Sourabh Dwivedi

Production of antimicrobial silver nanoparticles in water extracts of the fungus Amylomyces rouxii strain KSU-09.

A fungal strain, KSU-09, isolated from the roots of date palm (Phoenix dactylifera), was identified as Amylomyces rouxii based on sequence analysis of the internal transcribed spacer (ITS) region of its rRNA genes. Mycelia-free water extracts obtained from mycelium suspended in water for 72h facilitated the production of stable, predominantly monodispersed and spherical silver nanoparticles (AgNPs) in the size range of 5-27nm upon addition of 1mM silver nitrate, as determined by the XRD, AFM and TEM. The AgNPs exhibited antimicrobial activity against Shigella dysenteriae type I, Staphylococcus aureus, Citrobacter sp., Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Fusarium oxysporum. Thus, the strain KSU-09 could be used for simple, non-hazardous and efficient synthesis of antimicrobial AgNPs.

ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7 cancer cells and their antibacterial activity.

Liver and breast cancer are the most traumatic diseases because they affect the major organs of the body. Nanomedicine recently emerged as a better option for the treatment of these deadly diseases. As a result, many nanoparticles have been used to treat cancer cell lines. Of the various nanoparticles, zinc oxide exhibits biocompatibility. Therefore, the aim of the present study was to investigate the activity of zinc oxide nanoparticles (ZnO-NPs) against HepG2 and MCF-7 cells. The NPs (∼13±2 nm) were prepared via a non-protonated chemical route and were well-characterized through standard techniques. The study showed that treatment with NPs is notably effective against the proliferation of HepG2 and MCF-7 cancer cells in a dose-dependent manner. The MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide, a tetrazole) assays revealed the concentration-dependent cytotoxic effects of NPs in range of 2.5-100 μg/ml. HepG2 and MCF-7 cells were exposed to ZnO-NPs and exhibited a significant reduction in their cell viability (95% and 96%; p<0.05) in response to a very low concentration (25 μg/ml) of the ZnO-NPs; this finding was confirmed with FACS (fluorescence-activated cell sorting) data. The reduction in cell viability in response to NP treatment induces cytotoxicity in the cultured cells. The quantitative RT-PCR (real-time polymerase chain reaction) results demonstrate that the exposure of HepG2 cells to ZnO-NPs results in significant upregulation of the mRNA expression level of Bax, p53, and caspase-3 and the down regulation of the anti-apoptotic gene Bcl-2. The NPs were also tested against five pathogenic bacteria through the disk diffusion method, and their antibacterial activities were compared with that of ZnO salt.

Reactive Oxygen Species Mediated Bacterial Biofilm Inhibition via Zinc Oxide Nanoparticles and Their Statistical Determination

The formation of bacterial biofilm is a major challenge in clinical applications. The main aim of this study is to describe the synthesis, characterization and biocidal potential of zinc oxide nanoparticles (NPs) against bacterial strain Pseudomonas aeruginosa. These nanoparticles were synthesized via soft chemical solution process in a very short time and their structural properties have been investigated in detail by using X-ray diffraction and transmission electron microscopy measurements. In this work, the potential of synthesized ZnO-NPs (∼10–15 nm) has been assessed in-vitro inhibition of bacteria and the formation of their biofilms was observed using the tissue culture plate assays. The crystal violet staining on biofilm formation and its optical density revealed the effect on biofilm inhibition. The NPs at a concentration of 100 µg/mL significantly inhibited the growth of bacteria and biofilm formation. The biofilm inhibition by ZnO-NPs was also confirmed via bio-transmission electron microscopy (Bio-TEM). The Bio-TEM analysis of ZnO-NPs treated bacteria confirmed the deformation and damage of cells. The bacterial growth in presence of NPs concluded the bactericidal ability of NPs in a concentration dependent manner. It has been speculated that the antibacterial activity of NPs as a surface coating material, could be a feasible approach for controlling the pathogens. Additionally, the obtained bacterial solution data is also in agreement with the results from statistical analytical methods.

Aloe vera extract functionalized zinc oxide nanoparticles as nanoantibiotics against multi-drug resistant clinical bacterial isolates

ZnO nanoparticles (ZnONPs) were synthesised through a simple and efficient biogenic synthesis approach, exploiting the reducing and capping potential of Aloe barbadensis Miller (A. vera) leaf extract (ALE). ALE-capped ZnO nanoparticles (ALE-ZnONPs) were characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM) analyses. XRD analysis provided the average size of ZnONPs as 15 nm. FTIR spectral analysis suggested the role of phenolic compounds, terpenoids and proteins present in ALE, in nucleation and stability of ZnONPs. Flow cytometry and atomic absorption spectrophotometry (AAS) data analyses revealed the surface binding and internalization of ZnONPs in Gram +ve (Staphylococcus aureus) and Gram -ve (Escherichia coli) cells, respectively. Significant antibacterial activity of ALE-ZnONPs was observed against extended spectrum beta lactamases (ESBL) positive E. coli, Pseudomonas aeruginosa, and methicillin resistant S. aureus (MRSA) clinical isolates exhibiting the MIC and MBC values of 2200, 2400 μg/ml and 2300, 2700 μg/ml, respectively. Substantial inhibitory effects of ALE-ZnONPs on bacterial growth kinetics, exopolysaccharides and biofilm formation, unequivocally suggested the antibiotic and anti-biofilm potential. Overall, the results elucidated a rapid, environmentally benign, cost-effective, and convenient method for ALE-ZnONPs synthesis, for possible applications as nanoantibiotics or drug carriers.

Synthesis of stable cadmium sulfide nanoparticles using surfactin produced by Bacillus amyloliquifaciens strain KSU-109

In this study, a surfactin was extracted from a novel surfactant producing bacterial strain Bacillus amyloliquifaciens KSU-109, isolated from rhizosphere of date palm (Phoenix dactylifera), and characterized based on 16Sr RNA and sfp genes using Blastn, Blastx and phylogenetic analyses. The study was performed to obtain a renewable bioresource for surfactin production, and its application in nanotechnology as a non-hazardous and environmentally compatible nanoparticle (NP) stabilizer. The strain KSU-109 produced the surfactin with an average yield of 160 mg/L with strong surfactant activity, reducing the surface tension of the medium from 72 mN/m to 29.3 mN/m. The surfactin preparation was used for synthesizing the cadmium sulfide nanoparticles (CdS-NPs) by mixing 0.005% surfactin with 1mM Cd(NO(3))(2) in 1:1 ratio (v/v) and 10mM Na(2)S solution at pH 7.2 and ambient temperature, which were stable up to 120 days. The surfactin stabilized CdS-NPs were characterized using XRD, TEM, and spectroscopic techniques. The data revealed a significant role of surfactin as a stabilizer and capping agent, which also causes phase transition to yield the cubic/hexagonal CdS-NPs of average size of 3-4 nm. The results elucidated the significance of biocompatible and biodegradable surfactin as an effective and inexpensive stabilizing agent for developing stable CdS nanoparticles.

Biomimetic Synthesis of Selenium Nanospheres by Bacterial Strain JS-11 and Its Role as a Biosensor for Nanotoxicity Assessment: A Novel Se-Bioassay

Selenium nanoparticles (Se-NPs) were synthesized by green technology using the bacterial isolate Pseudomonas aeruginosa strain JS-11. The bacteria exhibited significant tolerance to selenite (SeO3 2−) up to 100 mM concentration with an EC50 value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO3 2− to insoluble red elemental selenium (Se0) at 37°C. Characterization of red Se° product by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) released by strain JS-11 in culture supernatant along with the known redox agents like NADH and NADH dependent reductases are responsible for biomimetic reduction of SeO3 2− to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO3 2− to elemental red Se0, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel detection and quantification of nanoparticles (NPs) cytotoxicity in a 96 well format. Thus, it has been concluded that the reducing power of the culture supernatant of strain JS-11 could be effectively exploited for developing a simple and environmental friendly method of Se-NPs synthesis. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity assessment, contemplating the inhibition of SeO3 2− bioreduction process in NPs treated bacterial cell culture supernatant, as a toxicity end point.

Microwave Accelerated Green Synthesis of Stable Silver Nanoparticles with Eucalyptus globulus Leaf Extract and Their Antibacterial and Antibiofilm Activity on Clinical Isolates

A simple and rapid microwave assisted method of green synthesis of silver nanoparticles (AgNPs) was developed using aqueous leaf extract of Eucalyptus globulus(ELE), and their antibacterial and antibiofilm potential investigated. With this aim, the aqueous solutions of ELE and AgNO3(1 mM) were mixed (1:4 v/v), and microwave irradiated at 2450 Mhz, for 30 sec. The instant color change of the ELE-AgNO3 mixture from pale yellow to dark brown indicated ELE-AgNPs synthesis. The intensity of peak at 428 nm in UV-Vis spectra, due to the surface plasmon resonance of AgNPs, varied with the amount of ELE, AgNO3 concentration, pH and time of incubation. The biosynthesized ELE-AgNPs were characterized by UV-visible spectroscopy, XRD, TEM, SEM-EDX, FTIR and TGA analyses. The size of ELE-AgNPs was determined to be in range of 1.9–4.3 nm and 5-25 nm, with and without microwave treatment, respectively. SEM exhibited the capping of AgNPs with the ELE constituents, and validated by FTIR analysis. The FTIR data revealed the presence of plant organic constituents and metabolites bound to ELE-AgNPs, which contributes for their stability. The antimicrobial activity of ELE-AgNPs was assessed by growth and biofilm inhibition of extended spectrum β-lactamase (ESBL) producing Pseudomonas aeruginosa, Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) clinical bacterial isolates. The results demonstrated that S. aureus were more sensitive to ELE-AgNPs than E. coli and P. aeruginosa. MRSA exhibited higher sensitive than MSSA, whereas P. aeruginosa were more sensitive than E. coli to ELE-AgNPs treatment. Also, significant (83 ± 3% and 84 ± 5%) biofilm inhibition was observed in case of S. aureus and P. aeruginosa, respectively. The results elucidated environmentally friendly, economical and quick method for production of colloidal bio-functionalized ELE-AgNPs, for effectual clinical applications, as broad spectrum antibacterial agents and biofilm inhibitors.

Statistical analysis of gold nanoparticle-induced oxidative stress and apoptosis in myoblast (C2C12) cells.

Nanoscale gold particles (Au-NPs) with a diameter below 20nm are notably important candidates for various important applications because of their extraordinary quantum size effects. Their high surface area-to-volume ratio facilitates their very high reactivities; therefore, they can be utilised in different ways in biomedical applications. For example, these nanoparticles can penetrate into cells and bind with proteins or DNA and are therefore potential nanostructures employed for sensing and detecting various biological identities. In the present work, we synthesised Au-NPs via a colloidal process using chloroauric acid (HAuCl4·4H2O) and trisodium citrate dihydrate (N3C6H5O7) as a reducing agent. The shape evolution and the structural properties of these NPs were investigated in detail using TEM and high resolution HR-TEM investigations. Different doses of Au NPs have been applied to treat C2C12 myoblast cells in a 24-h incubation period, and a dose-dependent study has also been performed. The cells were cultivated in DMEM with FBS and antibiotics (strepto-penicillin) at 37°C in a 5% humidified environment of CO2 and 95% air. Cell viability analysis using MTT assays revealed that increased concentration of Au NPs (100-1000 ng/mL) resulted in a decreased density of cells. The amount of reactive oxygen species (ROS) in C2C12 cells analysed with Au-NPs (in a dose-dependent manner), and the RT-PCR data demonstrated the up-regulation of caspase-3 and caspase-7 genes in C2C12 cells after treatment with Au-NPs. These results have been confirmed by detailed confocal microscopy (CLSM) studies. In addition, the quantitative analysis of the Au-NPs was also confirmed by statistical analytical parameters, such as precision, accuracy, linearity, limits of detection (LOD) and limit of quantitation (LOQ), quantitative recoveries and relative standard deviation (RSD), and the analyses again exhibited a significant and large effect of Au NPs on C2C12 cells.

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0±1.38), Ni (150.8±0.78), Cu (99.3±0.56) and Cr (64.0±0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr>Ni>Cu>Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O(2)•(-)) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity.

Synthesis, characterization and toxicological evaluation of iron oxide nanoparticles in human lung alveolar epithelial cells.

The present investigation was aimed to characterize the synthesized iron oxide nanoparticles (Fe3O4-NPs) and to assess their cytotoxicity and oxidative stress in human lung alveolar epithelial cells (A-549). Fe3O4-NPs were characterized by X-ray diffraction, transmission electron microscopy, dynamic light scattering, and atomic force microscopy. The morphology of the Fe3O4-NPs was found to be variable with a size range of 36nm. A-549 cells were exposed to Fe3O4-NPs (10-50μg/ml concentrations) for 24h. Post exposure, cytotoxicity assays (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT; neutral red uptake, NRU; and cellular morphology) and oxidative stress (lipid peroxidation, LPO and glutathione, GSH) were evaluated. Further, intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were also studied. MTT and NRU assays revealed a concentration-dependent decrease in the cell viability of A-549 cells. Fe3O4-NPs exposed cells also altered the normal morphology of the cells. Furthermore, the cells showed significant induction of oxidative stress. This was confirmed by the increase in LPO and ROS generation, and the decrease in the GSH level and MMP. Our results demonstrated that Fe3O4-NPs induced cytotoxicity is likely to be mediated through the oxidative stress and ROS generation in A-549 cells.