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Mixed metal oxide nanoparticles in fight against bacteria

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Antibiotic resistance has impelled the research for new agents that can inhibit bacterial growth without showing cytotoxic effects on humans and other species. We describe the synthesis and physicochemical characterization of nanostructured ZnMgO whose antibacterial activity was compared to its pure counterparts ; nano-ZnO and nano-MgO. Among the three oxides, ZnO nanocrystals were found to be the most effective antibacterial agents since both Gram-positive (B. subtilis) and Gram-negative (E. coli) bacteria were completely eradiated at concentration of 1 mg/mL. MgO nanocubes only partially inhibited bacterial growth whereas ZnMgO nanoparticles revealed high specific antibacterial activity to Gram-positive bacteria at this concentration. Furthermore, our preliminary toxicological study pointed out that nano-ZnO is toxic when applied to human HeLa cells, while nano-MgO and the mixed oxide did not induce any cell damage. Overall, our results suggested that nanostructured ZnMgO, may reconcile efficient antibacterial efficiency while being a safe new therapeutic for bacterial infections.

Over the past decade, many potential antibacterial agents – including nanometer sized metal oxides– have been researched. Some of these agents were found to be cytotoxic against bacteria but not against mammalian cells making, thus, medical applications possible (Taylor and Webster 2011). The use of inorganic nanoparticles has attracted lots of interest mostly because of their reliable antimicrobial activity found to be effective at low concentrations (Anagnostakos et al. 2008). This is due to their high specific surface area which allows a broad range of reactions with the bacterial surface. Nano-MgO was shown to exhibit bacteriocidal activity which is highly dependent on the particle size and concentration (Huang et al. 2005 ; Makhulf et al. 2005) and to act against both Gram-positive and Gram-negative bacteria (Koper et al. 2002 ; Krishnamoorthy et al. 2012 ; Makhulf et al. 2005). These nanoparticles are considered as a promising novel antibacterial agent, being harmless to mammalian cells and the environment. For instance, nano-MgO – alone or in combination with other microbials – was proposed as a bacteriocide for treatment of food products in order to improve microbiological food safety (Jin and He 2011). Nanostructured ZnO is a highly efficient antibacterial agent at significantly low concentrations showing, thus, an advantage compared to nano-MgO. It was also found to act against both, Gram-positive and Gram-negative bacteria (Apperlot et al. 2009 ; Brayner et al. 2006 ; Padamavathy and Vijayaraghavan 2008 ; Stoimenov et al. 2002 ; Zhang et al. 2007). The cytotoxic effect was found to be size-dependent : the smaller the particle size, the greater the efficiency in inhibiting bacterial growth (Apperlot et al. 2009). Despite numerous benefits regarding ZnO antibacterial effects, some recent reports point out that nano-ZnO may exhibit toxic effects on human cells (Lai et al. 2008 ; Lyon et al. 2007 ;). Our aim was to combine the strong antibacterial activity of ZnO with safe-to-use antibacterial activities of MgO. We focused on E. coli (Gram-negative) and B. subtilus (Gram-positive) cultures and compared antibacterial activities of ZnMgO nanoparticles to those of nano-MgO and nano-ZnO. Finally, toxicity on mammalian cells was investigated for all three oxides. Obtained data give the first indication that nano-ZnMgO can be used as antibacterial agents and point to some synergistic effects of its pure metal oxide components.

For the production of nanoparticles we used the metal combustion technique. This synthesis approach, that comprises the oxidation of metal to occur in the gas phase, results typically in particles morphology that is presented in Fig.1A. MgO particles adopt extraordinary cubic shape with the mean size 50 nm (blue), ZnO are represented by nano-tetrapods and nano-rods with lengths of either nanorods or tetrapod arms between 150-200 nm and the corresponding diameters 10 nm (green) whereas ZnMgO nanocrystals exhibit shapes that are found in TE micrographs of both of its pure components (red). The corresponding X-ray diffraction patterns revealed the presence of both crystal phases in ZnMgO powder ; cubic and wurtzite indicating that phase separation takes place during the synthesis of the mixed metal oxide.

GIF Figure 1
TEM images (A) and X-ray diffraction patterns of (B) ZnO (green), MgO (blue) and ZnMgO (red).

The antibacterial activity of ZnO, MgO and ZnMgO nanopowders was studied against two bacterial strains E. coli (Gram-negative) and B. subtilis Gram-positive) (Fig.2). Bacterial viability quantified after 24 h incubation with nanoparticles shows that ZnMgO completely inactivated the growth of B. subtilis, while about 80 % E. coli survived this treatment. This indicates not only the high antibacterial activity of ZnMgO system but it implies also its selectivity against Gram-positive bacteria. This may be explained by differences in cell wall structure between Gram-positive and G-negative bacteria implying that ZnMgO exhibits higher binding affinity to Gram-positive bacteria cell wall.

GIF Figure 2
Growth curves of E. coli (A) and B. subtilis (B) in Luria-Bertani (LB) medium in the presence of 1 mg/mL : ZnO (green), MgO (blue) or ZnMgO (red) nanoparticles.

In addition, TEM images of B. subtilis treated with ZnMgO nanoparticles showed an extensive injury of the bacterial cell membrane and complex morphological changes (Fig. 3). The inhomogeneous appearance of the cytoplasm might be a result of the leakage of the cell content.

Figure 3
TEM images of untreated B. subtilis (A) and treated B. subtilis with ZnO (B), MgO (C) and ZnMgO (D) nanoparticules (for 5 h, at concentration 1 mg/mL).

Human HeLa cells were used to test the cytotoxic effects of the nanopowders on mammalian cells. The cells were treated for 24 h, at optimal conditions for antibacterial activity. Optical microscopy revealed no changes with respect to the morphology and density of HeLa cells after cells incubations with nano-MgO or nano-ZnMgO whereas the ZnO treatment led to the death of all cells. To support these findings, a quantification of cell death was performed on treated HeLa cells using a flow cytometry analysis. This analysis confirmed no cytotoxicity of nano-ZnMgO on HeLa cells.

Overall this study showed not only the selective antibacterial efficiency of nano-ZnMgO against Gram-positive bacteria, but also the biocompatibility of this oxide. However, complementary study is needed to elucidate the molecular mechanism by which mixed ZnMgO damages Gram-positive bacteria.

“Selective antibacterial effects of mixed ZnMgO nanoparticles”
J. Vidic, S. Stankic, F. Haque, D. Ciric, R. Le Goffic, A. Vidy, J. Jupille, B. Delmas.