Synergistic effect of ZnO-PEI NP and Ampicillin.

<p><i>H. pylori</i> was incubated with ZnO-PEI NP and Ampicillin either alone or in different combinations and O.D. was measured at 600 nm after 24 h of treatment. Ampicilin and ZnO-PEI NP are abbreviated as “A” and “Z”, respectively, followed by a number representing the concentration (µg/ml) used. Error bars represent standard deviation. Results are represented as mean ± SD. * P<0.05, *** P<0.001.</p

Toxicity of ZnO-PEI NP against different human cells: (a) PBMC, (b) RBC, (c) AGS cells.

<p>In (b) tube “2” is the positive control (100% hemolysis); tube “4” is the control, where NP is absent and tubes “3” and “1” represent RBC at different concentrations of NP. * P<0.05, ** P<0.01.</p

Morphological transition and rRNA degradation.

<p><i>H. pylori</i> incubated without or with ZnO-PEI NP for 3 h was visualized by (a) SEM and (b) TEM. (c) RNA was extracted from ZnO-PEI NP treated and untreated <i>H. pylori</i> and electrophoresed on 7M urea-6% polyacrylamide gel. In (a), (b) and (c) the left panel shows the control and the right panel shows treated cells. (d) 16S and 23S rRNA was estimated in the RNA samples by qRT-PCR (list of primers are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070776#pone.0070776.s010" target="_blank">Table S2</a>). * P<0.05.</p

Viability of ZnO-PEI NP treated <i>H. pylori</i> strain 26695 grown to the logarithmic phase and treated with different concentrations of ZnO-PEI NP; cells were removed for CFU assay after 24 h of treatment.

<p>Results are represented as mean ± SD, * P<0.05.</p

The Molecular Basis of Inactivation of Metronidazole-Resistant <i>Helicobacter pylori</i> Using Polyethyleneimine Functionalized Zinc Oxide Nanoparticles

<div><p>In view of the world wide prevalence of <i>Helicobacter pylori</i> infection, its potentially serious consequences, and the increasing emergence of antibiotic resistant <i>H. pylori</i> strains there is an urgent need for the development of alternative strategies to combat the infection. In this study it has been demonstrated that polyethyleneimine (PEI) functionalized zinc oxide (ZnO) nanoparticles (NPs) inhibit the growth of a metronidazole-resistant strain of <i>H. pylori</i> and the molecular basis of the anti-bacterial activity of ZnO-PEI NP has been investigated. The ZnO-PEI NP was synthesized using a wet chemical method with a core size of approximately 3–7 nm. Internalization and distribution of ZnO-PEI NP without agglomeration was observed in <i>H. pylori</i> cytosol by electron microscopy. Several lines of evidence including scanning electron microscopy, propidium iodide uptake and ATP assay indicate severe membrane damage in ZnO-PEI NP treated <i>H. pylori</i>. Intracellular ROS generation increased rapidly following the treatment of <i>H. pylori</i> with ZnO-PEI NP and extensive degradation of 16S and 23S rRNA was observed by quantitative reverse-transcriptase PCR. Finally, considerable synergy between ZnO-PEI NP and antibiotics was observed and it has been demonstrated that the concentration of ZnO-PEI NP (20 µg/ml) that is non-toxic to human cells could be used in combination with sub-inhibitory concentrations of antibiotics for the inhibition of <i>H. pylori</i> growth.</p></div

Schematic representation of the mechanism of antimicrobial activity of ZnO-PEI NP.

<p>Schematic representation of the mechanism of antimicrobial activity of ZnO-PEI NP.</p

Estimation of ROS.

<p>(a) ROS generation by ZnO (from acetate precursor) and ZnO-PEI NPs in aqueous solution was estimated by XTT assay. (b) <i>H. pylori</i> strain 26695 preloaded with the fluorescent dye H₂DCFDA was incubated with or without ZnO-PEI NP (100 μg/ml) for 30 min. (The control cells are shown to the left and the treated cells to the right). (c) (%) Increase in DCF fluorescence on ZnO-PEI treatment measured by fluorescence microscopy. * P<0.05.</p

Membrane damage of ZnO-PEI NP treated <i>H.pylori</i> strain 26695.

<p>Cells were incubated with or without ZnO-PEI NP (100 μg/ml) for 3 h and samples were removed for (a) propidium iodide-SYTO9 staining (untreated cells are shown in the left and treated cells in the right panel) and (b) ATP assay. * P<0.05.</p

Fluorescence quenching spectra of bis-ANS bound HSA in absence (top) and presence (the arrow indicating curves obtained with increasing concentration) of virstatin in phosphate buffer pH 7.2; [HSA] = 7.5 µM, [bis-ANS] = 5.0 µM and [virstatin] = 0.1 to 5.0 µM (with 10 increments of 0.1 µM, followed by another 5 of 1 µM).

<p>Fluorescence quenching spectra of bis-ANS bound HSA in absence (top) and presence (the arrow indicating curves obtained with increasing concentration) of virstatin in phosphate buffer pH 7.2; [HSA] = 7.5 µM, [bis-ANS] = 5.0 µM and [virstatin] = 0.1 to 5.0 µM (with 10 increments of 0.1 µM, followed by another 5 of 1 µM).</p

FT- IR spectra of free HSA (blue) and HSA -virstatin complex (red) in D₂O in the range of 1720 to 1560 cm^−<b>1</b><b>.</b>

<p>FT- IR spectra of free HSA (blue) and HSA -virstatin complex (red) in D₂O in the range of 1720 to 1560 cm^−<b>1</b><b>.</b></p