Curious Case of Bactericidal Action of ZnO

ZnO nanoparticles (NPs) are well known for their bactericidal properties. Various mechanisms are proposed for their bactericidal activity. An ambiguity still prevails to know which mechanism or property is mainly influencing the bactericidal activity of ZnO NPs.The antibacterial properties of ZnO NPs were investigated against both Gram-positive and Gram-negative bacteria. Different ZnO samples with different degrees of surface oxygen vacancies were prepared from ZnO 2 . The surface oxygen vacancy and thereby reactive oxygen species (ROS) production in aqueous ZnO solution are quantified by photoluminescence (PL) and electron paramagnetic resonance (EPR) spin trapping experiments, respectively. Systematic experiments have been performed to validate a precise antibacterial mechanism of ZnO particle.


Introduction
The rapid development of nanotechnology emerges in a diverse range of nanomaterials and nanoproducts [1].Various desired targets have been accomplished in order to employ materials in medicinal fields by maneuvering them at their atomic size scale [2,3] through nanotechnology.Unfortunately, many such benevolent materials develop toxicity.As toxicities are selective to biological systems, nanomaterials are well exploited for antibacterial applications.To combat bacterial infections metal and metal oxide NPs in various forms are well studied [4][5][6][7][8][9][10][11][12][13][14][15].
The exposure of such NPs in the environment demands a fundamental understanding about their mode and range of toxicity.Their mode or modes of action towards bacteria remained ambiguous.
ZnO NPs have been extensively used as antibacterial agents for water purification [16][17][18][19], biofilm prevention [20][21][22], sunscreen lotion [23,24], wound dressing [25], and so forth.Protection against intestinal bacterial infections by bulk ZnO was reported in late 1990s [26,27] though its bactericidal activity against a broad spectrum of bacteria (e.g., Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Streptococcus agalactiae, etc.) was revealed little late when ZnO NPs were exposed to bacterial solution [28][29][30][31][32].Although the antimicrobial properties of ZnO NPs have been utilized to kill bacteria in different issues, their mechanistic pathways are still imprecise.The mechanisms of antibacterial property of ZnO NPs so far proposed are as follows: (i) physical attack of ZnO NPs on the bacteria [18,33,34] (ZnO NPs can adhere to the bacterial cell wall surface and eventually pierce into cell leading to bacterial death by membrane disruption), (ii) oxidative stress generated by particles in solutions [35,36] (hydroxyl radials are the result of interaction of ZnO NPs with aqueous solution which causes oxidation of bacterial metabolic enzyme leading to bacterial death), and (iii) solubility of NPs in aqueous solution [37,38].But none of them is solely responsible on a "one fits all" basis for the desired activity as reported in the literature.The above-mentioned different mechanisms play a role in the bactericidal activity because of size, surface oxygen vacancy, and active surface area of the ZnO particles.A careful investigation is required to evaluate the influence of each such property towards the toxicity of bacteria.
The antibacterial properties of ZnO NPs were investigated against both Gram-positive and Gram-negative bacteria.During bactericidal activity study various properties (e.g., particle size, solubility, surface area, etc.) of ZnO samples were maintained unaltered when varying the other (e.g., reactive oxygen species (ROS) production, surface defect) through simple chemical route to correlate the antibacterial activity of ZnO with such property.Similarly, when solubility of ZnO samples was varied, ROS production was arrested by glutathione (GSH) in order to eliminate or establish the possibility of oxidative stress mechanism for their bactericidal action.These systematic studies state that bactericidal efficiency of ZnO is little higher for Grampositive Staphylococcus aureus ATCC 25923 compared to Gram-negative Escherichia coli MTCC 1302.It has been found that as the surface area increases the solubility of the same sized different ZnO samples in saline water increases and also the bactericidal activity.Interestingly, when ROS production was stopped by employing GSH, bactericidal activity for each of the samples decreases a little.When the amount of ROS production was raised by creating more surface defects in ZnO samples maintaining the same solubility, the bactericidal activity increases with the rise in ROS production.Surprisingly for these samples when ROS production in aqueous solution was blocked bactericidal activity decreases compared to when ROS production was not arrested.But level of antibacterial activity prevails similarly within those samples after ROS production was blocked.The whole understanding of all the observations says that each of these individual physicochemical properties of ZnO samples contributes individually towards the killing of bacteria and noticeably the contribution by ROS production due to surface defect in account of bactericidal activity is the leading part.

Synthesis of ZnO Submicron
Particles.Zinc acetate dihydrate (0.9855 gm) was added to 45 mL of diethylene glycol (DEG) with vigorous stirring for 0.5 h at room temperature.The mixture was refluxed at 180 ∘ C for 1 h, similar to the procedure described by Ghosh et al. [18].A milky white precipitate appeared at the end of the reaction indicating the formation of ZnO.The obtained milky precipitate was centrifuged, separated, and washed with ethanol several times by repeated sonication-centrifugation process.The precipitate so obtained was dried under vacuum at 60 ∘ C for 6 h and characterized by powder X-ray diffraction.

Synthesis of ZnO
2 .The pristine ZnO powder was stirred with 30 mL of aqueous KOH (1 M) solution for 2 h at room temperature and the solution was then washed with water and 50 mL of H 2 O 2 (30%) was added, and stirring continued for 24 h at 45 ∘ C. The precipitate was washed with water and dried under vacuum and characterized by powder X-ray diffraction, thermogravimetric study.

DCFH-DA Test.
A fluorescence experiment was carried out using a sensitive probe, 2  ,7  -dichlorofluorescein diacetate (DCFH-DA), for the detection of ROS by incubating the samples in 30 mL of 0.05 × 10 −3 M DCFH-DA solution for 2 h.The colourless DCFH-DA solution changes to green upon exposure with samples and imparts a fluorescence peak at ∼525 nm (excited at 485 nm) which indicates the samples produced ROS in aqueous solution.

Suppression of ROS Production from Aqueous ZnO by GSH
Treatment.Glutathione (-glutamylcysteinylglycine, GSH), a sulfhydryl (-SH) antioxidant, an antitoxin, is sued for the suppression of ROS as produced from aqueous ZnO solution.Being water soluble and having facile electron donating power, GSH first arrests ROS (i.e., hydroxyl radical, OH • ) by reducing them.Various aqueous ZnO samples were incubated with excess GSH till the solution became colorless and hence nonfluorescent (i.e., no peak at ∼525 nm when excited at 485 nm).

Minimum Inhibition Concentration (MIC).
The MIC is the lowest concentration at which a material exhibits antimicrobial activity and this was done by serial dilution techniques.Sterile test tubes were taken separately with 9.9 mL of saline water, inoculated with corresponding microorganisms (fresh culture, mid-log phase, OD600 ∼0.045-0.050),and diluted up to 10 8 CFU mL −1 .Different concentrations (5-200 g mL −1 ) of various ZnO samples were added to individual test tubes.After 4 h incubation at 37 ∘ C, each 0.1 mL of this solution was taken and plated in sterile nutrient agar plates and the plates were incubated at 37 ∘ C overnight and colonies were counted to determine the MIC.

Powder X-Ray Diffraction (XRD).
Powder XRD patterns of the prepared ZnO were recorded on Philips XRD "X" PERT PRO diffractometer using Cu-K radiation ( = 1.5438Å) as X-ray source.

Inductively Coupled Plasma-Optical Emission Spectrophotometry (ICPOES).
The amount of ZnO dissolved in known volume of saline water for different samples was estimated using Perkin-Elmer optima 2100 ICPOES at  = 213.8nm. ZnO-400 ZnO-500 Scheme 1: Flow chart of conversion of ZnO to ZnO 2 and again to different ZnO samples.

BET Surface
Area.Surface area of different samples was determined by NOVA surface area analyzer.

Scanning Electron Microscopy (SEM).
The morphological studies of the prepared ZnO were carried out in field emission scanning electron microscope (FE-SEM SIRION).A drop of well-dispersed particles in water was cast onto a piece of silicon wafer and air-dried.A thin gold coating was applied to avoid charging during scanning and a detailed microscopic study was carried out.

Electron Paramagnetic Resonance Spectroscopy (EPR).
Hydroxyl radicals were detected by EPR-spin trapping technique using spin trapper 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 0.02 M).Aqueous suspensions of different ZnO samples were drawn separately into quartz capillaries (one end closed) along with DMPO.The capillaries were placed in the EPR tube and spectra were recorded on a Bruker emx X-band EPR spectrometer.

Results and Discussion
In order to elucidate proper mechanism of antibacterial activity, ZnO samples with different degrees of physicochemical properties have been synthesized through a simple route as depicted in Scheme 1. Submicron size ZnO particles were synthesized by polyol method.The as-synthesized ZnO particles were converted to ZnO 2 by reacting with H 2 O 2 and heated in presence of H 2 gas at different temperatures.
In this method, various degrees of surface defects (oxygen vacancies) were obtained without much change in particle nature between the samples.The thermogravimetric analysis (TGA) and differential thermal analysis (DTA) of ZnO 2 show a sharp exothermic peak (Figure 1(a)), indicating decomposition of ZnO 2 to ZnO at ∼225 ∘ C and the reactions were carried out above this temperature.
XRD patterns were obtained for all the samples and are depicted in Figure 1(b).All the reflections were assigned with standard wurtzite structure of ZnO (JCPDS file number 36-1451) while for ZnO 2 the peaks are assigned with pure cubic phase of ZnO 2 (JCPDS file number 13-311).SEM pictures obtained for the samples along with ZnO 2 are shown in Figure 2. It reveals that the pristine ZnO particles are spherical and the size varies between 150 and 250 nm (Figure 2(a)).On treatment with H 2 O 2 , few smaller sized particles disintegrated (Figure 2(b)) and the larger particles (size 150-250 nm) remain intact even after heating at different temperatures (Figures 2(c), 2(d), 2(e), and 2(f)).The physicochemical properties of ZnO samples were tabulated along with MICs as shown in Table 1.To investigate the mechanism of antibacterial activity, MICs were determined with and without treating ZnO samples with GSH.
From pristine ZnO to ZnO-H, the surface area and solubility increase which may be attributed to the smaller fragmentation on treatment with H 2 O 2 and heating, but both solubility and surface area remain unaltered for other samples of ZnO-300 to ZnO-500 (Figure 3(a) and Table 1).
When bactericidal activity was evaluated without GSH treatment, MICs for ZnO to ZnO-500 decrease (Table 1 and Figure 3(b)).The surface area (and hence solubility) of ZnO to ZnO-H increases, but EPR peak-area due to ROS production does not differ much.Bactericidal activity of ZnO NPs increases along with increase in solubility reported earlier [37,38].Thus, the decrease in MIC for ZnO to ZnO-H could be accounted by the solubility factor.But the same argument does not fit to account the MIC values of ZnO-300 to ZnO-500.Solubility (Table 1) of ZnO-300 to ZnO-500 in saline was the same, but MIC value decreases or bactericidal activity increases from ZnO-300 to ZnO-500.So not only the solubility of ZnO samples but also the other factors are responsible for its bactericidal activity.
Similarly, when bactericidal activity was determined after GSH (i.e., without ROS production) treatment with ZnO samples, MIC values (Table 1; Figure 3(c)) for ZnO-300 to ZnO-500 were almost the same indicating oxidative stress due to the fact that ROS is the governing factor as other formed on the ZnO surface is not clear, although some investigations on the nature of the ZnO surface such as chemisorbed OH species after interaction with water produces ROS have been reported [39].
In addition, a little is known about the nature of defect sites on a wet metal oxide surface.The formation of ROS may be explained by assuming the formation of hydroxyl radicals by the reaction of water and (dissolved) oxygen over basic metal oxides [40]: where V O refers to an oxygen vacancy and "s" refers to surface species.The higher oxygen vacancy, the higher ROS production.This is further shown in the PL spectra of the samples (Figure 3(d)).The green emission between 440 and 700 nm is due to surface oxygen vacancy.The increase in PL peak area (Figure 3(e)) starting from ZnO to ZnO-500 indicates the increase in oxygen vacancy [41].When such areas are plotted against the corresponding ZnO samples, this shows almost a linear increase and the shape of the curve matches well with the EPR peak area as shown in Figure 3(f).This supports the above proposed mechanism for the ROS production through oxygen vacancy of ZnO.

Conclusions
The method adopted for the synthesis of ZnO and ZnO 2 is very simple and effective.Different physicochemical properties of ZnO have been varied to validate their influencing nature towards their bactericidal action.When oxidative stress has been nullified by arresting OH • (hydroxyl) radical with GSH, MICs of ZnO samples (from ZnO to ZnO-H) decrease with the increase of their surface area (particle size remains the same) and solubility.The effect of oxidative stress towards bactericidal action has been considered (i.e., without GSH treatment) and the MICs of ZnO samples decrease with the increase of the surface defects or ROS production (from ZnO-300 to ZnO-500).Throughout the experiments, particles sizes of ZnO samples were constant and the effect due to physical attack (i.e., direct interactions between the particles and bacteria) is the same and cannot be avoided.Bactericidal properties of ZnO are due to the combination of all depicted mechanisms.It can be concluded that out of all mechanisms oxidative stress developed in bacteria through ROS production of ZnO samples is the most influencing factor for its bactericidal activity.

Figure 3 :
Figure 3: Plots of (a) MICs (without GSH) versus solubility, (b) MICs (without GSH) versus EPR peak area, and (c) MICs (with GSH) versus solubility; (d) PL spectra; (e) EPR spectra; and (f) correlation plot of EPR peak area versus PL peak area of different ZnO samples.

Table 1 :
Different physicochemical properties and minimum inhibition concentration (MIC) values.