Effect of Morphology and Concentration on Crossover between

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Effect of Morphology and Concentration on Crossover between Antioxidant and Pro-oxidant Activity of MgO Nanostructures Soumik Podder,† Dipak Chanda,†,‡ Anoop Kumar Mukhopadhyay,‡ Arnab De,§ Bhaskar Das,§ Amalesh Samanta,§ John George Hardy,*,∥,⊥ and Chandan Kumar Ghosh*,† †

School of Materials Science and Nanotechnology, Jadavpur University, Kolkata 700032, India Advanced Mechanical and Materials Characterization Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700032, India § Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India ∥ Department of Chemistry, Lancaster University, Lancaster, Lancashire LA1 4YB, U.K. ⊥ Materials Science Institute, Lancaster University, Lancaster, Lancashire LA1 4YB, U.K.

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S Supporting Information *

ABSTRACT: The toxicity of nanomaterials can sometimes be attributed to photogenerated reactive oxygen species (ROS), but these ROS can also be scavenged by nanomaterials, yielding opportunities for crossover between the properties. The morphology of nanomaterials also influences such features due to defect-induced properties. Here we report morphology-induced crossover between pro-oxidant activity (ROS generation) and antioxidant activity (ROS scavenging) of MgO. To study this process in detail, we prepared three different nanostructures of MgO (nanoparticles, nanoplates, and nanorods) and characterized them by HRTEM. These three nanostructures effectively generate superoxide anions (O2•−) and hydroxyl radicals (•OH) at higher concentrations (>500 μg/mL) but scavenge O2•− at lower concentrations (40 μg/mL) with successful crossover at 200 μg/mL. Nanorods of MgO generate the highest levels of O2•−, whereas nanoparticles scavenge O2•− to the highest extent (60%). Photoluminescence studies reveal that such crossover is based on the suppression of F2+ and the evolution of F+, F2+, and F23+ defect centers. The evolution of these defect centers reflects the antibacterial activity of MgO nanostructures which is initiated at 200 μg/mL against Gram-positive S. aureus ATCC 29737 and among different bacterial strains including Gram-positive B. subtilis ATCC 6633 and M. luteus ATCC 10240 and Gram-negative E. coli ATCC K88 and K. pneumoniae ATCC 10031. Nanoparticles exhibited the highest antibacterial (92%) and antibiofilm activity (17%) against B. subtilis ATCC 6633 in the dark. Interestingly, the nitrogen-centered free radical DPPH is scavenged (100%) by nanoplates due to its large surface area (342.2 m2/g) and the presence of the F2+ defect state. The concentration-dependent interaction with an antioxidant defense system (ascorbic acid (AA)) highlights nanoparticles as potent scavengers of O2•− in the dark. Thus, our findings establish guidelines for the selection of MgO nanostructures for diverse therapeutic applications.

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resistant bacterial population.2 The pro-oxidant activity of metallic and semiconductor nanoparticles typically originates from light-induced oxidative stress, caused by reactive oxygen species (ROS), examples of which include: superoxide anions (O2•−),4 hydroxyl radicals (•OH),4 singlet oxygen (1O2),4 dissolution of cations,2,3,5,6 internalization of nanostructures5 resulting in disintegration of the cell membrane, inhibition of enzyme activity and DNA synthesis, interruption of energy transduction,3,7 etc. Generally, ROS damage biomolecules, e.g. proteins, vitamins, and lipids (lipid peroxidation) of microbial cells, due to the strong oxidation potential of ROS.2,8−12 In addition to antibacterial activity, ROS also introduce anticancer and antitumor activity, leading to the use of these types of materials to have potential therapeutic applica-

INTRODUCTION In recent years bacteria have become more resistant against antimicrobials, and if the current trend persists, there are projected to be ca. 10 million lives lost every year by 2050 caused by antimicrobial resistance related issues.1,2 Importantly, 80% of the antimicrobial-resistant bacterial infections originate from the hospital environment and contact with contaminated surfaces. In this context, metallic and semiconductor nanoparticles have gained the attention of researchers interested in developing potential replacements for antibiotics,3 as antibiotics induce environmental pressure in which accidentally surviving bacteria (due to degradation of antibiotics) will replicate with genes at a high rate that resist antibiotics by several mechanisms, e.g. further degradation of antibiotics, modification of drug target, expression of efflux pumps, etc., and conventional treatment, e.g. higher doses of drugs, even enhance these strategies to increase an antibiotic © XXXX American Chemical Society

Received: July 11, 2018

A

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry tion.4,12−18 ROS generation by metallic and semiconducting nanoparticles is a complex process which is affected by various characteristics of nanoparticles (including size,3,5,16 morphology,6,5,19 concentration,19 heterostructure,20 and environmental parameters such as light irradiation).9 In this context, the morphology dependence of pro-oxidant activity arises from the tunable density of states at the exposed active sites.21 Some reports have highlighted the defect-controlled pro-oxidant activity of various nanostructures.4,21,22 ROS are produced in vivo by the body’s metabolic process (e.g., degradation of food during the respiratory process) as byproducts.23 ROS levels are maintained at equilibrium by various competitive endogenous antioxidants (e.g., superoxide dismutase, peroxidase, catalase)24 that help to prevent deleterious effects in the body. Under stressed conditions ROS levels are elevated due to the disruption of the normal cellular metabolism; consequently, natural enzyme activity is disturbed.23 Stress and the associated increased ROS levels promote several neurological disorders, cardiovascular disease, Alzheimer’s disease, Parkinson’s disease,25 etc. Exogenous antioxidants (e.g., vitamins C and E and coenzymes) are often consumed to control levels of ROS,23,26 but their poor chemical stability, cell uptake, and bioavailability23 have encouraged researchers to find alternatives. Interestingly, oxide nanoparticles exhibit ROS scavenging ability24,27 similar to enzyme-mimetic activity.28 This scavenging activity is influenced by the surface properties,26 concentration,19 and redox potential24 of the nanoparticles. Pro- and antioxidant activities are important for various therapeutic applications, which has motivated the development of new nanoparticles with tailored properties. A variety of nanoscale entities (nanoceria, graphene quantum dots, low molecular weight, etc.) exhibit crossover between these two competitive activities, depending on the environmental conditions to which they are exposed, and such entities have a variety of potential applications.9,19,23 Magnesium oxide (MgO) is a nontoxic,29 environmentally benign, semiconducting, catalytically active30,31 antibacterial material.3,5,32,33 Studies support the UV and visible light controlled ROS mediated antibacterial activity of MgO;34 however, the mechanism of antibacterial activity in the dark is a matter of current debate. Recent studies reveal that the antibacterial activity of MgO is mediated by an oxygen centered radical pathway5,35 and the surface plane (111) orientation in MgO results in higher O2•− concentrations under dark conditions which are antibacterial.35 To the best of our knowledge, the effect of nanoparticle shape on the antibacterial activity of MgO has been unexplored. High concentrations (100 μg/mL) of MgO nanoparticles generate ROS5,34 in the dark; however, as yet, no results at lower concentrations have been reported. In this present study we have observed antioxidant (radical scavenging) activity at 40 μg/mL and, moreover, crossover between these two functionalities in the dark, suggesting that MgO nanoparticles have a variety of potential therapeutic applications in the light and dark. The focus of this study is the concentration-dependent crossover between anti- and prooxidant activities of MgO with various dimensions (nanoparticles, nanoplates, and nanorods) and concentrations in the dark. The concentration-dependent pro-oxidant activity of these nanostructures was assessed in the form of antibacterial activity against bacteria (Gram-positive B. subtilis ATCC 6633, M. luteus ATCC 10240, and S. aureus ATCC 29737 and Gramnegative E. coli ATCC K88 and K. pneumoniae ATCC 10031)

and biofilms of B. subtilis ATCC 6633, whereas the antioxidant activity was assessed by exposure to the well-known free radical DPPH (1,1-diphenyl-2-picrylhydrazyl), used in agriculture and the food industry. Finally the interaction between AA and MgO nanostructures in the dark was studied to understand the concentration-dependent crossover, which may prove helpful in regulating ROS production in cells.

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EXPERIMENTAL SECTION

Materials. Mg(CH3COO)2·4H2O, Mg(NO3)2·6H2O, cetyltrimethylammonium bromide (CTAB), thiourea, NaOH, Na2CO3, ethylenediaminetetraacetic acid (EDTA), hydroxylamine hydrochloride, trichloroacetic acid (TCA), H2O2, and FeCl3·6H2O were procured from Merck (India). 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), gallic acid (GA), ascorbic acid (AA), butylated hydroxytoluene (BHT), and terephthalic acid (TA, C8H6O4, 98%) were purchased from Sigma, and thiobarbituric acid (TBA), 2- deoxy-D-ribose, nitroblue tetrazolium chloride (NBT), nutrient broth (NB), nutrient agar (NA), crystal violet (CV), and black and white 96-well plates were obtained from Himedia (India). Filter paper (0.45 μm) was procured from Whatman. Ethanol was purchased from Merck (Germany). All reagents were used without any further purification. Characterization. The structural properties, i.e. crystallinity and phase of the samples, were characterized with an X-ray diffractometer (Ultima-III, Rigaku, Japan). The morphology and size of the samples were analyzed with a field emission scanning electron microscope (FESEM, S-4800, JEOL) and high-resolution transmission electron microscope (HRTEM, JEOL 2010). N2 adsorption−desorption isotherm measurements were carried out at 77 K with a Quantachrome Instruments version 4.0 apparatus, for which the samples were outgassed at 70 °C for 1.5 h before measurement. The specific surface area was determined by the Brunauer−Emmet−Teller (BET) method. The pore size distribution was derived from the desorption branch of the N2 isotherm by the Barret−Joyner−Halanda (BJH) method. Fourier transform infrared (FTIR) spectra were collected with an IR Prestige instrument (Shimadzu, Japan) in absorbance mode. Room- temperature micro-Raman spectra were recorded with a Raman spectrometer using 532 nm laser sources (Alpha 300, WITEC, Germany). Diffuse reflectance spectra were acquired with a UV−vis spectrophotometer (Shimadzu, Japan), while room-temperature photoluminescence spectra were collected with a photoluminescence (PL) spectrophotometer using a 100 W Xe lamp (FP-8300, JASCO). The ζ potential was measured by a ζ analyzer (Zetasizer NS Nano). Electron paramagnetic resonance (EPR) spectra were recorded in standard quartz EPR tubes using a JEOL JES-FA200 X-band spectrometer at room temperature. The X-ray photoelectron spectroscopy (PHI 5000, Versaprobe II, USA) technique was used with an Al Kα radiation source and the 284.5 eV C 1s signal as internal calibrator to identify the chemical states in MgO nanoparticles, nanoplates, and nanorods. Synthesis of MgO Nanoparticles. MgO nanoparticles were prepared by a wet chemical procedure.32 Briefly a Mg(CH3COO)2· 4H2O (0.1 M) solution was mixed slowly with NaOH (0.2 M) and the mixture was stirred for 15 min and immediately centrifuged (5000 rpm, 20 min) repeatedly. The solid precipitate was washed with DI water and ethanol and dried at 100 °C. Finally, it was calcined in air at 450 °C for 2 h to afford MgO nanoparticles as a white powder. Synthesis of MgO Nanoplate. MgO nanoplates were synthesized by a hydrothermal method.36 Briefly Mg(NO3)2·6H2O (0.3 M) and CTAB (2 mM) were mixed in DI water (100 mL) followed by dropwise addition of an NaOH solution (0.3 M) as a 1/2 mixture (v/ v). The entire mixture was continuously stirred for 1 h and then transferred into an autoclave heated to 200 °C for 4 h. The autoclave was cooled to room temperature, and the white precipitate was filtered, washed with DI and ethanol, and dried at 90 °C for 2 h. Finally the product was calcined in air at 450 °C for 2 h to obtain a white MgO nanoplate powder. Synthesis of MgO Nanorods. MgO nanorods were prepared by a hydrothermal method36 with some modifications. Briefly MgB

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry (NO3)2·6H2O (0.3 M) and thiourea (2 mM) were mixed in DI water (100 mL) into which NaOH (0.3 M, 200 mL) was added dropwise with continuous stirring for 1 h. The mixture was transferred in an autoclave heated at 150 °C for 4 h. The autoclave was cooled to room temperature, and the white precipitate was filtered, washed with DI water and ethanol, and dried at 90 °C for 2 h. Finally the product was calcined at 450 °C for 2 h to afford a white nanorod powder. Pro-oxidant Activity Measurements. The pro-oxidant activity of MgO nanostructures was assessed by measuring the generation of superoxide anion (O2•−) and hydroxyl radical (•OH). For O2•− generation, each MgO nanostructure (700 μg/mL) was incorporated into NBT (1 mM) with vigorous stirring in the dark followed by centrifugation (5000 rpm, 10 min) and filtration. Aliquots (3 mL) were collected at regular intervals of time, and the absorbance was recorded with a UV−vis spectrophotometer (JASCO V650) at 259 nm using quartz cuvettes with 1 cm path length.4 For concentrationdependent O2•− generation, each nanostructure at various concentrations (50, 100, 200, 500, and 700 μg/mL) was assessed for 4 h. For •OH generation, TA (2 mM) was added to an aqueous suspension of each of the MgO nanostructures (700 μg/mL) with constant stirring in the dark. The reaction product (5 mL) after successive centrifugation (5000 rpm, 10 min) and filtration (Whatman, 0.45 μm) was withdrawn at regular intervals of time, and 200 μL of the supernatant was transferred into black 96-well plates. The fluorescence intensity (λex 312 nm/λem 430 nm) was measured with a fluorescence plate reader (Spectramax M5, Molecular device).4 Antioxidant Activity Measurements. To measure the antioxidant activity of MgO nanostructures, 200 μL aliquots of either control or sample solution were placed in white 96-well plates and the absorbance (As) was recorded using a microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific, USA) at the characteristic wavelength. Ultrapure water was used as control. For superoxide (O2•−) scavenging, 300 μL of MgO nanostructures having different concentrations (20, 40, 100, 250, and 500 μg/mL) were separately mixed with Na2CO3 (50 mM, 300 μL), NBT (24 mM, 120 μL), EDTA (0.1 mM, 120 μL), and hydroxylamine hydrochloride (120 μL) and the reaction mixture was incubated at 25 °C for 15 min. The scavenging was measured as the percentage change in absorbance of the reaction mixture at 560 nm37 and calculated as % inhibition =

was further diluted to 100, 200, 400, 600, 800, 1200, and 1600 μg/ mL. Each inoculum (2 × 106 CFU/mL) was spotted in agar plates containing those concentrations and incubated at 37 °C for 24 h. The minimum concentration of the drug that inhibits bacterial growth was recorded as MIC. In the turbidity method, 200 μL of nanoparticles, nanoplates, and nanorods were added to 1 mL of fresh inoculums (∼1.5 × 108 CFU/mL) in 2 mL Eppendorf tubes and incubated at 37 °C for 24 h in an orbital shaker (150 rpm). Bacteria cells without nanostructures were used as a control. The absorbance of the bacterial suspension was recorded using a UV−vis spectrophotometer (JASCO V650) at 600 nm. The antibacterial potency was expressed as the change in absorbance at 600 nm.20 All antibacterial activity tests were performed in triplicate. Anti-Biofilm Activity Measurement. Biofilm inhibition of B. subtilis ATCC 6633 was quantified by a colorimetric detection process.39 Specifically, fresh inoculums (1 × 106 CFU/mL, 200 μL) was pipetted out into white 96-well plates, onto which nanoparticle, nanoplate, and nanorod solutions (1.6 g/L) were added and incubated at 37 °C for 4 days. Control wells were marked without sample. Each incubated well was rinsed three times with sterile distilled water to remove any free bacteria. CV (10% (v/v), 400 μL) was pipetted out into each well and left for 1 h to stain the biofilm followed by washing with sterile distilled water. Quantification of the biofilm was assessed by eluting CV with 95% ethanol (400 μL) for 10 min. Finally the mixture (200 μL) was pipetted out into another white 96-well plate and the absorbance was recorded at 570 nm with a UV− vis spectrophotometer (SpectraMax M5, Molecular Devices). Biofilm inhibition was expressed as change in absorbance at 570 nm and calculated as % inhibition =

Acontrol

× 100

DPPH Scavenging Measurements. For DPPH scavenging, an ethanolic solution of DPPH (0.1 mM, 200 μL) was mixed with 200 μL of MgO nanostructures at different concentrations (20, 40, 100, 250, and 500 μg/mL) and incubated in the dark at room temperature for 30 min. Aliquots (200 μL) of either control or sample solution were put into white 96-well plates, and the absorbance (As) was recorded using a microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Fisher Scientific, USA) at 517 nm.37 Ultrapure water was used as a control. The free radical scavenging activity was expressed as the percentage of change in absorbance of DPPH at its characteristic wavelength.

Ac − A s × 100 Ac

where Ac and As are the absorbances of control and sample, respectively. AA and BHT were used as standards. Hydroxyl (•OH) scavenging was assessed by mixing 80 μL of MgO nanostructures with different concentrations (20, 40, 100, 250, 500 μg/mL) with FeCl3·6H2O (100 mM, 80 μL), H2O2 (1.25 mM, 80 μL), 2-deoxy-D-ribose (2.5 mM, 80 μL), and AA (100 mM, 80 μL). The whole mixture was incubated at 37 °C for 1 h, mingled with TBA (0.5% (diluted with 0.025 M sodium hydroxide), 40 μL) and TCA (2.8%, 40 μL), and heated at 100 °C for 30 min. The colored product was cooled to room temperature. The •OH scavenging was measured by the percentage change in absorbance of the final product at 532 nm:37 % inhibition =

Acontrol − A sample

% inhibition =

Ac − A s × 100 Ac

where Ac and As are the absorbances of control and sample, respectively. AA and BHT were used as standards. Nanotoxicity of MgO: Morphological Impact on Antioxidant Defense System. Oxidative damage on AA by MgO nanostructures was monitored by UV−vis spectroscopy.9 Specifically, each nanostructure (200, 700 μg/mL) was mixed with AA (5 mM). The solution was kept for 1 h in the dark, and the sample (5 mL) was collected after centrifugation (5000 rpm, 10 min) and filtration. The oxidative damage on AA was quantified by measuring its absorbance at the characteristic wavelength λmax (265 nm). AA without MgO was marked as control. Loss of AA was calculated using the following formula: % loss = [(absorbance of control) − (absorbance of sample)]/(absorbance of control) × 100.

Ac − A s × 100 Ac

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where Ac and As are the absorbances of control and sample, respectively. GA was used as a standard. Antibacterial Activity Measurement. The antibacterial activity of MgO nanostructures was evaluated by minimum inhibitory concentration (MIC) tests and turbidity methods against Grampositive bacteria, i.e. B. subtilis ATCC 6633, M. luteus ATCC 10240, and S. aureus ATCC 29737, and Gram-negative bacteria, i.e. E. coli ATCC K88 and K. pneumoniae ATCC 10031. sMIC evaluation was carried out by the agar dilution method.38 Specifically nanoparticles, nanoplates, and nanorods of MgO were dissolved in DMSO (2%) and sterile water to make a final concentration of 10000 μg/mL, which

RESULTS AND DISCUSSION Physical Characterization of MgO Nanostructures. The MgO nanostructures produced having a face-centeredcubic phase and lattice parameter a ≈ 2.33 Å were confirmed by XRD (shown in Figure S1A). FESEM and TEM studies on the rodlike structures (Figure S1B,C) reveal their lengths to be ∼1.75−4 μm and diameters to be ∼70 nm, while the platelike structures (Figure S1D,E) have well-defined uniform and C

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry homogeneous nanoplates of average thickness ∼12.5 nm and width ∼50−70 nm. TEM micrographs of MgO nanoparticles (Figure S1F) demonstrate that they consist of spherically shaped nanoparticles (diameter ∼36−50 nm). Selected area electron diffraction patterns with spots, superimposed on rings, explore the polycrystalline nature of the synthesized sample (inset of Figure S1C,E,F). The difference in the SEM and TEM micrographs is attributed to agglomeration, which could be diminished by ultrasonication of the TEM sample preparation. Surface areas obtained from BET measurements are 113.2, 342.2, and 47.02 m2/g. Pore diameters, analyzed by the BJH method, are ∼6.07, 4.91, and 3.49 nm for nanorods, nanoplates, and nanoparticles, respectively (Figure S2A−C). The highest surface area of the nanoplates can likely be attributed to its two-dimensional nature.40 FTIR spectra of the synthesized samples (shown in Figure S3A) consist of intense bands at 862 and 418 cm−1 corresponding to the stretching vibration of Mg−O bonds.41 Figure S3B demonstrates typical Raman spectra of the MgO nanostructures, which shows a characteristic band at 1060 cm−1, assigned to the surface phonon mode.42,43 UV−vis diffuse reflectance spectra of the MgO nanostructures (shown in Figure S3C−E) illustrate two reflection minima at 220 and 287 nm, which can be attributed to defect-mediated electronic excitation. Optical band gap values, calculated from reflectance spectra, are 3.73, 3.61, and 3.80 eV for nanorods, nanoplates, and nanoparticles, respectively. The lower band gap, observed for our synthesized nanostructures in comparison with that of bulk MgO (7.8 eV), is attributed to the presence of defects.29,43 Such an observation is in good agreement with a previous investigation by Jain et al.44 and could be resolved further by luminescence spectroscopy, as MgO nanostructures often show luminescence depending on defects from anionic or cationic vacancies or low-coordinated sites at the surface.45 Luminescence spectra (shown in Figure S3F) consist of defect-related violet emission at 388 and green emission at 527 nm, followed by additional emission peaks, obtained at 470 nm for nanorods, at 431 nm for nanoplates, and 411 nm for nanoparticles. Violet and green emissions are assigned to doubly (V2+ O ) and singly charged (V+O) oxygen vacancies, denoted as F2+ and F+ centers, respectively, owing to the encounter of free electrons in the conduction band with F2+ and F+ centers, respectively, described by the equations F2+ + e ↔ F+ + hν and F+ + e ↔ F + hν.46 The formation of F+ and F2+ centers can be expressed by the equations OO = V +O + 2+ OO = VO +

1 O2 (g) + e− 2 1 O2 (g) + 2e− 2

the equation F23+ + e ↔ F22+ + hν. The higher intensity of indigo emission indicates higher F23+ concentrations in MgO nanoparticles in comparison with MgO nanoplates. These defects result from incomplete oxidation of Mg(OH)2 into MgO nanostructures.29 This can be ascertained from a DTA/ TGA plot of these three nanostructures (Figure S4A−C). For nanorods the endothermic peak for transformation of the hydroxyl group of Mg(OH)2 to the oxide ion of MgO (Figure S4A) is observed at 394 °C, which is lower than that for nanoparticles (400 °C) as observed in Figure S4C.41 It is noteworthy that the maximum shift of the endothermic peak (362 °C) is observed in the case of nanoplates (Figure S4B). This displacement in endothermic peak observed from Figure S4 is ascribed to incomplete oxidation of MgO.45 O2•− and •OH Generation and Scavenging by MgO Nanostructures in the Dark: Concentration-Dependent Crossover. Defects in nanostructured materials can facilitate electron transport and electron sstorage.9 Trapped electrons can be transferred to surface-adsorbed O2 molecules to produce O2•−. O2•−-induced antimicrobial activity5,47 of various MgO nanostructures such as microrods, nanoparticles, and microspheres has been examined by researchers.31,48−50 While O2•− generation by most of the nanostructures was studied under light irradiation, a previous study shows that ZnO nanoparticles could facilitate O2•− generation in the dark due to the migration of trapped electrons at the surface of ZnO.4 A recent study by Hao et al. also illustrated that oxygen vacancies in MgO nanostructures can produce O2•− in the dark.35 Our synthesized nanostructures contain various defects, as observed from PL spectra (Figure S3F); we have investigated O2•− generation in the dark at a fixed time interval of 4 h over a wide range of concentrations (50, 100, 200, 500, and 700 μg/mL) (Figure S5) using the nitro blue tetrazolium chloride (NBT) assay.4 A decrease in the absorption maxima (λmax 259 nm) of NBT due to the decomposition of its molecular structure to formazone was used to quantify O2•− generation (Figure 1).

(1)

(2)

The violet emission exhibits similar intensity for all samples, illustrating identical F2+ defect concentrations within the nanostructures. Nanorods exhibit the highest intensity for green emission, followed by nanoplates and nanoparticles: i.e., the concentration of F+ centers is in the order nanorods > nanoplates > nanoparticles. Pathak et al. reported the defect centers at 2.58 eV below the conduction band, formed due to clustering of F2+ and F0 defect centers, resulting in intense blue emissions at 470 nm which is described by the equation F2+ + e ↔ F2 + hν.46 Indigo emissions at 431 and 411 nm are attributed to electronic transitions between conduction bands and F23+ states following electron encounter, as described by

Figure 1. Concentration-dependent NBT degradation in the presence of MgO nanoparticles, nanoplates, and nanorods after 4 h.

The concentration-dependent degradation of NBT by our MgO nanostructures was plotted as ln(C/C0) vs concentration, where C0 and C represent the molar concentrations of NBT without and with varying amounts of MgO. Significant generation is observed in the presence of MgO nanorods within this time interval. The figure also demonstrates an initial sharp increment of O2•− generation with concentration, D

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

quantities of •OH were not produced), observing •OH generation in the order nanorods, nanoparticles > nanoplates. Defects in nanostructures can store electrons which eventually scavenge oxygenated free radicals, 9 i.e. an antioxidant property is imparted to them, and the search for nanomaterials with antioxidant properties is a topic of intense current research interest as they reduce oxidative stress, caused by overproduction of O2•− and •OH.24 To investigate defectinduced antioxidant activity within MgO nanostructures, a series of experiments were performed to illustrate the O2•−and •OH-scavenging properties of our samples. The in vitro O2•−-scavenging activity of our MgO nanostructures was investigated at different concentrations (20−500 μg/mL).37 In contrast to in vivo enzymatic O2•− scavenging by dismutation reaction, nonenzymatic scavenging of O2•− was investigated with the NBT assay. As represented in Figure 4,

followed by continuous enhancement until it eventually plateaus. Importantly, no measurable O2•− generation was observed for nanoplates or nanoparticles below a concentration of 500 μg/mL. In this context, O2•− generation kinetics (NBT absorption spectra at different time intervals as shown in Figure S6), evaluated at a higher concentration (700 μg/mL), is presented in Figure 2.

Figure 2. O2•− generation kinetics of nanoparticles, nanoplates, and nanorods of MgO (inset shows rate constant of O2•− generation from these nanostructures).

Interestingly, all of these nanostructures generate significant amounts of O2•− at higher concentrations and the rate of generation is found to be in the order nanorods > nanoplates > nanoparticles (shown in the inset of Figure 2). Hydroxyl radicals (•OH) are another physiologically relevant reactive oxygenated species which have a strong effect on the antibacterial activity of nanomaterials, and therefore we studied its production by our MgO nanostructures. We tested • OH generation by MgO nanostructures using TA as the probe molecule. The fluorescence peak of 2-hydroxyterepthalic acid, formed due to reaction between TA and •OH, was used for the quantitative analysis of •OH generation. • OH is also known to be a derivative of O2•−;4 consequently we evaluated •OH generation kinetics (FL spectra of TA at different time intervals shown in Figure S7) at a nanomaterial concentration of 700 μg/mL, presented in Figure 3 (the kinetics was not studied at low concentrations, as measurable

Figure 4. O2•−-scavenging activity of nanoparticles, nanoplates, and nanorods of MgO.

nanoparticles exhibit the highest O2•− scavenging activity of ∼60% at a concentration of 40 μg/mL, followed by nanoplates and nanorods (∼40%). Notably, O2− scavenging decreases monotonously with increasing concentration, particularly in the case of nanorods. There are two plausible mechanisms for O2•−scavenging. MgO nanostructures can either scavenge O2•− directly or degrade NBT with O2•− generated by MgO nanostructures. To gain insight into the mechanism, we used experiments described by Kimura.51,52 O2•− scavenging is determined by two phenomena and depends on the ratio of activities of the phenomena, where the concentration dependence of the scavenging follows a sigmoid nature that we have observed for our nanomaterials (particularly in the case of nanorods), confirming that the MgO nanostructures have the potential to scavenge O2•− at low concentrations, while no • OH-scavenging activity was observed by any of the nanostructures. Study of Concentration-Dependent Crossover between Pro-oxidant and Antioxidant Activity of MgO Nanostructures. The MgO nanostructures exhibit prooxidant as well as antioxidant activity, and the origin of these properties can be explained in the following way: electron− hole pairs are generated within metal oxide semiconductors by excitation of electrons in the presence of light of the appropriate wavelength and the excited electrons after reacting

Figure 3. Kinetics of hydroxyl radical (•OH) generation from nanoparticles, nanoplates, and nanorods of MgO showing increasing production of •OH over time. E

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry with molecular oxygen produce O2•−. However, such photoexcitation of electrons does not happen in the dark; hence, O2•− generation in the dark is attributed to electron migration from surface defect states to 2π* antibonding orbitals of O2 depending on the redox potential of the defect states.53 Our optical studies on MgO nanostructures reveal F+, F2+, and F23+ defect states and their corresponding redox potentials are calculated to be −0.73, −0.44, and −0.20 eV on the NHE scale (detailed calculations are given in Scheme S1): i.e., these states have redox potentials higher than that of the O2/O2•− reaction (−0.20 eV),8 illustrating the feasibility of the redox reaction by electrons trapped at these defects. The generation of O2•− can be described by the equation O2 + e−(trapped at F+/F+2 /F32 + defects) → O2•−

vitamin C,55 tea catechin oxypolymers,56 etc., it is very rare in inorganic metal oxide semiconducting nanostructures. Recently, nanoceria was noticed to have such competitive properties depending on the relative ratio of Ce3+ and Ce4+ ions.57 The crossover of competitive activities induced by light has been studied for graphene quantum dots,9 but here we report the concentration-dependent crossover between antioxidant and pro-oxidant activity for the first time for MgO nanostructures among various metal oxide nanostructures. Chakraborty et al. reported concentration-dependent crossover which includes antioxidant properties at low concentration and pro-oxidant activity for high concentrations of vitamin C conjugated nanoparticles, but the investigation was focused on targeted tumor cell delivery.23 Here we report defect state induced concentration-dependent crossover for the first time. Thus, the concentration-dependent crossover can be explained in the following way: the O2•− generation (O2•−generation) and scavenging (O2•−scavenging) ability of MgO nanostructures, described by the eq 3,7 respectively, can be expressed by

(3)

The redox potential (+4.71 eV, on the NHE scale) of the valence band of MgO nanostructures is higher than that of the H2O/•OH reaction (+2.2 eV, on the NHE scale), but no acceptor level hole production was noticed from luminescence spectroscopy. Thus, in contrast to hole-mediated direct •OH generation, we propose O2•−-induced secondary generation of • OH, which can be described by the equations4,54 O2 •

•−

•

+ H 2O → HO2 + OH

−

(4)

•

HO2 + HO2 → H 2O2

H 2O2 + O2

•−

O2•−(generation) = k(O−2 generation)Defect(F+ and F2+

(5) •

→ O2 + OH + OH

−

(8)

O2•−(scavenging) = k(O−2 scavenging)Defect(F2 +)

(9)

where k(O−2 generation) (k(O−2 scavenging)) is a proportionality constant, which depends on types of defects. Defect(F+ and F2+ and F23+) and Defect(F2+) represent the corresponding defect concentrations. To gain insight into the concentration dependence of these defects, we studied concentrationdependent photoluminescence spectra of our nanostructures (shown in Figure S8). It is evident that, at lower concentrations, emission from F2+ predominates over other processes, while at higher concentrations, significant emissions are noticed to originate from F+ and F2+ and F23+. Therefore, at lower concentrations, MgO nanostructures exhibit antioxidant activity due to F 2+ defect centers, while at higher concentrations nanostructures become aggregated, which results in the suppression of F2+ defects, followed by the predominance of F+ and F2+ and F23+ defects, resulting in prooxidant activity. The EPR spectra (shown in Figure S14) of the MgO nanostructures reveal the presence of singly ionized defect states, as the defects have g values (g = 2.00232) close to that of free electrons.58,59 The following order of EPR intensity is observed: nanoparticles > nanorods > nanoplates. Interestingly, this variation is found to be similar to F2+-related luminescence intensity (Figure S8A−C), and other defect states such as F2+ and F23+ (clusters of F2+ and F+) could not be detected by EPR. In order to elucidate other defect states, the nanostructures were characterized by XPS. Careful analyses of the highresolution XPS spectra of Mg 2s states (shown in Figure S15) reveal that the spectra of all the samples have an intense peak at 87.5 eV,60 attributed to Mg 2s electrons bound to O2−, and a weak peak at 89.13 eV, which is assigned to Mg 2s electrons surrounded by F23+ defects. Analysis illustrates 22%, 37%, and 50% F23+ defects in nanorods, nanoparticles, and nanoplates, respectively, and the trend corroborates the variation of F23+related luminescence intensity (Figure S8A−C). In general, agglomeration of the nanostructures is controlled by two interactions: long range and short range. Long-range interactions control collisions between nanostructures in

(6)

•−

O2 , thus formed, reacts with water solvents to form hydroperoxyl radicals (•HO2) and hydroxyl ions (OH−) (eq 4), and •HO2 recombines to form H2O2 (eq 5). Reaction between H2O2 and O2•− gives rise to •OH generation (eq 6). Singlet oxygen does not form in the dark, as it requires holes. The redox potential of F2+ was observed to be +0.11 eV on the NHE scale (Scheme S1). That is, F2+ defect sites may act as electron acceptors to scavenge O2•−, and hence their antioxidant activity is attributed to F2+ defect states and can be described by the equation O2•− → O2 + e−(trapped at F 2 + defects)

and F2 3 +)

(7)

•−

As O2 /O2 are mobile in water and get easily dissolved in water, the influence of surface area is not as significant for the generation/scavenging of O2 (Figures 2 and 4, respectively). Consequently, O2•− generation (scavenging) by the nanostructures is likely to be dependent on electron donation (acceptance) potency of F23+, F2+, and F+ (F2+) defect states, respectively, attributed to their redox potential. Our experimental evidence corroborates this mechanism: i.e., nanorods with the highest number of F+ defect sites (Figure S8A) exhibit the highest O2•− generation (Figure 2), while nanoparticles with the highest number of F2+ defects (Figure S8C) possess the highest O2•− scavenging activity (Figure 4). Again •OH is produced via an O2•−-mediated pathway (eqs 4−6), and so the strongly negative redox potential of MgO nanostructures is a prerequisite for such conversion. Nanorods and nanoparticles possess higher levels of F+ and F2+ defect states (−0.73 and −0.44 eV), respectively (Figure S8A,C) in comparison to nanoplates (Figure S8B), which facilitates •OH generation via an electron-mediated pathway from these nanostructures (Figure 3). Therefore, the influence of surface area is not too significant for •OH production. Though the simultaneous existence of such competitive antioxidant and pro-oxidant phenomena was noticed in F

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Inorganic Chemistry

charge transfer from O2•− radicals into F2+ defect states; hence, it supports the suppression of F2+ defect states at higher concentration. Thus, the concentration-dependent crossover has the potential to become a novel tool for cancer cell therapy with minimal adverse effects on normal cells (shown in Scheme 1).

colloidal form, while short-range interactions influence physical processes such as nanostructure surfaces in contact with each other during collisions. According to Derjaguin−Landau− Verwey−Overbeak theory, the latter is determined by van der Waals attractions and electrostatic double-layer repulsion.61,62 It is evident from concentration-dependent photoluminescence spectra that, at lower concentrations, emission from F2+ predominates over others, while significant emissions originate from F+, F2+, and F23+ at higher concentration. Analysis shows that F2+ defects scavenge O2•−, while other defects generate O2•− due to their suitable redox potential. It has been investigated earlier by researchers that F2+ defect states are associated with strong electric fields.63,64 In the lowconcentration regime, not only are there fewer collisions between particles but also the electric field associated with F2+ defects inhibits the agglomeration of nanostructures. At higher concentrations not only does the probability of collisions increase but also fewer F2+ defects become converted into F+ by O2•− and OH− ions. Such conversion suppresses electrostatic double-layer repulsion between nanostructures during collision in colloidal form, and consequently nanostructures become agglomerated. Agglomeration is associated with suppression of F2+ defect states, while other defects are not affected significantly by agglomeration. Such radical-induced agglomeration has also been noticed by other researchers.65 All MgO nanostructures exhibit O2•− scavenging activity in the concentration range of 20−500 μg/mL (Figure 4); the activity initially increases with concentration and finally decreases beyond a concentration of 40 μg/mL. The decreasing tendency of DPPH scavenging activity with increasing concentration of Se nanoparticles has also been identified.66 At a concentration of 40 μg/mL, scavenging activity has been observed in the following order: nanoparticles (∼60%) > nanoplates (∼40%), nanorods (∼40%). F2+ defect states are O2•−-scavenging centers since the strong electric field60,61 with positive redox potential (+0.11 eV on the NHE scale) (Scheme S1) easily scavenges negatively charged O2•− radicals. The intensity of the violet emission which is the fingerprint of the F2+ defect state follows the same trend (Figure S8A−C), corroborating scavenging activity. Nanoparticles with the highest F2+ defect states exhibit the highest scavenging activity, whereas similar F2+ defect states are noticed for nanoplates and nanorods. The decrease in O2•− scavenging at higher concentrations (>40 μg/mL) is attributed to the suppression of F2+ defect states due to the agglomeration effect. As mentioned above, agglomeration is mediated by F2+ defects and other defect states (e.g., F2+ and F23+) present in all the synthesized nanostructures generate O2•− due to electron donation properties, attributed to negative redox potential (−0.20 and −0.44 eV, respectively). As the different nanostructures have different F2+ and F23+ concentrations, they exhibit different O2•− generation rates. Concentration-dependent photoluminescence spectra illustrate the higher intensity of F2+-related emission (Figure S8A− C) in the low-concentration range (20−40 μg/mL). Thus, O 2•− scavenging activity predominates for all of the nanostructures in this concentration range. At higher concentration (>40 μg/mL), F2+ defect states become suppressed due to agglomeration effects (discussed in the previous section) and consequently reduce O2•− scavenging. Thus, O2•− generation (i.e., pro-oxidant activity) dominates at higher concentrations. The appearance of the F+-related peak at higher concentrations (700 μg/mL) could be attributed to

Scheme 1. Cancer Cell Therapy and Free Radical Scavenging in Normal Cells by MgO

MgO can impart oxidative stress via production of O2•− and OH to kill cancer cells in close proximity without any illumination and simultaneously low concentrations of MgO found at distant regions from cancer cell will scavenge free radicals, thereby protecting normal cells from released oxidative stress. This proposed mechanism may complement PDT and stressful chemotherapy of cancer cells in the foreseeable future. Exploring O2•−- and •OH-Induced Therapeutic Application of MgO: Antibacterial and Antibiofilm Activity. Primary production of O2•− and secondary generation of •OH by our MgO nanostructures indicate their ability to have antibacterial activity. MgO nanostructures have been tested for antibacterial activity against five bacterial strains, including Gram-positive B. subtilis 6633, M. luteus 10240, and S. aureus 29737 and Gram-negative E. coli K88 and K. pneumoniae 10031. E. coli (Gram-negative) and S. aureus (Gram-positive) have been chosen here because they are the most common pathogenic bacteria. B. subtilis, M. luteus, and K. pneumoniae have been selected because these bacteria are known to form robust biofilms on mammalian skin, gastrointestinal tracts, intestines, etc. Agar plates with each inoculum (2 × 106 CFU/ mL) were incubated with various concentrations of MgO nanostructures for 24 h at 37 °C, and the MIC of each type of nanostructure was studied (Table 1).The MIC values of the MgO nanostructures were found to be comparable with those of other metal oxide nanoparticles.67,68 Interestingly, the MgO nanoparticles have significant antibacterial activity against all five strains tested, whereas nanorods show low activity against B. subtilis, M. luteus, and S. aureus and nanoplates show low activity against B. subtilis and S. aureus. Nanoparticles and nanorods are more active against B. subtilis than nanoplates, whereas nanorods and nanoplates exhibit higher antibacterial activity against S. aureus in comparison to nanoparticles. Nanorods produce higher quantities of O2•− and •OH but do •

G

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Inorganic Chemistry Table 1. MIC Test Results of Nanoparticles, Nanoplates, and Nanorods of MgO against Five Different Bacteria MIC (μg/mL) nanoparticles nanoplates nanorods

B. subtilis 6633

M. luteus 10240

S. aureus 29737

E. coli K88

K. pneumoniae ATCC 10031

400 ± 0.005 800 ± 0.0054 400 ± 0.0043

600 ± 0.017 not found 800 ± 0.023

400 ± 0.0034 200 ± 0.0078 200 ± 0.007

1200 ± 0.004 not found not found

1200 ± 0.012 not found not found

Figure 5. Growth kinetics of (a) B. subtilis 6633, (b) S. aureus 29737, and (c) E. coli K88 after incubation with nanoparticles, nanoplates, and nanorods, respectively. (d) Biofilm inhibition efficiency against B. subtilis 6633 after 4 days in the dark.

as model Gram-positive bacteria. The bacterial growth kinetics in the presence of the MgO nanostructures in the dark is shown in Figure 5a−c. Outstanding bactericidal activity was observed for all MgO nanostructures against B. subtilis 6633. Though nanostructures generate O2•− and •OH differently, no significant difference in bactericidal activity was observed. The inhibition efficiency after 4 h of incubation was calculated to be 92, 88, and 90% for nanoparticles, nanoplates, and nanorods, respectively. In the case of S. aureus 29737, the growth kinetics indicates bacteriostatic activity20 and we observed the following order of bacteriostatic activity: nanoparticles > nanorods > nanoplates. As the antibacterial activity of metal oxides is necessarily a surface phenomenon,67 the difference in antibacterial properties, i.e. bactericidal activity against B.subtilis 6633 and bacteriostatic activity against S. aureus 29737, is attributed to their intrinsic surface potentials giving different electrostatic stresses.69 In the case of E. coli K88, nanoparticles inhibit bacterial population and this is attributed to its high negative surface potential (−23.6 mV) that gives very weak electrostatic interaction69s with nanoparticles as well as O2•−. In both methods of antibacterial activity, growth inhibition for E. coli is weaker than that for B. subtilis and S. aureus, which may be the cumulative effect of difference in cell wall and membrane structures, cell physiology, metabolism, and degree of contact.5 We propose antibacterial activity against E. coli K88 is mostly caused by •OH radicals, which is consistent with the established mechanism where the cytoplasm of E. coli cells

not show the highest antibacterial activity, and nanoparticles show superior antibacterial activity, suggesting that there is a secondary influence other than O2•−- and •OH-induced oxidative stress, where surface potential and the shape of the particle may play a vital role. We propose that electrostatic interactions between MgO nanostructures and bacteria make a significant contribution to the antibacterial activity of the materials, which depends on the surface potential of the nanostructures: −12.6 mV for nanoplates, −6.84 mV for nanoparticles, and −6.02 mV for nanorods, respectively (Figure S9). The antibacterial activity of MgO nanostructures is attributed to the combined effects of oxidative and electrostatic stress. Table 1 also demonstrates that the toxic effect toward bacterial cells starts at ∼200 μg/mL, which is the approximately the same concentration necessary for prooxidant activity of the MgO nanostructures, and this value is consistent with a previous report.5 The higher MIC values of nanoparticles against E. coli and K. pneumoniae is attributed to their higher negative surface charge that causes weaker electrostatic stress. Such differences in antibacterial activity of MgO nanoparticles, evaluated by the zone of inhibition (ZOI) technique, has also been observed by Bindhu et al. against Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria.32 For better insight into the antibacterial activity, the bacterial growth kinetics was evaluated by turbidity methods (accredited to produce the most accurate result among various conventional techniques20) was using B. subtilis 6633 and S. aureus 29737 as model Gram-negative bacteria and E. coli K88 H

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Inorganic Chemistry was damaged by MgO nanoparticles.5 In addition, bacterial inhibition efficiency is found to be proportional to •OH concentration for other bacteria strains, indicating •OHmediated antibacterial activity of the synthesized MgO nanostructures. Since some fraction of O2•− becomes scavenged by nanostructures, we believe that they would not contribute significantly to the antibacterial activity. Bacteria do not always exist in planktonic forms; sometimes they form surface-adherent structured community a few layers thick, called biofilms. Biofilms impart a major challenge for treatment, as they exhibit increased tolerance against antibiotics and some become resistant to host immune systems as well.2,33 Several mechanisms have been proposed for the resistive behavior of biofilms against antibiotics. First, bacteria become bound stiffly to living and nonliving surfaces, resisting themselves from host immune systems in vivo or biocides in vitro. Biofilms inhibit nutrient intake and slow cellular processes; hence, antibiotics become ineffective toward biofilms.70 Moreover, a biofilm’s exopolymeric matrix and bacterial dead cells act as a barrier for diffusion of antibiotics; hence, live bacteria become protected.2 Generally, biofilms form in/on catheters2 or faucets or create intense adhered communities in the lungs of individuals with cystic fibrosis or chronic obstructive pulmonary disease. For the sake of morphology-controlled toxicity in the broader sense, the biofilm inhibition efficiency of MgO nanostructures has also been examined against B. subtilis 6633, as all of the nanostructures show the highest antibacterial activity against B. subtilis 6633. This particular bacterium often forms biofilms on gastrointestinal tracts; thus, the present study gives us an insight into potential treatments for biofilms of B. subtilis 6633. Antibiofilm activity was measured at various concentrations (400−2000 μg/mL) using a CV assay based colorimetric detection protocol (Figure S10). In this context it may be stated that the present assay quantifies the amount of extracellular polymeric substances, generated by biofilms, and indirectly monitors biofilm biomasses.39 Here, we have calculated biofilm inhibition efficiency according to the method suggested by Jaiswal et al.71 Though 10−12% biofilm inhibition efficiency is noticed at lower concentrations (400− 800 μg/mL) of nanoparticle and nanoplate samples (Figure S10), no significant biofilm inhibition was observed for nanorods (Figure S10). At higher concentrations (2000 μg/ mL), the highest biofilm inhibition efficiency was observed for nanoparticles, followed by nanorods and nanoplates (Figure 5D). The biofilm inhibition efficiency of MgO nanostructures is comparable with that of Ag nanoparticles against S. epidermis (10−15%).70 Therefore, the simultaneous presence of antibacterial and antibiofilm activity in MgO nanostructures at high concentrations (particularly for nanoparticles) leads us to conclude that they could promote healing of chronic wounds, particularly gastrointestinal wounds. Interestingly, nanoparticles exhibit the highest antibacterial and biofilm inhibition, indicating that these two activities are primarily related to O2•− generation.72 However, biofilm inhibition is found to be several-fold less than antibacterial efficiency, in line with reports that materials with simultaneous antibacterial and biofilm inhibition may not be effective against mature biofilms.73 DPPH Scavenging by MgO Nanostructures. MgO nanostructures are known to have significant O2•− scavenging ability; thus, to explore the antioxidant activity of MgO nanostructures further, we have also evaluated their antioxidant

behavior using a DPPH free radical scavenging assay. DPPH, a synthetic nitrogen-based lipophilic-free radical, is widely used to assess antioxidant activities of various natural and inorganic materials in a concentration-dependent manner. The concentration−response curve for the MgO nanostructures (Figure 6)

Figure 6. DPPH radical scavenging of MgO nanoparticles, nanoplates and nanorods.

illustrates that all the nanostructures show DPPH scavenging activity in a concentration-dependent fashion. It is also noted that the DPPH scavenging ability of nanoplates monotonically increases with concentration, while nanoparticles and nanorods do not show significant changes in scavenging activity with respect to concentration. Such concentration-independent scavenging activity had previously been observed by Lee et al. for gold@platinum nanoparticles.74 At the highest concentration (500 μg/mL), DPPH scavenging activity was observed in the order nanoplates > nanorods > nanoparticles. Nanoplates offer 100% DPPH scavenging ability at this concentration. The excellent DPPH scavenging ability of MgO nanoplatelike nanostructures illustrates their potential for inflammatory treatment,24,25 related to nonhealing chronic wounds.75 The half-maximum inhibitory concentrations, defined as the concentration of a material with 50% scavenging efficiency (IC50 (μg/mL)),25 are found to be 126.47, 71.04, and 16.61 for MgO nanoparticles, nanorods, and nanoplates, respectively. The observed variation of IC50 is also found to be consistent with scavenging activity for MgO nanostructures at a concentration of 500 μg/mL. We compared the IC50 values corresponding to our samples with those of other conventional antioxidants (see Table S1). The table clearly demonstrates that all nanostructures exhibit low IC50 values, indicating their higher potency in comparison to Thymol,25 Carvacol,25 DVHA flower extract,37 etc. Among the MgO nanostructures, the nanoplates possess IC50 values similar to those of Trolox and vitamin C. DPPH scavenging activity, similar to O 2•− generation, generally depends on electron donation. While MgO nanorods exhibit O2•− generation, the highest DPPH scavenging activity was observed for MgO nanoplates. Such a difference may be attributed to electron transfer compatibility between electron-donating (defects in MgO) and -accepting orbitals (O2 or DPPH) and adsorption of the radicals on nanostructures. As DPPH has a larger size than O2, it would be reasonable to assume that nanostructures with higher surface area are able to adsorb more DPPH; hence, higher DPPH I

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Inorganic Chemistry Scheme 2. Mechanistic Pathway of MgO+-DPPH− Formation for DPPH Scavenging

scavenging activity would be expected from nanostructures with greater surface area, as we observed. DPPH is scavenged by electron transfer from F23+, F2+, and F+ defect sites of MgO nanostructures to the central nitrogen atom of DPPH free radicals.76,77 After electron transfer, DPPH is negatively charged, while MgO is positively charged. Since DPPH is insoluble in water, it interacts with the defect sites of the surface of the MgO nanostructure by electrostatic forces and the complex MgO+-DPPH− is formed (Scheme 2). The formation of this complex is surface area dependent. Hence, DPPH scavenging activity is found to be proportional to the surface area of the nanostructures, and similar trends have been noticed previously by others.76 Thus, MgO nanostructures, particularly nanoplates, have high potency toward DPPH scavenging. Nanotoxicity of MgO: Morphological Effect on Antioxidant Defense System. Our study demonstrates that MgO nanostructures have scavenging activity against intracellular O2•− at low concentrations, indicating their ability to protect cells from toxicity elicited by O2•−. In addition, DPPH scavenging activity has also been identified in MgO nanostructures: i.e., the MgO nanostructures are very good antioxidants. Though antioxidant activities have been noticed in MgO nanostructures, their interaction with biogenic antioxidants (e.g., ascorbic acid, uric acid, polyphenol, etc.) is required for real world applications. We selected ascorbic acid (AA) as a model antioxidant, as it is a very well known antioxidant protecting the human body from chronic diseases and is widely used in foods, cosmetic products, etc. At a low concentration of 200 μg/mL, which is the crossover concentration between antioxidant and pro-oxidant activities, and at the higher concentration 700 μg/mL, the interaction was monitored by the absorption peak of AA, measured at 265 nm by UV−vis spectroscopy (Figure S11) in the dark.9 We observed a loss of AA (in percent) in the presence of MgO nanostructures after 1 h exposure (Figure 7). AA is oxidized via a two-step oxidation process to finally yield dehydroascorbic acid (DHA) by the enzyme ascorbic acid oxidase (AAO). Thus, our study reveals AAO-mimetic activity of MgO nanostructures. It is clear from Figure 7 that nanoparticles cause relatively weaker degradation (∼37%) at lower concentration; however, nanoplates (∼97%) and nanorods (∼87%) give much higher degradation. Since AA is highly water soluble, oxidation of AA is believed to be dependent purely on defect concentration. F23+ has a lower negative redox potential in comparison to F+ and F2+ defect

Figure 7. Oxidation of AA by nanoparticles, nanoplates, and nanorods at two different concentrations (200 and 700 μg/mL) quantified by percent loss of AA amount.

sites (Figure S13); thus, it has a lower tendency to reduce AA. Nanoplates are highly enriched with F23+ defect sites in comparison to nanoparticles and nanorods. Consequently, nanoplates oxidize AA to the ascorbyl radical (AA*), an intermediate stable free radical of the two-step oxidation process of AA, used as a noninvasive marker of oxidative stress78 (Figure 7). Thus, this defect-induced AA oxidation overwhelms any effect of the surface area of the nanostructures. At higher concentration, all of the nanostructures significantly degrade ascorbic acid (80% for nanoparticles and 93% for nanoplates and nanorods), also corroborating pro-oxidant activity.

■

CONCLUSIONS The MgO nanostructures potentially generate O2•− at higher concentrations, while no significant trace is found at lower concentrations. Nanorods can generate O2•− to the highest extent. The nanostructures successfully scavenge O2•− at low concentrations, and this property decreased with increasing concentration. Nanoparticles scavenge O2•− to the highest extent (∼60%) followed by nanoplates and nanorods. Additionally, a linear decrement of O2•− scavenging with increasing concentrations was observed for nanorods. These nanostructures are unable to scavenge •OH regardless of concentration but significantly produce •OH at higher concentrations. The concentration-dependent and morphology-induced crossover between ROS scavenging and generation is attributed to the suppression of F2+ defect states, which is responsible for O2•− scavengin,g and the evolution of J

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Inorganic Chemistry F23+, F2+, and F+ states, which is responsible for O2•− generation. At lower concentrationsthe F2+ state (electron acceptor level) predominates, while at higher concentration F23+, F2+, and F+ states (electron donor level) become more active due to suppression of aggregated F2+ states. This crossover defines the appropriate concentration (200 μg/mL) for minimum antibacterial potency of all three MgO nanostructures. Nanoparticles exhibit the highest activity, followed by nanorods and nanoplates, and this activity is a result of O2•−-mediated oxidative stress and electrostatic stress. Biofilm inhibition is also affected in a similar fashion. The concentration-dependent crossover also unveils morphologyindependent DPPH scavenging behavior at lower concentrations, but at higher concentrations nanoplates exhibited the highest protecting ability against DPPH radicals due to the largest surface area. Additionally, this crossover also defines the antioxidant behavior of nanoparticles when they interact with ascorbic acid and the strongest oxidant nature of nanoplates. The dual activity and concentration-dependent crossover in MgO could be implemented simultaneously in cancer therapeutic agents and chemotherapy-induced free radical scavengers.

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Dipak Chanda: 0000-0003-0147-4972 Arnab De: 0000-0001-9483-9533 Bhaskar Das: 0000-0003-1589-0299 John George Hardy: 0000-0003-0655-2167 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Department of Science & Technology, Government of India, for financial assistance for S.P. through the DST-INSPIRE program (Grant No. DST/INSPIRE Fellowship/2014/82). We thank the Council of Scientific & Industrial Research, Government of India, for financial support for D.C. in the form of a project fellowship (Sanction No. CSC0135). We also thank the UGC, Government of India, for financial support for A.D. in the form of a National Fellowship. We thank the UGC-UPE-II program of Jadavpur University, Kolkata, India, for providing access to facilities including fluorescence spectroscopy and ζ a potential measurement apparatus. We thank Prof. Kausikisankar Pramanik, Department of Chemistry, Jadavpur University, Kolkata, India, for providing access to EPR spectroscopy facilities. We thank Dr. K. Muraleedharan (Director, CSIR-Central Glass and Ceramic Research Institute (CGCRI)) in Kolkata for insightful discussions and support during the preparation of this paper. We thank the staff of the Advanced Mechanical and Materials Characterization Division (AMMCD), Advanced Materials Characterization Unit (AMCU), and Materials Characterization and Instrumentation Division (MCID) at the CSIRCentral Glass and Ceramic Research Institute (CGCRI)) in Kolkata for their support with measurements. We thank Dr. Ambarish Sanyal of the Advanced Material Characterization Unit (AMCU), CGCRI, Kolkata, for providing access to XPS facilities. We acknowledge the support of a Lancaster University Faculty of Science and Technology Early Career Internal Grant and a Royal Society Research Grant (RG160449) to facilitate international collaborative research activities for J.G.H.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01938. XRD data, FESEM and HRTEM images with SAED pattern of MgO nanostructures, N2 adsorption− desorption isotherms of MgO nanostructures, BJH pore size distribution of MgO nanostructures, FTIR and Raman spectra, UV−vis diffuse reflectance and PL spectra of MgO nanostructures, DTA/TGA plots of MgO nanostructures, NBT absorption spectra in the presence of MgO nanostructures for different concentrations after 4 h exposure in the dark, absorption spectra of NBT reduction in the presence of MgO nanostructures at different time intervals in the dark, fluorescence spectra of TA with MgO nanostructures at different time intervals in the dark, PL spectra of MgO nanostructures at different concentrations, ζ potential distribution for MgO nanostructures, biofilm inhibition efficiency (%) against B. subtilis 6633 by nanoparticles, nanoplates, and nanorods of MgO at different concentrations after 4 days of incubation, UV−vis spectra of ascorbic acid (AA) in the presence of nanoparticles, nanoplate,s and nanorods of MgO at different concentrations for 1 h in the absence of light, position of various defect centers in the MgO nanostructures in an energy band diagram, position of various defect centers in MgO on the NHE scale, activity of commonly known natural antioxidants and MgO nanostructures in DPPH scavenging, and band engineering prospect of MgO nanostructures (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for J.G.H.: [email protected]. *E-mail for C.K.G.: [email protected]. ORCID

Soumik Podder: 0000-0002-8599-4635 K

DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.8b01938 Inorg. Chem. XXXX, XXX, XXX−XXX