spectra of PPy-NTs and the nanocomposites with different Ag-NP loading are depicted in Fig. 4. Polypyrrole exhibits its characteristic bands at 1547 and 1470 cm−1, which are assigned to C_C
where AS, AP and AN are the absorbance of the sample, positive control and negative control, respectively. 3. Results and discussion 3.1. Morphological analysis The morphology of the nanocomposites was analyzed by means of SEM (Fig. 1) and HRTEM (Fig. 2). Both the electron microscopy techniques confirm the nanotubular morphology of PPy and nanoparticle nature of Ag. SEM micrographs of PPy-NTs and PPy-NT:Ag-NP nanocomposites with 6 and 15 wt.% of AgNO3 concentrations are depicted in Fig. 1 (a), (b) and (c), respectively. In Fig. 1 (b) and (c), the Ag-NPs are displayed as white dots and an increase in concentration of AgNPs is observed as the concentration of AgNO3 increases from 6 to 15 wt.%. However, the AgNO3 concentration does not have any effect on PPy-NTs as Ag-NPs are grown within the pre-synthesized PPy-NT matrix by in-situ reduction of AgNO3 by NaBH4. From the SEM micrographs it is observed that the Ag-NPs are distributed uniformly within the polymeric nanotubular matrix. Ag-NPs tend to agglomerate to
Fig. 4. FTIR spectra of (a) PPy-NTs and PPy-NT:Ag-NP nanocomposites with (b) 6 wt.%, (c) 9 wt.%, (d) 12 wt.% and (e) 15 wt.% of Ag-NPs.
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suppressed by the absorption band of PPy-NTs. As the amount of silver in the nanocomposites is increased up to 15 wt.%, blue shifting of the band to 460 nm is observed which is attributed to the increased absorption of Ag-NPs as a result of which the band is shifted towards the absorption of Ag-NPs. 3.5. Antimicrobial activity of the nanocomposite
Fig. 5. UV–vis absorption spectra of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
stretching and C\\N stretching, respectively [22]. The C\\H in-plane vibration is observed at 1037 cm−1 [23]. Strong peaks near 1176 and 910 cm−1 represent the doped state of polypyrrole, which shifts towards higher wave number side in the case of nanocomposites [24, 25]. The C\\H out-of-plane ring deformation vibration appears around 800–770 cm− 1 for both PPy-NTs and nanocomposites. Compared to the pure polypyrrole, the characteristic bands at 1547 and 1470 cm−1 shift to 1556 and 1481 cm−1, respectively in the case of the nanocomposites but the C\\H in-plane and out-of-plane band positions remain unchanged. This is ascribed to the difference in electron density as Ag makes the electron cloud of PPy more delocalized, which indicates that Ag-NPs segregate near the C_C and C\\N in the nanocomposites [26]. 3.4. Optical property analysis The UV–vis absorption spectrum of PPy-NT:Ag-NP nanocomposites is depicted in Fig. 5. PPy-NTs exhibit π–π* absorption band at 485 nm while, Ag-NPs show the absorbance in the range of 410–440 nm due to surface plasmon resonance depending on the particles size [27,28]. Therefore, the single broad absorbance band of PPy-NTs:Ag-NPs nanocomposites in the range of 310–550 nm is attributed to the overlapping of the π-π* transition of polypyrrole and surface plasmon resonance absorption of Ag-NPs. Due to the scanty intensity of the absorbance band of silver nanoparticles it became difficult to detect separately and was
The antibacterial activity of PPy-NT:Ag-NP nanocomposites against the gram positive (S. aureus) and gram negative (E. coli) bacteria has been estimated and presented in Fig. 6. Zones of inhibition (clear areas where no bacteria grows) are observed against both E. coli and S. aureus when incubated for 24 h with the nanocomposites on bacteria-inoculated agar plates. The generation of these zones of inhibition is ascribed to antibacterial activity of the nanocomposites. It is observed that the bactericidal performance of the nanocomposites increases with the concentration of Ag-NPs in the nanocomposites. Maximum zone of inhibition is measured around 23 mm with 15 wt.% of silver against both the organisms. In contrast the antibacterial activity of pristine PPy-NTs cannot be estimated by the diffusion plate method as it does not produce any zone of inhibition when in contact with E. coli and S. aureus bacteria. Therefore the antibacterial activity of the nanocomposites is mainly influenced by the existence of Ag-NPs which are active biocidal agents as compared to the PPy-NTs. The mechanism of the bactericidal effect of silver nanoparticles against the bacteria is not very well-understood. Ag-NPs may attach to the bacterial cell wall and form ‘pits’ thereby causing structural changes in the cell membrane, such as increasing the permeability of the cell membrane, resulting in cell death [29]. This mainly depends on the free surface area of the Ag-NPs. The Ag-NPs are dispersed and encapsulated within the PPy-NT matrix, which prevents their agglomeration. As a result of which the larger surface area of the nanoparticles is exposed, giving rise to the increased bacterial cell attachment resulting in improved antimicrobial action. It is also possible that Ag+ ions originating from AgNPs can coordinate with electron donating groups for instance thiols, carboxylates, amides, indoles, etc. damaging and deactivating the DNA and cellular enzymes [30,31]. Additionally, Ag+ ions may produce excess OH• radical by interacting with intercellular Fe–S cluster resulting in cell death [32]. On the other hand, during the oxidative polymerization of pyrrole, positive charges are produced along the backbone chains of polypyrrole. The electrostatic adherence between the positively charged polypyrrole molecule and negatively charged bacteria cell membrane may affect the membrane permeability, disturb internal osmotic imbalances, and lead to microbial growth inhibition. Furthermore, the nanotubular structure of polypyrrole can penetrate the cell wall and release the active silver through the nanopores enhancing the antibacterial activity of the nanocomposites [33].
Fig. 6. Photograph image of the zone of inhibition of PPy-NT:Ag-NP nanocomposites with different Ag-NP concentrations by modified Kirby–Bauer test against (a) E. coli and (b) S. aureus.
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Table 1 MIC values of PPy-NT:Ag-NP nanocomposites against E. coli and S. aureus with different loading concentrations of Ag-NPs. Sample
6 wt.% Ag-NPs 9 wt.% Ag-NPs 12 wt.% Ag-NPs 15 wt.% Ag-NPs
MIC value (mg/ml) E. coli
S. aureus
0.625 0.3125 0.156 0.078
0.625 0.625 0.3125 0.156
The MIC value which is defined as the minimum concentration of an antimicrobial that is able to restrict the growth of a microbe after whole night incubation has been investigated by broth dilution method and presented in Table 1 [34]. Concentration of PPy-NT:Ag-NP nanocomposites below the MIC value fail to inhibit the growth of bacteria. Different concentrations of the nanocomposites with varying Ag content (10 mg/ ml–0.0195 mg/ml) have been subjected to E. coli and S. aureus to investigate the value of MIC. After 16 h of incubation in Luria Bertani media in 96 well microtiter plate, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to the solution followed by 45 minute incubation in the dark. Live bacterial cells are capable of producing succinate dehydrogenase (enzyme) which reacts with MTT and forms formazan complexes with purple appearance. So the well having minimum concentration of sample where the complex does not form i.e. which does not appear purple is considered as the MIC of the compound. It is observed that the MIC values for both E. coli and S. aureus decrease with increase in the concentration of Ag-NPs in the
nanocomposites as expected. The lowest MIC value is calculated to be 0.078 mg/ml and 0.15625 mg/ml for the nanocomposites having 15 wt.% of Ag-NPs against E. coli and S. aureus, respectively. The lower MIC value against E. coli is attributed to the thinner cell wall of the gram negative bacteria as compared to that of S. aureus. Therefore, the penetration of the cell wall becomes easier which increases the antimicrobial efficiency of the nanocomposites against E. coli. 3.6. Hemolysis activity study The fundamental requirement of a biomaterial is that it must be biocompatible and not generate any toxic effects. The biocompatibility is generally a surface property, and can be determined based on the adverse host response intensity. In the present work we have investigated the biocompatibility of PPy-NT:Ag-NP nanocomposites via hemolysis assay. The percentage of hemolysis caused by the nanocomposites with different silver loading is depicted in Fig. 7. It is observed that the percent hemolysis increases with increase in sample concentration as well as the amount of Ag-NPs in the nanocomposites. Thus the concentration and surface to volume ratio of silver nanoparticles greatly influence the hemolysis activity of the nanocomposites. It has been reported that the hemolysis activity of Ag-NPs is not due to leaching of Ag+ ions from nanoparticles but due to the toxicity of the nanoparticles. Ag-NPs are capable of forming depressions and pits on the RBC membrane that could ultimately result in hemolysis by forming pores on the membrane and osmotic lysis [35,36]. All the nanocomposites exhibit hemolysis below 5% up to a concentration of 2.5 mg/ml which is permissible for biomaterials. Therefore the enhanced hemo-compatibility of the PPy-
Fig. 7. Hemolysis activity of PPy-NT:Ag-NP nanocomposites with (a) 6 wt.%, (b) 9 wt.%, (c) 12 wt.% and (d) 15 wt.% of Ag-NPs.
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NT:Ag-NP nanocomposites is attributed to the biocompatible nature of PPy-NTs as polypyrrole is less toxic compared to silver. 4. Conclusions In summary, we synthesized nanocomposites of PPy-NTs and AgNPs by in-situ reduction of AgNO3 in the existence of PPy-NTs. Morphological studies demonstrate that PPy-NTs act as capping agent and prevent the agglomeration of Ag-NPs. It is found that the loading of Ag-NPs can be controlled by changing the amount of metal precursor silver nitrate during the in-situ reduction process. XRD results confirm the formation of the nanocomposites as they exhibit the characteristic peaks of both PPy and Ag. The shifting of C_C and C\\N peaks of the nanocomposites in the FTIR spectra indicates that Ag-NPs interact and segregate near these bonds in the nanocomposites. The single broad UV–vis absorbance band of PPy-NT:Ag-NP nanocomposites in the range of 310–550 nm is attributed to the overlapping of the π–π* transition of polypyrrole and surface plasmon resonance absorption of Ag-NPs. The absence of a separate absorbance band of silver is attributed to the suppression of scanty absorption of Ag-NPs by the absorbance of PPy-NTs. It is observed that the antibacterial activity of the nanocomposites against E. coli and S. aureus bacteria can be facilely tuned by controlling the silver concentration in the nanocomposites. The antibacterial activity of the PPy-NT:Ag-NP nanocomposites may be attributed to the interaction of Ag-NPs and/or Ag+ ions originating at Ag-NPs with the bacteria. The limited antibacterial properties of the PPy-NTs may be ascribed to their tubular structure and electrostatic interaction between the positively charged polypyrrole molecules and negatively charged bacterial cell membrane. The lowest MIC values are calculated as 0.078 mg/ml and 0.15625 mg/ml for the nanocomposites having 15 wt.% of silver against E. coli and S. aureus, respectively. It is observed that the hemolysis activity of the nanocomposites increases with the concentration of Ag-NPs. Acknowledgment The authors gratefully acknowledge the help extended for TEM measurements by the Electron Microscopy Division, SAIF, Shillong. References [1] J. Xu, J. Hu, B. Quan, Z. Wei, Decorating polypyrrole nanotubes with Au nanoparticles by an in situ reduction process, Macromol. Rapid Commun. 30 (2009) 936–940. [2] Y. Shen, Y. Lin, C.-W. Nan, Interfacial effect on dielectric properties of polymer nanocomposites filled with core/shell-structured particles, Adv. Funct. Mater. 17 (2007) 2405–2410. [3] X. Lu, W. Zhang, C. Wang, T.-C. Wen, Y. Wei, One-dimensional conducting polymer nanocomposites: synthesis, properties and applications, Prog. Polym. Sci. 36 (2011) 671–712. [4] K. Ramanathan, M.A. Bangar, M. Yun, W. Chen, A. Mulchandani, N.V. Myung, Individually addressable conducting polymer nanowires array, Nano Lett. 4 (2004) 1237–1239. [5] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobials, Biotechnol. Adv. 27 (2009) 76–83. [6] R. Kumar, H. Münstedt, Silver ion release from antimicrobial polyamide/silver composites, Biomaterials 26 (2005) 2081–2088. [7] T. Yao, C. Wang, J. Wu, Q. Lin, H. Lv, K. Zhang, K. Yu, B. Yang, Preparation of raspberry-like polypyrrole composites with applications in catalysis, J. Colloid Interface Sci. 338 (2009) 573–577. [8] M.K. Corbierre, N.S. Cameron, M. Sutton, S.G. Mochrie, L.B. Lurio, A. Rühm, R.B. Lennox, Polymer-stabilized gold nanoparticles and their incorporation into polymer matrices, J. Am. Chem. Soc. 123 (2001) 10411–10412. [9] M.R. Gizdavic-Nikolaidis, J.R. Bennett, S. Swift, A.J. Easteal, M. Ambrose, Broad spectrum antimicrobial activity of functionalized polyanilines, Acta Biomater. 7 (2011) 4204–4209.
13
[10] E. Park, H. Kim, J. Song, H. Oh, H. Song, J. Jnag, Synthesis of silver nanoparticles decorated polypyrrole nanotubes for antimicrobial application, Macromol. Res. 20 (2012) 1096–1101. [11] S. Fujii, A. Aichi, K. Akamatsu, H. Nawafune, Y. Nakamura, One-step synthesis of polypyrrole-coated silver nanocomposite particles and their application as a coloured particulate emulsifier, J. Mater. Chem. 17 (2007) 3777–3779. [12] P. Dallas, D. Niarchos, D. Vrbanic, N. Boukos, S. Pejovnik, T. Christos, D. Petridis, Interfacial polymerization of pyrrole and in situ synthesis of polypyrrole/silver nanocomposites, Polymer 48 (2007) 2007–2013. [13] J. Zhang, X. Liu, L. Zhang, B. Cao, S. Wu, Reactive template synthesis of polypyrrole nanotubes for fabricating metal/conducting polymer nanocomposites, Macromol. Rapid Commun. 34 (2013) 528–532. [14] J.-J. Lin, W.-C. Lin, R.-X. Dong, S.-H. Hsu, The cellular responses and antibacterial activities of silver nanoparticles stabilized by different polymers, Nanotechnology 23 (2012) 065102–065113. [15] J.-J. Lin, W.-C. Lin, S.-D. Li, C.-Y. Lin, S.-H. Hsu, Evaluation of the antibacterial activity and biocompatibility for silver nanoparticles immobilized on nano silicate platelets, Appl. Mater. Interfaces 5 (2012) 433–443. [16] J. Choi, V. Reipa, V.M. Hitchins, P.L. Goering, R.A. Malinauskas, Physicochemical characterization and in vitro hemolysis evaluation of silver nanoparticles, Toxicol. Sci. 123 (2011) 133–143. [17] M.J. Ki, S. Shin, Toxic effects of silver nanoparticles and nanowires on erythrocyte rheology, Food Chem. Toxicol. 67 (2014) 80–86. [18] T. Dai, X. Yang, Y. Lu, Controlled growth of polypyrrole nanotubule/wire in the presence of a cationic surfactant, Nanotechnology 17 (2006) 3028–3034. [19] M.T. Yilmaz, Minimum inhibitory and minimum bactericidal concentrations of boron compounds against several bacterial strains, Turk. J. Med. Sci. 12 (2012) 1423–1429. [20] Q.Y. Zhu, R.R. Holt, S.A. Lazarus, T.J. Orozco, C.L. Keen, Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte haemolysis, Exp. Biol. Med. 227 (2002) 321–329. [21] P. Boomi, H.G. Prabu, J. Mathiyarasu, Synthesis and characterization of polyaniline/Ag-Pt nanocomposite for improved antibacterial activity, Colloids Surf. B: Biointerfaces 103 (2013) 9–14. [22] P.-G. Su, L.-N. Huang, Humidity sensors based on TiO2 nanoparticles/polypyrrole composite thin films, Sens. Actuators B 123 (2007) 501–507. [23] D. Zhang, X. Zhang, Y. Chen, P. Yu, C. Wang, Y. Ma, Enhanced capacitance and rate capability of graphene/polypyrrole composite as electrode material for supercapacitors, J. Power Sources 196 (2011) 5990–5996. [24] J. Upadhyay, A. Kumar, Engineering polypyrrole nanotubes by 100 MeV Si9+ ion beam irradiation: enhancement of antioxidant activity, Mater. Sci. Eng. C 33 (2013) 4900–4904. [25] P. Xu, X. Han, C. Wang, B. Zhang, X. Wang, H.-L. Wang, Facile synthesis of polyaniline-polypyrrole nanofibers for application in chemical deposition of metal nanoparticles, Macromol. Rapid Commun. 29 (2008) 1392–1397. [26] H. Zhang, X. Zhong, J.-J. Xu, H.-Y. Chen, Fe3O4/polypyrrole/Au nanocomposites with core/shell/shell structure: synthesis, characterization, and their electrochemical properties, Langmuir 24 (2008) 13748–13752. [27] J. Upadhyay, A. Kumar, Investigation of structural, thermal and dielectric properties of polypyrrole nanotubes tailoring with silver nanoparticles, Compos. Sci. Technol. 97 (2014) 55–62. [28] S. Pal, Y.K. Tak, J.M. Song, Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli, Appl. Environ. Microbiol. 73 (2007) 1712–1720. [29] V. Sambhy, M.M. MacBride, B.R. Peterson, A. Sen, Silver bromide nanoparticle/polymer composites: dual action tunable antimicrobial materials, J. Am. Chem. Soc. 128 (2006) 9798–9808. [30] H. Kong, J. Jang, Antibacterial properties of novel poly(methyl methacrylate) nanofiber containing silver nanoparticles, Langmuir 24 (2008) 2051–2056. [31] M. Ignatova, D. Labaye, S. Lenoir, D. Strivay, R. Jerome, C. Jerome, Immobilization of silver in polypyrrole/polyanion composite coatings: preparation, characterization, and antibacterial activity, Langmuir 19 (2003) 8971–8979. [32] J.R. Morones-Ramirez, J.A. Winkler, C.S. Spina, J.J. Collins, Silver enhances antibiotic activity against Gram-negative bacteria, Sci. Transl. Med. 190 (2013) 190ra81. [33] A. Varesano, C. Vineis, A. Aluigi, F. Rombaldoni, C. Tonetti, G. Mazzuchetti, Antibacterial efficacy of polypyrrole in textile applications, Fibers Polym. 14 (2013) 36–42. [34] I. Wiegand, K. Hilpert, R.E. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175. [35] P.V. Asharani, S. Sethu, S. Vadukumpully, S. Zhong, C.T. Lim, M.P. Hande, S. Valiyaveettil, Investigations on the structural damage in human erythrocytes exposed to silver, gold, and platinum nanoparticles, Adv. Funct. Mater. 20 (2010) 1233–1242. [36] D. Drescher, T. Büchner, D. McNaughton, J. Kneipp, SERS reveals the specific interaction of silver and gold nanoparticles with hemoglobin and red blood cell components, Phys. Chem. Chem. Phys. 15 (2013) 5364–5373.
