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Limited availability of antimicrobials and emergence of microbial multi drug resistance (MDR) have posed clinical challenges and highlighted the demand for alternative, effective antimicrobial strategies . Nanoscale materials are increasingly used as new paradigm for infectious diseases which have become a global threat to public health . Owing to tuneable and unique physical and chemical attributes, nanomaterials have shown therapeutic promises . In the past two decades, among various nanomaterials, ZnO nanostructures have gained attention in biomedicine due to their excellent biocompatibility, low cost and low toxicity . However, the clinical translation of nanotechnology requires appropriate method for synthesis and thorough understanding of physicochemical attributes and molecular mechanism of action. Traditional antimicrobial compounds mediate antimicrobial action via generation of reactive oxygen species (ROS) (a lethal stress), which plays a central role in killing of microbes regardless of the specific targets of compounds . Although, basal ROS levels have specific roles in cellular signalling and maintaining cellular homeostasis , excessive ROS production beyond cellular antioxidant capacity induces oxidative stress, which eventually leads to cell death . These active ROS can directly cause oxidative damage and cells are less probable to develop antimicrobial resistance. Nanomaterials are also known for their ability to trigger ROS and induce cell killing. With advancement in nanotechnology, ROS generation is one of the key factors in metallic nanoparticle-induced toxicity and modulation of cell signalling cascades responsible for cell death . The amount of ROS generated by metallic nanoparticles is dependent on particle shape, size, surface area, and chemistry . Quantum dots (QDs) with high surface area to volume ratio have shown tremendous potential to eliminate a wide range of multi drug resistant (MDR) pathogens by augmenting intracellular ROS . Compounds with ability to modulate ROS generation represent a class of drug candidates that warrant further detailed investigation for further clinical translation. In this study, we synthesized ZnO quantum dots (QDs) using cost-effective, wet chemical route and investigated the role of ROS generated in mediating cellular toxicity in a human pathogenic yeast, Candida albicans. Spherical, monodispersed and stable ZnO QDs of ~4-6 nm size was prepared (Fig. 1A), which exhibited dose dependent toxicity against Candida albicans as evident from spot assay and broth microdilution assay. The minimum inhibitory concentration (MIC90) of ZnO QDs required for 90% growth inhibition of Candida cells was 200 μg/mL. The effect of ZnO QDs on cellular and molecular targets were assessed by monitoring the production of endogenous ROS in the absence and presence of ascorbic acid, a natural antioxidant. Endogenous ROS levels were evaluated by using an oxidant-sensitive fluorescent dye, dichlorofluorescein diacetate (DCFDA) . This cell-permeant dye diffuses into the cells to be converted into stable and colorless dichlorofluorescein, which subsequently gets oxidized by ROS to produce green fluorescent 2,7-dichlorofluorescein (DCF). As compared to untreated control cells (without ZnO QDs), Candida cells treated with 100 and 200 µg/ml ZnO QDs exhibited increase in fluorescence inside the cells, which implied enhanced intracellular ROS as depicted by Rf intensity (Fig 1B). Presence of ROS in control untreated and ZnO QDs treated cells was confirmed via images of green fluorescent cells taken in confocal microscope (Fig. 1C, panels a, b and c). The H2O2-treated cells were used as positive control (Fig. 1B and 1C, panel d). Our results demonstrated a dose-dependent increase in endogenous ROS levels which were significantly elevated by around 29% in the presence of 100 μg/mL (subinhibitory concentration) ZnO QDs and by 53% i.e. almost more than double in the presence of 200 μg/mL (MIC90) ZnO QDs. The dose-dependent antifungal effects exerted by ZnO QDs (Fig. 1D, left panel) is possibly contributed by endogenous ROS. In order to elucidate the ZnO QDs-mediated antifungal action, the direct involvement of ROS in cell toxicity was further investigated in the presence of antioxidant. Without ZnO QDs treatment, antioxidant inhibited basal ROS production and lowered ROS levels (indicated by green bars in Fig. 1B). In addition, 5 mM antioxidant further reduced ROS levels in Candida cells treated with 100 μg/mL and 200 μg/mL ZnO QDs, respectively (Fig.1B and 1C). The antifungal effect of ZnO QDs in the presence and absence of antioxidant was assessed using spot assay as described elsewhere . The antioxidant itself did not exert any inhibitory effect on the growth of the cells (Fig. 1D, right upper panel). On the other hand, ZnO QDs exerted dose dependent inhibition of Candida cells (Fig. 1D, left middle and lower panels) in absence of antioxidant. The reduced cell viability in the presence of ZnO QDs, indicate that ROS production is an important mechanism for ZnO QDs mediated toxicity. However, reversal of endogenous ROS to basal levels by antioxidant, could not completely restore the growth of the fungal cells grown in presence of ZnO QDs (Fig. 1D, right middle and lower panels) identical to those without ZnO QDs (Fig. 1D, top panels). Our results indicated that scavenging ROS by antioxidant was unable to offer complete protection against cell killing mediated by ZnO QDs and it is speculated that intracellular ROS alone is not the only mechanism for antifungal action mediated by ZnO QDs. Intracellular ROS probably exerts a concerted action along with other cellular mechanisms for ZnO QDs mediated cellular toxicity and thus, paves way for further investigations on underlying mechanisms. The elucidation of this mechanism will be helpful for facilitating the future research progress for further medical adoption of ZnO QDs in biomedicine as a broad spectrum, new-generation antimicrobial agent.
How to Cite
Reactive Oxygen species, ROS scavenger, antioxidant, ZnO quantum dots, antimicrobial, pathogenic fungus, Candida albicans, nanomedicine
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