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1.  Synthesis, optimization, and characterization of silver nanoparticles from Acinetobacter calcoaceticus and their enhanced antibacterial activity when combined with antibiotics 
Background
The development of nontoxic methods of synthesizing nanoparticles is a major step in nanotechnology to allow their application in nanomedicine. The present study aims to biosynthesize silver nanoparticles (AgNPs) using a cell-free extract of Acinetobacter spp. and evaluate their antibacterial activity.
Methods
Eighteen strains of Acinetobacter were screened for AgNP synthesis. AgNPs were characterized using various techniques. Reaction parameters were optimized, and their effect on the morphology of AgNPs was studied. The synergistic potential of AgNPs on 14 antibiotics against seven pathogens was determined by disc-diffusion, broth-microdilution, and minimum bactericidal concentration assays. The efficacy of AgNPs was evaluated as per the minimum inhibitory concentration (MIC) breakpoints of the Clinical and Laboratory Standards Institute (CLSI) guidelines.
Results
Only A. calcoaceticus LRVP54 produced AgNPs within 24 hours. Monodisperse spherical nanoparticles of 8–12 nm were obtained with 0.7 mM silver nitrate at 70°C. During optimization, a blue-shift in ultraviolet-visible spectra was seen. X-ray diffraction data and lattice fringes (d =0.23 nm) observed under high-resolution transmission electron microscope confirmed the crystallinity of AgNPs. These AgNPs were found to be more effective against Gram-negative compared with Gram-positive microorganisms. Overall, AgNPs showed the highest synergy with vancomycin in the disc-diffusion assay. For Enterobacter aerogenes, a 3.8-fold increase in inhibition zone area was observed after the addition of AgNPs with vancomycin. Reduction in MIC and minimum bactericidal concentration was observed on exposure of AgNPs with antibiotics. Interestingly, multidrug-resistant A. baumannii was highly sensitized in the presence of AgNPs and became susceptible to antibiotics except cephalosporins. Similarly, the vancomycin-resistant strain of Streptococcus mutans was also found to be susceptible to antibiotic treatment when AgNPs were added. These biogenic AgNPs showed significant synergistic activity on the β-lactam class of antibiotics.
Conclusion
This is the first report of synthesis of AgNPs using A. calcoaceticus LRVP54 and their significant synergistic activity with antibiotics resulting in increased susceptibility of multidrug-resistant bacteria evaluated as per MIC breakpoints of the CLSI standard.
doi:10.2147/IJN.S48913
PMCID: PMC3826770  PMID: 24235826
Ag nanoparticles; lattice fringes; disc-diffusion; minimum inhibitory concentration; synergistic activity
2.  Biosynthesis of silver nanoparticles using Acacia leucophloea extract and their antibacterial activity 
The immense potential of nanobiotechnology makes it an intensely researched field in modern medicine. Green nanomaterial synthesis techniques for medicinal applications are desired because of their biocompatibility and lack of toxic byproducts. We report the toxic byproducts free phytosynthesis of stable silver nanoparticles (AgNPs) using the bark extract of the traditional medicinal plant Acacia leucophloea (Fabaceae). Visual observation, ultraviolet–visible spectroscopy, and transmission electron microscopy (TEM) were used to characterize the synthesized AgNPs. The visible yellow-brown color formation and surface plasmon resonance at 440 nm indicates the biosynthesis of AgNP. The TEM images show polydisperse, mostly spherical AgNP particles of 17–29 nm. Fourier transform infrared spectroscopy revealed that primary amines, aldehyde/ketone, aromatic, azo, and nitro compounds of the A. leucophloea extract may participate in the bioreduction and capping of the formed AgNPs. X-ray diffraction confirmed the crystallinity of the AgNPs. The in vitro agar well diffusion method confirmed the potential antibacterial activity of the plant extract and synthesized AgNPs against the common bacterial pathogens Staphylococcus aureus (MTCC 737), Bacillus cereus (MTCC 1272), Listeria monocytogenes (MTCC 657), and Shigella flexneri (MTCC 1475). This research combines the inherent antimicrobial activity of silver metals with the A. leucophloea extract, yielding antibacterial activity-enhanced AgNPs. This new biomimetic approach using traditional medicinal plant (A. leucophloea) barks to synthesize biocompatible antibacterial AgNPs could easily be scaled up for additional biomedical applications. These polydisperse AgNPs green-synthesized via A. leucophloea bark extract can readily be used in many applications not requiring high uniformity in particle size or shape.
doi:10.2147/IJN.S61779
PMCID: PMC4035312  PMID: 24876776
AgNPs; antibacterial activity; Acacia leucophloea; biogenic; FTIR; XRD; TEM
3.  Enhanced antibacterial and anti-biofilm activities of silver nanoparticles against Gram-negative and Gram-positive bacteria 
Nanoscale Research Letters  2014;9(1):373.
Silver nanoparticles (AgNPs) have been used as antibacterial, antifungal, antiviral, anti-inflammtory, and antiangiogenic due to its unique properties such as physical, chemical, and biological properties. The present study was aimed to investigate antibacterial and anti-biofilm activities of silver nanoparticles alone and in combination with conventional antibiotics against various human pathogenic bacteria. Here, we show that a simple, reliable, cost effective and green method for the synthesis of AgNPs by treating silver ions with leaf extract of Allophylus cobbe. The A. cobbe-mediated synthesis of AgNPs (AgNPs) was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Furthermore, the antibacterial and anti-biofilm activity of antibiotics or AgNPs, or combinations of AgNPs with an antibiotic was evaluated using a series of assays: such as in vitro killing assay, disc diffusion assay, biofilm inhibition, and reactive oxygen species generation in Pseudomonas aeruginosa, Shigella flexneri, Staphylococcus aureus, and Streptococcus pneumonia. The results suggest that, in combination with antibiotics, there were significant antimicrobial and anti-biofilm effects at lowest concentration of AgNPs using a novel plant extract of A. cobbe, otherwise sublethal concentrations of the antibiotics. The significant enhancing effects were observed for ampicillin and vancomycin against Gram-negative and Gram-positive bacteria, respectively. These data suggest that combining antibiotics and biogenic AgNPs can be used therapeutically for the treatment of infectious diseases caused by bacteria. This study presented evidence of antibacterial and anti-biofilm effects of A. cobbe-mediated synthesis of AgNPs and their enhanced capacity against various human pathogenic bacteria. These results suggest that AgNPs could be used as an adjuvant for the treatment of infectious diseases.
doi:10.1186/1556-276X-9-373
PMCID: PMC4127560  PMID: 25136281
Allophylus cobbe; Antibacterial activity; Anti-biofilm activity; Antibiotics; Silver nanoparticles; Sublethal concentrations
4.  Potential Theranostics Application of Bio-Synthesized Silver Nanoparticles (4-in-1 System) 
Theranostics  2014;4(3):316-335.
In this report, we have designed a simple and efficient green chemistry approach for the synthesis of colloidal silver nanoparticles (b-AgNPs) that is formed by the reduction of silver nitrate (AgNO3) solution using Olax scandens leaf extract. The colloidal b-AgNPs, characterized by various physico-chemical techniques exhibit multifunctional biological activities (4-in-1 system). Firstly, bio-synthesized silver nanoparticles (b-AgNPs) shows enhanced antibacterial activity compared to chemically synthesize silver nanoparticles (c-AgNPs). Secondly, b-AgNPs show anti-cancer activities to different cancer cells (A549: human lung cancer cell lines, B16: mouse melanoma cell line & MCF7: human breast cancer cells) (anti-cancer). Thirdly, these nanoparticles are biocompatible to rat cardiomyoblast normal cell line (H9C2), human umbilical vein endothelial cells (HUVEC) and Chinese hamster ovary cells (CHO) which indicates the future application of b-AgNPs as drug delivery vehicle. Finally, the bio-synthesized AgNPs show bright red fluorescence inside the cells that could be utilized to detect the localization of drug molecules inside the cancer cells (a diagnostic approach). All results together demonstrate the multifunctional biological activities of bio-synthesized AgNPs (4-in-1 system) that could be applied as (i) anti-bacterial & (ii) anti-cancer agent, (iii) drug delivery vehicle, and (iv) imaging facilitator. To the best of our knowledge, there is not a single report of biosynthesized AgNPs that demonstrates the versatile applications (4-in-1 system) towards various biomedical applications. Additionally, a plausible mechanistic approach has been explored for the synthesis of b-AgNPs and its anti-bacterial as well as anti-cancer activity. We strongly believe that bio-synthesized AgNPs will open a new direction towards various biomedical applications in near future.
doi:10.7150/thno.7819
PMCID: PMC3915094  PMID: 24505239
Bio-synthesis; Silver nanoparticle; Green Chemistry; Olax scandens; Multifunctional activities; Antibacterial; anti-cancer.
5.  Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria 
Biological synthesis of nanoparticles is a growing innovative approach that is relatively cheaper and more environmentally friendly than current physicochemical processes. Among various microorganisms, fungi have been found to be comparatively more efficient in the synthesis of nanomaterials. In this research work, extracellular mycosynthesis of silver nanoparticles (AgNPs) was probed by reacting the precursor salt of silver nitrate (AgNO3) with culture filtrate of Aspergillus flavus. Initially, the mycosynthesis was regularly monitored by ultraviolet-visible spectroscopy, which showed AgNP peaks of around 400–470 nm. X-ray diffraction spectra revealed peaks of different intensities with respect to angle of diffractions (2θ) corresponding to varying configurations of AgNPs. Transmission electron micrographs further confirmed the formation of AgNPs in size ranging from 5–30 nm. Combined and individual antibacterial activities of the five conventional antibiotics and AgNPs were investigated against eight different multidrug-resistant bacterial species using the Kirby–Bauer disk-diffusion method. The decreasing order of antibacterial activity (zone of inhibition in mm) of antibiotics, AgNPs, and their conjugates against bacterial group (average) was; ciprofloxacin + AgNPs (23) . imipenem + AgNPs (21) > gentamycin + AgNPs (19) > vancomycin + AgNPs (16) > AgNPs (15) . imipenem (14) > trimethoprim + AgNPs (14) > ciprofloxacin (13) > gentamycin (11) > vancomycin (4) > trimethoprim (0). Overall, the synergistic effect of antibiotics and nanoparticles resulted in a 0.2–7.0 (average, 2.8) fold-area increase in antibacterial activity, which clearly revealed that nanoparticles can be effectively used in combination with antibiotics in order to improve their efficacy against various pathogenic microbes.
doi:10.2147/IJN.S49284
PMCID: PMC3754765  PMID: 23986635
mycosynthesis; silver nanoparticles; antibacterial agents; Aspergillus flavus
6.  Phytoextracts-Synthesized Silver Nanoparticles Inhibit Bacterial Fish Pathogen Aeromonas hydrophila 
Indian Journal of Microbiology  2013;53(4):438-446.
Fish disease is a major stumbling block towards sustainable growth of the fisheries sector. Aeromonas hydrophila, which is a major infectious aquatic pathogen is reportedly the causative agent of ulcers, fin-rot, tail-rot, hemorrhagic septicemia in fish, and has reportedly developed resistance against many of the available antibiotics. In this context, the inhibitory function of silver nanoparticles (AgNPs) against A. hydrophila was studied to evaluate its possible application in aquaculture as alternative to antibiotics. AgNPs were synthesized using the leaf extracts of subtropical plants Mangifera indica (Mango), Eucalyptus terticornis (Eucalyptus), Carica papaya (Papaya) and Musa paradisiaca (Banana). The absorbance maxima, size range and shape of the AgNPs as characterized by the UV–Vis spectroscopy, high resolution transmission electron microscopy (HR-TEM), and energy dispersive X-ray spectroscopy (EDX) were, Mangifera—442, 50–65 nm, ovular; Eucalyptus—465, 60–150 nm, oval; Carica—442, 25–40 nm, round, irregular; and Musa—454, 10–50 nm, round, irregular, respectively. Well-diffusion of these AgNPs for their antimicrobial characteristics exhibited that, the papaya leaf extract synthesized AgNPs had maximum antimicrobial activity at 153.6 μg/ml concentrations, and that from the eucalyptus leaves was least effective. As observed, the potency of the nanoparticles enhanced with the decrease in particle size, from 60–150 nm in eucalyptus to 25–40 nm in papaya. Due to its purely natural sourcing, phytosynthesized AgNPs can be applied as alternative to antibiotics and other biocides as a cost-effective and eco-friendly therapeutic agent against A. hydrophila stimulated diseases in aquatic animals.
doi:10.1007/s12088-013-0409-9
PMCID: PMC3779295  PMID: 24426148
Aeromonas hydrophila; Silver nanoparticle; Phytosynthesis; Anti-microbial activity
7.  Enhanced stability and antibacterial efficacy of a traditional Chinese medicine-mediated silver nanoparticle delivery system 
Silver nanoparticles (AgNPs) are widely used as antibacterial products in various fields. Recent studies have suggested that AgNPs need an appropriate stabilizer to improve their stability. Some antibacterial traditional Chinese medicines (TCMs) contain various reductive components, which can not only stabilize AgNPs but also enhance their antimicrobial activity. In this study, we developed a series of novel AgNPs using a TCM extract as a stabilizer, reducing agent, and antimicrobial agent (TCM-AgNPs). A storage stability investigation of the TCM-AgNPs suggested a significant improvement when compared with bare AgNPs. Further, conjugation of TCMs onto the AgNP surface resulted in stronger antimicrobial potency on antibacterial evaluation using Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus with minimum inhibitory concentration 50% (MIC50) ratios (and minimum bactericidal concentration 90% [MBC90] ratios) of AgNPs to respective TCM-AgNPs as assessment indices. Among these, P. cuspidatum Sieb. et-conjugated AgNPs (P.C.-AgNPs) had the advantage of a combination of TCMs and AgNPs and was studied in detail with regard to its synthesis and characterization. The extraction time, reaction temperature, and concentrations of AgNO3 and Polygonum cuspidatum Sieb. et extract were critical factors in the preparation of P.C.-AgNPs. Further, the results of X-ray diffraction and Fourier transform infrared spectroscopy indicated successful preparation of P.C.-AgNPs. In representative studies, P.C.-AgNPs showed a well-defined spherical shape, a homogeneous small particle size (36.78 nm), a narrow polydispersity index (0.105), and a highly negative zeta potential (−23.6 mV) on transmission electron microscopy and dynamic light scattering. These results indicate that TCM-AgNPs have a potential role as antibacterial agents in the clinic setting.
doi:10.2147/IJN.S71670
PMCID: PMC4251751  PMID: 25473286
traditional Chinese medicine; extract; silver nanoparticles; stability; antibacterial; Polygonum cuspidatum Sieb. et
8.  Facile method for the synthesis of silver nanoparticles using 3-hydrazino-isatin derivatives in aqueous methanol and their antibacterial activity 
Introduction
A new method for preparation of silver nanoparticles (AgNPs) based on using hydrazino-isatin derivatives in an aqueous methanol reaction medium is reported here. AgNPs were prepared using silver nitrate solubilized in a water core as the source of silver ions and 3-hydrazino-isatin derivatives (3-hydrazino-isatin [IsH] and 1-benzyl-3-hydrazino-isatin [BIsH]) solubilized in methanol core as a reducing agent. The proposed method is effective, rapid, and convenient. X-ray diffraction (XRD), energy dispersive X-ray analysis, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for characterization of the AgNPs. The TEM micrographs confirmed that the nanopowders consist of well-dispersed agglomerates of grains with a narrow size distribution of 18–21 nm and 17–20 nm. The AgNPs, as well as BIsH, showed high antimicrobial and bactericidal activity against the Gram-positive Bacillus subtilis and Gram-negative Micrococcus luteus and Proteus vulgaris, as well as antifungal activities against Saccharomyces cerevisiae. On the other hand, they were not effective against the Gram-negative Escherichia coli.
Purpose
A simple, effective, rapid, and convenient chemical reduction method for the synthesis of AgNPs in an aqueous methanol reaction medium using hydrazino-isatin derivatives and studying their antibacterial effect.
Results
IsH and BIsH are remarkably powerful reductants for Ag+ ions in an aqueous methanol medium, which could be considered as a simple chemical reduction method for formation of AgNPs. The AgNP formation depends on the solubility of the hydrazino-isatin derivatives. BIsH gave more AgNPs than IsH, as observed from XRD. The formation of AgNPs is attributed to the adsorption of hydrazine derivatives and/or interparticle interaction on the surface of AgNP through electrostatic interactions between the lone pair electrons of the hydrazino group (C=N-NH2) and the positive surface of AgNPs. AgNPs and BIsH showed high antimicrobial and bacterial activity.
Conclusion
In summary, it is shown that IsH and BIsH are remarkably powerful reductants for Ag+ ions in an aqueous methanol medium. BIsH gave more AgNPs than IsH, as observed from XRD due to better solubility of the BIsH than IsH in aqueous-methanol. The formation of AgNPs is attributed to the adsorption of hydrazine derivatives and/or interparticle interaction on the surface of AgNPs through electrostatic interactions between the lone pair electrons of the hydrazino group (C=N-NH2) and the positive surface of AgNPs. The AgNps as well as BIsH ligand showed high antimicrobial and bactericidal activity.
doi:10.2147/IJN.S58571
PMCID: PMC3949700  PMID: 24623975
silver nanoparticles; 1-benzyl-3-hydrazinoisatin; SEM; TEM; antimicrobial
9.  In vivo synthesis of nanomaterials in plants: location of silver nanoparticles and plant metabolism 
Nanoscale Research Letters  2014;9(1):101.
Metallic nanoparticles (MeNPs) can be formed in living plants by reduction of the metal ions absorbed as soluble salts. It is very likely that plant metabolism has an important role in MeNP biosynthesis. The in vivo formation of silver nanoparticles (AgNPs) was observed in Brassica juncea, Festuca rubra and Medicago sativa. Plants were grown in Hoagland's solution for 30 days and then exposed for 24 h to a solution of 1,000 ppm AgNO3. In the leaf extracts of control plants, the concentrations of glucose, fructose, ascorbic acid, citric acid and total polyphenols were determined. Total Ag content in plant fractions was determined by inductively coupled plasma atomic emission spectroscopy. Despite the short exposure time, the Ag uptake and translocation to plant leaves was very high, reaching 6,156 and 2,459 mg kg−1 in B. juncea and F. rubra, respectively. Ultrastructural analysis was performed by transmission electron microscopy (TEM), and AgNPs were detected by TEM X-ray microanalysis. TEM images of plant fractions showed the in vivo formation of AgNPs in the roots, stems and leaves of the plants. In the roots, AgNPs were present in the cortical parenchymal cells, on the cell wall of the xylem vessels and in regions corresponding to the pits. In leaf tissues, AgNPs of different sizes and shapes were located close to the cell wall, as well as in the cytoplasm and within chloroplasts. AgNPs were not observed in the phloem of the three plant species. This is the first report of AgNP synthesis in living plants of F. rubra. The contents of reducing sugars and antioxidant compounds, proposed as being involved in the biosynthesis of AgNPs, were quite different between the species, thus suggesting that it is unlikely that a single substance is responsible for this process.
MSC 2010
92 Biology and other natural sciences; 92Cxx Physiological, cellular and medical topics; 92C80 Plant biology
doi:10.1186/1556-276X-9-101
PMCID: PMC3973990  PMID: 24581279
Festuca rubra; Medicago sativa; Brassica juncea; Silver; Nanoparticles; Biosynthesis; Plant metabolites
10.  Green Synthesis of Silver Nanoparticles Using Extract of Oak Fruit Hull (Jaft): Synthesis and In Vitro Cytotoxic Effect on MCF-7 Cells 
A green synthetic approach by using oak fruit hull (Jaft) extract for preparation of silver nanoparticles (AgNPs) was developed and optimized. Parameters affecting the synthesis of AgNPs, such as temperature, extract pH, and concentration of extract (ratio of plant sample to extraction solvent), were investigated and optimized. Optimum conditions for the synthesis of silver nanoparticles are as follows: Ag+ concentration, 1 mM; extract concentration, 40 g/L (4% w/v); pH = 9 and temperature, 45°C. Biosynthesized silver nanoparticles were characterized using UV-visible absorption spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). TEM and DLS analyses have shown the synthesized AgNPs were predominantly spherical in shape with an average size of 40 nm. The cytotoxic activity of the synthesized AgNPs and Jaft extract containing AgNPs against human breast cancer cell (MCF-7) was investigated and the half maximal inhibitory concentrations (IC50) were found to be 50 and 0.04 μg/mL at 24 h incubation, respectively. This eco-friendly and cost-effective synthesis method can be potentially used for large-scale production of silver nanoparticles.
doi:10.1155/2015/846743
PMCID: PMC4313055
11.  Green synthesis of silver nanoparticles by Chrysanthemum morifolium Ramat. extract and their application in clinical ultrasound gel 
Eco-friendly green synthesis with plant extracts plays a very important role in nanotechnology, without any harmful chemicals. In this report, the synthesis of water-soluble silver nanoparticles was developed by treating silver ions with Chrysanthemum morifolium Ramat. extract at room temperature. The effect of the extract on the formation of silver nanoparticles was characterized by ultraviolet and visible absorption spectroscopy, X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The ultraviolet and visible absorption spectroscopy results show a strong resonance centered on the surface of silver nanoparticles (AgNP) at 430 nm. The Fourier transform infrared spectroscopy spectral study demonstrates Chrysanthemum morifolium Ramat. extract acted as the reducing and stabilizing agent during the synthesis. The X-ray diffraction analysis confirmed that the synthesized AgNP are single crystallines, corresponding with the result of transmission electron microscopy. Water-soluble AgNP, with an approximate size of 20 nm–50 nm were also observed in the transmission electron microscopy image. The bactericidal properties of the synthesized AgNP were investigated using the agar-dilution method and the growth-inhibition test. The results show the AgNP had potent bactericidal activity on Staphylococcus aureus and Escherichia coli, as well as a strong antibacterial activity against gram-negative bacteria, as compared to gram-positive bacteria with a dose-dependent effect, thus providing a clinical ultrasound gel with bactericidal property for prevention of cross infections.
doi:10.2147/IJN.S43289
PMCID: PMC3653761  PMID: 23687447
silver nanoparticles; green synthesis; Chrysanthemum morifolium Ramat.; bactericidal activity; ultrasound gel
12.  Biofabricated Silver Nanoparticles Act as a Strong Fungicide against Bipolaris sorokiniana Causing Spot Blotch Disease in Wheat 
PLoS ONE  2014;9(5):e97881.
The present study is focused on the extracellular synthesis of silver nanoparticles (AgNPs) using culture supernatant of an agriculturally important bacterium, Serratia sp. BHU-S4 and demonstrates its effective application for the management of spot blotch disease in wheat. The biosynthesis of AgNPs by Serratia sp. BHU-S4 (denoted as bsAgNPs) was monitored by UV–visible spectrum that showed the surface plasmon resonance (SPR) peak at 410 nm, an important characteristic of AgNPs. Furthermore, the structural, morphological, elemental, functional and thermal characterization of bsAgNPs was carried out using the X-ray diffraction (XRD), electron and atomic microscopies, energy dispersive X-ray (EDAX) spectrometer, FTIR spectroscopy and thermogravimetric analyzer (TGA), respectively. The bsAgNPs were spherical in shape with size range of ∼10 to 20 nm. The XRD and EDAX analysis confirmed successful biosynthesis and crystalline nature of AgNPs. The bsAgNPs exhibited strong antifungal activity against Bipolaris sorokiniana, the spot blotch pathogen of wheat. Interestingly, 2, 4 and 10 µg/ml concentrations of bsAgNPs accounted for complete inhibition of conidial germination, whereas in the absence of bsAgNPs, conidial germination was 100%. A detached leaf bioassay revealed prominent conidial germination on wheat leaves infected with B. sorokiniana conidial suspension alone, while the germination of conidia was totally inhibited when the leaves were treated with bsAgNPs. The results were further authenticated under green house conditions, where application of bsAgNPs significantly reduced B. sorokiniana infection in wheat plants. Histochemical staining revealed a significant role of bsAgNPs treatment in inducing lignin deposition in vascular bundles. In summary, our findings represent the efficient application of bsAgNPs in plant disease management, indicating the exciting possibilities of nanofungicide employing agriculturally important bacteria.
doi:10.1371/journal.pone.0097881
PMCID: PMC4026416  PMID: 24840186
13.  Green Synthesis of Silver Nanoparticles, Their Characterization, Application and Antibacterial Activity † 
Our research focused on the production, characterization and application of silver nanoparticles (AgNPs), which can be utilized in biomedical research and environmental cleaning applications. We used an environmentally friendly extracellular biosynthetic technique for the production of the AgNPs. The reducing agents used to produce the nanoparticles were from aqueous extracts made from the leaves of various plants. Synthesis of colloidal AgNPs was monitored by UV-Visible spectroscopy. The UV-Visible spectrum showed a peak between 417 and 425 nm corresponding to the Plasmon absorbance of the AgNPs. The characterization of the AgNPs such as their size and shape was performed by Atom Force Microscopy (AFM), and Transmission Electron Microscopy (TEM) techniques which indicated a size range of 3 to 15 nm. The anti-bacterial activity of AgNPs was investigated at concentrations between 2 and 15 ppm for Gram-negative and Gram-positive bacteria. Staphylococcus aureus and Kocuria rhizophila, Bacillus thuringiensis (Gram-positive organisms); Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium (Gram-negative organisms) were exposed to AgNPs using Bioscreen C. The results indicated that AgNPs at a concentration of 2 and 4 ppm, inhibited bacterial growth. Preliminary evaluation of cytotoxicity of biosynthesized silver nanoparticles was accomplished using the InQ™ Cell Research System instrument with HEK 293 cells. This investigation demonstrated that silver nanoparticles with a concentration of 2 ppm and 4 ppm were not toxic for human healthy cells, but inhibit bacterial growth.
doi:10.3390/ijerph10105221
PMCID: PMC3823307  PMID: 24157517
biosynthesis; silver nanoparticles; plant extracts; cytotoxicity bacteria; antibacterial activity
14.  Study on antibacterial activity of silver nanoparticles synthesized by gamma irradiation method using different stabilizers 
Nanoscale Research Letters  2014;9(1):162.
Colloidal solutions of silver nanoparticles (AgNPs) were synthesized by gamma Co-60 irradiation using different stabilizers, namely polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), alginate, and sericin. The particle size measured from TEM images was 4.3, 6.1, 7.6, and 10.2 nm for AgNPs/PVP, AgNPs/PVA, AgNPs/alginate, and AgNPs/sericin, respectively. The influence of different stabilizers on the antibacterial activity of AgNPs was investigated. Results showed that AgNPs/alginate exhibited the highest antibacterial activity against Escherichia coli (E. coli) among the as-synthesized AgNPs. Handwash solution has been prepared using Na lauryl sulfate as surfactant, hydroxyethyl cellulose as binder, and 15 mg/L of AgNPs/alginate as antimicrobial agent. The obtained results on the antibacterial test of handwash for the dilution to 3 mg AgNPs/L showed that the antibacterial efficiency against E. coli was of 74.6%, 89.8%, and 99.0% for the contacted time of 1, 3, and 5 min, respectively. Thus, due to the biocompatibility of alginate extracted from seaweed and highly antimicrobial activity of AgNPs synthesized by gamma Co-60 irradiation, AgNPs/alginate is promising to use as an antimicrobial agent in biomedicine, cosmetic, and in other fields.
doi:10.1186/1556-276X-9-162
PMCID: PMC3977955  PMID: 24708872
Silver nanoparticles; Gamma irradiation; Antibacterial; E. coli; Handwash
15.  Gene expression profiles in primary duodenal chick cells following transfection with avian influenza virus H5 DNA plasmid encapsulated in silver nanoparticles 
In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV) that induced cytokine expression, the hemagglutinin (H5) gene of AIV, A/Ck/Malaysia/5858/04 (H5N1) and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5) and formulated using green synthesis of silver nanoparticles (AgNPs) with poly(ethylene glycol) and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL)-18, IL-15, and IL-12â.
doi:10.2147/IJN.S39074
PMCID: PMC3582479  PMID: 23459681
silver nanoparticles; avian influenza; hemagglutinin; transfection; primary cells
16.  Silver Nanoparticles Compromise Neurodevelopment in PC12 Cells: Critical Contributions of Silver Ion, Particle Size, Coating, and Composition 
Background
Silver exposures are rising because of the increased use of silver nanoparticles (AgNPs) in consumer products. The monovalent silver ion (Ag+) impairs neurodevelopment in PC12 cells and zebrafish.
Objectives and methods
We compared the effects of AgNPs with Ag+ in PC12 cells for neurodevelopmental end points including cell replication, oxidative stress, cell viability, and differentiation. First, we compared citrate-coated AgNPs (AgNP-Cs) with Ag+, and then we assessed the roles of particle size, coating, and composition by comparing AgNP-C with two different sizes of polyvinylpyrrolidone-coated AgNPs (AgNP-PVPs) or silica nanoparticles.
Results
In undifferentiated cells, AgNP-C impaired DNA synthesis, but to a lesser extent than an equivalent nominal concentration of Ag+, whereas AgNP-C and Ag+ were equally effective against protein synthesis; there was little or no oxidative stress or loss of viability due to AgNP-C. In contrast, in differentiating cells, AgNP-C evoked robust oxidative stress and impaired differentiation into the acetylcholine phenotype. Although the effects of AgNP-PVP showed similarities to those of AgNP-C, we also found significant differences in potencies and differentiation outcomes that depended both on particle size and coating. None of the effects reflected simple physical attributes of nanoparticles, separate from composition or coating, as equivalent concentrations of silica nanoparticles had no detectable effects.
Conclusions
AgNP exposure impairs neurodevelopment in PC12 cells. Further, AgNP effects are distinct from those of Ag+ alone and depend on size and coating, indicating that AgNP effects are not due simply to the release of Ag+ into the surrounding environment.
doi:10.1289/ehp.1002337
PMCID: PMC3018497  PMID: 20840908
acetylcholine; developmental neurotoxicity; dopamine; in vitro; metal neurotoxicity; nanoparticles; PC12 cells; silver
17.  Synthesis and in vitro antineoplastic evaluation of silver nanoparticles mediated by Agrimoniae herba extract 
A rapid synthesis of silver nanoparticles (AgNPs) using Agrimoniae herba extract as reducing agent and stabilizer (A. herba-conjugated AgNPs [AH-AgNPs]) were designed, characterized, and evaluated for antitumor therapy feasibility. In this study, critical factors in the preparation of silver nanoparticles, including extraction time, reaction temperature, the concentration of AgNO3, and A. herba extract amount, were investigated using ultraviolet-visible spectroscopy. AH-AgNPs with well-defined spherical shape, homogeneous distributional small size (30.34 nm), narrow polydispersity index (0.142), and high negative zeta potential (−36.8 mV) were observed by transmission electron microscopy and dynamic light scattering. Furthermore, the results of X-ray diffraction and Fourier-transform infrared spectroscopy further indicated successful preparation of AH-AgNPs. Acceptable long-term storage stability of AH-AgNPs was also confirmed. More importantly, AH-AgNPs displayed significantly higher antiproliferative effect against a human lung carcinoma cell line (A549 cells) compared with A. herba extract and bare AgNPs prepared by sodium citrate. The half-maximal inhibitory concentrations of AH-AgNPs, bare AgNPs, and A. herba extract were 38.13 μg · mL−1, 184.87 μg · mL−1, and 1.147 × 104 μg · mL−1, respectively. It is suggested that AH-AgNPs exhibit a strong antineoplastic effect on A549 cells, pointing to feasibility of antitumor treatment in the future.
doi:10.2147/IJN.S58732
PMCID: PMC3998862  PMID: 24790429
rapid synthesis; Agrimoniae herba extract; silver nanoparticles; A549 cells; antitumor
18.  Growth Kinetics and Mechanistic Action of Reactive Oxygen Species Released by Silver Nanoparticles from Aspergillus niger on Escherichia coli 
BioMed Research International  2014;2014:753419.
Silver Nanoparticles (AgNPs), the real silver bullet, are known to have good antibacterial properties against pathogenic microorganisms. In the present study AgNPs were prepared from extracellular filtrate of Aspergillus niger. Characterization of AgNPs by UV-Vis spectrum reveals specific surface plasmon resonance at peak 416 nm; TEM photographs revealed the size of the AgNPs to be 20–55 nm. Average diameter of the produced AgNPs was found to be 73 nm with a zeta potential that was −24 mV using Malvern Zetasizer. SEM micrographs showed AgNPs to be spherical with smooth morphology. EDS revealed the presence of pure metallic AgNPs along with carbon and oxygen signatures. Of the different concentrations (0, 2.5, 5, 10, and 15 μg/mL) used 10 μg/mL were sufficient to inhibit 107 CFU/mL of E. coli. ROS production was measured using DCFH-DA method and the the free radical generation effect of AgNPs on bacterial growth inhibition was investigated by ESR spectroscopy. This paper not only deals with the damage inflicted on microorganisms by AgNPs but also induces cell death through the production of ROS released by AgNPs and also growth kinetics of E. coli supplemented with AgNPs produced by A. niger.
doi:10.1155/2014/753419
PMCID: PMC4083831  PMID: 25028666
19.  Optimization and Characterization of Silver Nanoparticle by Endophytic Fungi Penicillium sp. Isolated from Curcuma longa (Turmeric) and Application Studies against MDR E. coli and S. aureus 
Development of ecofriendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest in nanotechnology because of its tremendous impetus in modulating metals into nanosize to their potential use for human benefits. In this study an endophytic fungus, Penicillium sp., isolated from healthy leaves of Curcuma longa (turmeric) was subjected to extracellular biosynthesis of silver nanoparticles (AgNps) and their activity against MDR E. coli and S. aureus. The biosynthesized AgNps optimization was studied and characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Then produced AgNps were tested against MDR E. coli and S. aureus. The endophytic fungus Penicillium sp. from healthy leaves of C. longa (turmeric) was found to be a good producer of AgNps. Parametric optimization showed maximum absorbance of 420–425 nm at pH-7, 25°C with 1 mM AgNO3 concentration and 15–20 g of wet biomass. Further TEM revealed the formation of spherical, well-dispersed nanoparticles with size ranging between 25 and 30 nm and FTIR shows the bands at 1644 and 1538 cm−1 corresponding to the binding vibrations of amide I and II bands of proteins, respectively. Antibacterial activity against MDR E. coli and S. aureus showed good results showing maximum zone of inhibition of 17 mm and 16 mm, respectively, at 80 µL of AgNps.
doi:10.1155/2014/408021
PMCID: PMC3930180  PMID: 24639625
20.  Green synthesis of silver nanoparticles using Ganoderma neo-japonicum Imazeki: a potential cytotoxic agent against breast cancer cells 
Background
Silver nanoparticles (AgNPs) are an important class of nanomaterial for a wide range of industrial and biomedical applications. AgNPs have been used as antimicrobial and disinfectant agents due their detrimental effect on target cells. The aim of our study was to determine the cytotoxic effects of biologically synthesized AgNPs using hot aqueous extracts of the mycelia of Ganoderma neo-japonicum Imazeki on MDA-MB-231 human breast cancer cells.
Methods
We developed a green method for the synthesis of water-soluble AgNPs by treating silver ions with hot aqueous extract of the mycelia of G. neo-japonicum. The formation of AgNPs was characterized by ultraviolet-visible absorption spectroscopy, X-ray diffraction, dynamic light scattering, and transmission electron microscopy. Furthermore, the toxicity of synthesized AgNPs was evaluated using a series of assays: such as cell viability, lactate dehydrogenase leakage, reactive oxygen species generation, caspase 3, DNA laddering, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling in human breast cancer cells (MDA-MB-231).
Results
The ultraviolet-visible absorption spectroscopy results showed a strong resonance centered on the surface of AgNPs at 420 nm. The X-ray diffraction analysis confirmed that the synthesized AgNPs were single-crystalline, corresponding with the result of transmission electron microscopy. Treatment of MDA-MB-231 breast cancer cells with various concentrations of AgNPs (1–10 μg/mL) for 24 hours revealed that AgNPs could inhibit cell viability and induce membrane leakage in a dose-dependent manner. Cells exposed to AgNPs showed increased reactive oxygen species and hydroxyl radical production. Furthermore, the apoptotic effects of AgNPs were confirmed by activation of caspase 3 and DNA nuclear fragmentation.
Conclusion
The results indicate that AgNPs possess cytotoxic effects with apoptotic features and suggest that the reactive oxygen species generated by AgNPs have a significant role in apoptosis. The present findings suggest that AgNPs could contribute to the development of a suitable anticancer drug, which may lead to the development of a novel nanomedicine for the treatment of cancers.
doi:10.2147/IJN.S51881
PMCID: PMC3833323  PMID: 24265551
AgNPs; Ganoderma neo-japonicum; human breast cancer cells; cytotoxicity; caspase-3 activity; DNA fragmentation
21.  Antibacterial properties of silver nanoparticles in three different sizes and their nanocomposites with a new waterborne polyurethane 
Silver nanoparticles (AgNPs) are strong bactericidal agents but they are also cytotoxic. Embedding them in a polymer matrix may reduce their cytotoxic effect. In the present study, AgNPs in three average sizes were tested for their antibacterial activities and cytotoxicity. Nanocomposites from a new waterborne polyetherurethane (PEU) ionomer and AgNPs were prepared without the use of any crosslinker. It was observed that the antibacterial activity of AgNPs against Escherichia coli started at the effective concentration of 0.1–1 ppm, while that against Staphylococcus aureus started at higher concentrations of 1–10 ppm. Cytotoxicity of AgNPs was observed at the concentration of 10 ppm. AgNPs with smaller average size showed greater antibacterial activity as well as cytotoxicity. The PEU synthesized in this study showed high tensile strength, and the addition of AgNPs at all sizes further increased its thermal stability. The delicate surface features of nanophases, however, were only observed in nanocomposites with either small-or medium-sized AgNPs. PEU-Ag nanocomposites had a strong bacteriostatic effect on the growth of E. coli and S. aureus. The proliferation of endothelial cells on PEU-Ag nanocomposites was enhanced, whereas the platelet adhesion was reduced. The expression of endothelial nitric oxide synthase gene was upregulated on PEU-Ag containing small-sized AgNPs (30 ppm) or medium-sized AgNPs (60 ppm). This effect was not as remarkable in nanocomposites from large-sized AgNPs. Overall, nanocomposites from the PEU and 60 ppm of the medium-sized (5 nm) AgNPs showed the best biocompatibility and antibacterial activity. Addition of smaller or larger AgNPs did not produce as substantial an effect in PEU, especially for the larger AgNPs.
doi:10.2147/IJN.S14572
PMCID: PMC3010153  PMID: 21187943
silver nanoparticles; polyurethane; antibacterial activity; biocompatibility
22.  Fungus-Mediated Green Synthesis of Silver Nanoparticles Using Aspergillus terreus 
The biosynthesis of nanoparticles has received increasing attention due to the growing need to develop safe, cost-effective and environmentally friendly technologies for nano-materials synthesis. In this report, silver nanoparticles (AgNPs) were synthesized using a reduction of aqueous Ag+ ion with the culture supernatants of Aspergillus terreus. The reaction occurred at ambient temperature and in a few hours. The bioreduction of AgNPs was monitored by ultraviolet-visible spectroscopy, and the AgNPs obtained were characterized by transmission electron microscopy and X-ray diffraction. The synthesized AgNPs were polydispersed spherical particles ranging in size from 1 to 20 nm and stabilized in the solution. Reduced nicotinamide adenine dinucleotide (NADH) was found to be an important reducing agent for the biosynthesis, and the formation of AgNPs might be an enzyme-mediated extracellular reaction process. Furthermore, the antimicrobial potential of AgNPs was systematically evaluated. The synthesized AgNPs could efficiently inhibit various pathogenic organisms, including bacteria and fungi. The current research opens a new avenue for the green synthesis of nano-materials.
doi:10.3390/ijms13010466
PMCID: PMC3269698  PMID: 22312264
silver nanoparticles; Aspergillus terreus; biosynthesis; NADH
23.  Biosynthesis, Antimicrobial and Cytotoxic Effect of Silver Nanoparticles Using a Novel Nocardiopsis sp. MBRC-1 
BioMed Research International  2013;2013:287638.
The biosynthesis of nanoparticles has been proposed as a cost effective environmental friendly alternative to chemical and physical methods. Microbial synthesis of nanoparticles is under exploration due to wide biomedical applications, research interest in nanotechnology and microbial biotechnology. In the present study, an ecofriendly process for the synthesis of nanoparticles using a novel Nocardiopsis sp. MBRC-1 has been attempted. We used culture supernatant of Nocardiopsis sp. MBRC-1 for the simple and cost effective green synthesis of silver nanoparticles. The reduction of silver ions occurred when silver nitrate solution was treated with the Nocardiopsis sp. MBRC-1 culture supernatant at room temperature. The nanoparticles were characterized by UV-visible, TEM, FE-SEM, EDX, FTIR, and XRD spectroscopy. The nanoparticles exhibited an absorption peak around 420 nm, a characteristic surface plasmon resonance band of silver nanoparticles. They were spherical in shape with an average particle size of 45 ± 0.15 nm. The EDX analysis showed the presence of elemental silver signal in the synthesized nanoparticles. The FTIR analysis revealed that the protein component in the form of enzyme nitrate reductase produced by the isolate in the culture supernatant may be responsible for reduction and as capping agents. The XRD spectrum showed the characteristic Bragg peaks of 1 2 3, 2 0 4, 0 4 3, 1 4 4, and 3 1 1 facets of the face centered cubic silver nanoparticles and confirms that these nanoparticles are crystalline in nature. The prepared silver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi. Cytotoxicity of biosynthesized AgNPs against in vitro human cervical cancer cell line (HeLa) showed a dose-response activity. IC50 value was found to be 200 μg/mL of AgNPs against HeLa cancer cells. Further studies are needed to elucidate the toxicity and the mechanism involved with antimicrobial and anticancer activity of the synthesized AgNPs as nanomedicine.
doi:10.1155/2013/287638
PMCID: PMC3727093  PMID: 23936787
24.  Biosynthesis and characterization of silver nanoparticles using panchakavya, an Indian traditional farming formulating agent 
Synthesis of silver nanoparticles (AgNPs) with biological properties is of vast significance in the development of scientifically valuable products. In the present study, we describe simple, unprecedented, nontoxic, eco-friendly, green synthesis of AgNPs using an Indian traditional farming formulating agent, panchakavya. Silver nitrate (1 mM) solution was mixed with panchakavya filtrate for the synthesis of AgNPs. The nanometallic dispersion was characterized by surface plasmon absorbance measuring 430 nm. Transmission electron microscopy showed the morphology and size of the AgNPs. Scanning electron microscopy–energy-dispersive spectroscopy and X-ray diffraction analysis confirmed the presence of AgNPs. Fourier transform infrared spectroscopy analysis revealed that proteins in the panchakavya were involved in the reduction and capping of AgNPs. In addition, we studied the antibacterial activity of synthesized AgNPs. The synthesized AgNPs (1–4 mM) extensively reduced the growth rate of antibiotic resistant bacteria such as Aeromonas sp., Acinetobacter sp., and Citrobacter sp., according to the increasing concentration of AgNPs.
doi:10.2147/IJN.S58932
PMCID: PMC3983015  PMID: 24741307
green synthesis; panchakavya; antibacterial; environment; nanoparticles
25.  Green Synthesis of Silver Nanoparticles through Reduction with Solanum xanthocarpum L. Berry Extract: Characterization, Antimicrobial and Urease Inhibitory Activities against Helicobacter pylori 
A green synthesis route for the production of silver nanoparticles using methanol extract from Solanum xanthocarpum berry (SXE) is reported in the present investigation. Silver nanoparticles (AgNps), having a surface plasmon resonance (SPR) band centered at 406 nm, were synthesized by reacting SXE (as capping as well as reducing agent) with AgNO3 during a 25 min process at 45 °C. The synthesized AgNps were characterized using UV–Visible spectrophotometry, powdered X-ray diffraction, and transmission electron microscopy (TEM). The results showed that the time of reaction, temperature and volume ratio of SXE to AgNO3 could accelerate the reduction rate of Ag+ and affect the AgNps size and shape. The nanoparticles were found to be about 10 nm in size, mono-dispersed in nature, and spherical in shape. In vitro anti-Helicobacter pylori activity of synthesized AgNps was tested against 34 clinical isolates and two reference strains of Helicobacter pylori by the agar dilution method and compared with AgNO3 and four standard drugs, namely amoxicillin (AMX), clarithromycin (CLA), metronidazole (MNZ) and tetracycline (TET), being used in anti-H. pylori therapy. Typical AgNps sample (S1) effectively inhibited the growth of H. pylori, indicating a stronger anti-H. pylori activity than that of AgNO3 or MNZ, being almost equally potent to TET and less potent than AMX and CLA. AgNps under study were found to be equally efficient against the antibiotic-resistant and antibiotic-susceptible strains of H. pylori. Besides, in the H. pylori urease inhibitory assay, S1 also exhibited a significant inhibition. Lineweaver-Burk plots revealed that the mechanism of inhibition was noncompetitive.
doi:10.3390/ijms13089923
PMCID: PMC3431837  PMID: 22949839
silver nanoparticles; Solanum xanthocarpum; anti-Helicobacter pylori activities; urease inhibitory activities; TEM; agar dilution method

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