Zinc oxide spherical-shaped nanostructures: investigation of surface

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Zinc oxide spherical-shaped nanostructures: investigation of surface reactivity and interactions with microbial and mammalian cells Diana Beatrice Visinescu, Mohammed Dyia Hussien, Raluca Negrea, Jose Maria Calderon-Moreno, Simona Schomi, Cristian Dumitru Ene, Ruxandra Barjega, Carmen Mariana Chifiriuc, Marcela Popa, Miruna Stan, and Oana Carp Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02528 • Publication Date (Web): 19 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Zinc oxide spherical-shaped nanostructures: investigation of surface reactivity and interactions with microbial and mammalian cells Diana Visinescu,a* Mohammed Dyia Hussien,b Jose Calderon Moreno,a Raluca Negrea,c Ruxandra Barjega,d Simona Somacescu,a Cristian D. Ene,a Mariana Carmen Chifiriuc,b* Marcela Popa,b Miruna S. Stane and Oana Carpa* a

“Ilie Murgulescu” Institute of Physical Chemistry, Romanian Academy, 202 Splaiul

Independentei, 060021 Bucharest, Romania; E-mail: [email protected]; [email protected] b

Department of Botanic-Microbiology, Faculty of Biology and Research Institute of the

University of Bucharest (ICUB), University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania; E-mail: [email protected] c

National Institute of Materials Physics, Atomistilor 105bis, 77125, Magurele, Ilfov, Romania

d

National Institute for Lasers, Plasma and Radiation Physics, 409 Atomistilor, POBox MG-36, 077125 Bucharest, Romania e

Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania

1 ACS Paragon Plus Environment

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ABSTRACT. Two ZnO materials of spherical hierarchical morphologies, with hollow (ZnOHS) and solid cores (ZnOSS), were obtained through the hydrolysis of zinc acetylacetonate in 1,4butanediol. The nature of the defects and surface reactivity for the two ZnO materials were investigated through photoluminescence (PL), X-ray photoelectron (XPS) and electronic paramagnetic resonance (EPR) spectroscopy proving the co-existence of shallow and deep defects and, also, the presence of polyol by-products adsorbed on the outer layers of the ZnO samples. The EPR spectroscopy coupled with spin-trapping technique showed that the surface of the ZnO samples generates reactive oxygen species (ROS) like hydroxyl (•OH) and singlet oxygen (1O2) as well as carbon-centered radicals. The ZnO materials exhibited a wide spectrum of antimicrobial activity, being active against Gram positive, Gram negative and fungi strains, both in planktonic and, more important, adherent growth state. The decrease of antimicrobial efficiency in the presence of a ROS-scavenger (mannitol) and the decrease of the cell viability with ROS level, suggests that one of the mechanism that governs both the antimicrobial and cytotoxic activity on human liver cells is ROS mediated. However, at active antimicrobial concentrations, the biocompatibility of the tested materials is very good.

INTRODUCTION The emergence of multidrug-, extensively drug- and pandrug-resistant bacterial and fungal strains prompted the search and development of new and efficient antimicrobial agents active on a broad spectrum of pathogens, through mechanisms which are different from those of conventional antibiotics. Nanomaterials represent the new “smart” antimicrobial agents,1-4 as their biotoxicity could be modulated by controlling the morpho-structural characteristics.5-10 Consequently, the development of rational synthetic methods through a judicious choice of the reactants and solvents together with a proper set-up of the synthesis parameters open large


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possibilities in formulating nanomaterials-based biocides with relevant and versatile antimicrobial properties.11 In particular, zinc oxide (ZnO) is a well-known biocompatible and non-toxic material, being intensively exploited in biomedical applications like drug delivery systems, cancer therapy, biological fluorescent imaging or biosensors.12-15 ZnO-based materials are also very promising biocidal agents with an efficiency that starts to compete the current antimicrobials.3,16-21 Several biotoxicity mechanisms were proposed, like the induction of microbial cell wall and membrane dysfunction,22-24 the internalization of ZnO nanoparticles and inhibition of different intracellular structures,22,25 the release of the active zinc(II) ions26-28 or induction of oxidative stress that generates reactive oxygen species (ROS).29 The intrinsic ROS production is considered to be the dominant mechanism being favored by the photo-excitability of zinc oxide to the UV radiation exposure, as proved by the higher antibacterial activity of ZnO in UV light.22,30 The redox reactions that take place on the ZnO nanoparticles surface play a crucial role in generation of oxygen-based radicals and H2O2 (that actually penetrate and damage the cell membrane).8,9,31,32 Several studies revealed that, depending on the concentration of the oxygen vacancies, ROS are generated even in dark.33 The hierarchical structures of ZnO nanoparticles, with large and porous surface areas, as well as an optimum configuration of the extrinsic/intrinsic defects (in terms of type and density) represent promising solutions to achieve enhanced antimicrobial responses.34 Moreover, such architectures allow an easier separation and recycling, being more effective in preventing further aggregation than their corresponding nano-building blocks.35 A simple method to obtain ZnO nanostructures consists in the hydrolysis of metal salts in polyol solvents, in the presence or absence of additives.36,37 The resulted oxide particles have organophilic surfaces (polyol by-


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products adsorbed on the ZnO surface), being easily available for further functionalization with antibiotics or essential oils without additional post-synthesis processing.22,38,39 Surprisingly, only a limited number of antimicrobial activity studies have been carried out on zinc oxide nanostructures obtained through polyol-assisted methods22,38,39 and almost all antimicrobial tests were routinely developed using planktonic inoculums. In our previous report,21 crystalline and radially distributed aggregates of zinc oxide were obtained through a simple hydrolysis of zinc acetylacetonate in 1,4-butanediol (1,4-BD) in which the reaction parameters (zinc source concentration, reaction time and temperature) direct the structural characteristics of the materials: crystallite/aggregate sizes and their assembly motifs, surface area and connected porosity. Herein, we thoroughly investigated the effect of zinc oxide peculiarities on their biological activity by using two types of ZnO spherical aggregates with hollow (ZnOHS) and solid (ZnOSS) cores obtained through polyol method. In this regard, we focused our study on their surface chemistry that was investigated through photoluminescence (PL), X-ray photoelectron (XPS) and electron paramagnetic resonance spectroscopy (EPR) coupled with spin trapping techniques. The qualitative and quantitative assessment of the antimicrobial activity towards several Gram positive and negative bacterial, as well as fungal strains was performed by estimating the minimal inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC). In order to couple the therapeutic effectiveness with a minimal toxicity on human cells, in vitro biocompatibility tests were also performed.

EXPERIMENTAL SECTION 2.1. Materials and synthesis. The reagents of analytical grade were used without further purification. Microbial strains. The antimicrobial activity of the obtained compounds was


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assayed on Gram negative, Gram positive and fungal reference and clinical strains. The Gram negative strains included Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ICUB 230, Klebsiella pneumoniae ICUB 40, Enterobacter cloacae ICUB 36. The Gram positive strains were represented by methicillin-susceptible Staphylococcus aureus ATCC 6538, methicillin-susceptible Staphylococcus aureus (MSSA) ATCC 25925, MSSA ICUB1, methicillin-resistant Staphylococcus aureus (MRSA) ATCC BAA-1026, MRSA ICUB1, Staphylococcus saprophyticus ATCC 15305, Enterococcus faecalis ATCC 29212, Enterococcus faecium VA-R17, Bacillus subtilis ATCC 6633. The tested fungal strains were Candida albicans ATCC 26790 and Cryptococcus neoformans ATCC 204092. Human cell culture. The cytotoxicity assay was performed on the human liver cells Hep G2 (ATCC HB8065). Culture media and reagents: dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA), sterile nutrient broth (Liofilchem, Italy), phosphate buffered saline (PBS) (Sigma-Aldrich, USA), cold methanol (Chempur, Poland), complete Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, USA), fetal bovine serum (Gibco, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA), 2-propanol (Sigma-Aldrich, USA), Griess reagent. Synthesis. The two types of ZnO aggregates were obtained through a polyol-assisted precipitation, as a result of the hydrolysis of Zn(acac)2 at 90o C (ZnOHS) and 120o C (ZnOSS). Thus, 0.1M (ZnOHS) and 0.25M (ZnOSS) of zinc acetylacetonate is dissolved in a known volume of 1,4-butanediol. The mixture was heated under stirring at the reaction temperature, in a roundbottom flask fitted with a reflux column, for 4 (ZnOHS) and 6 hours (ZnOSS), respectively. The precipitation of ZnO occurred after 30 minutes. After cooling at room temperature, the solid phases were collected by centrifugation and washed with ethanol.


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2.2. Characterization. FTIR spectroscopy. FTIR spectra (KBr pellets) were recorded with a FTIR Brucker Tensor V-37 spectrophotometer. UV-Vis spectroscopy. UV-Vis spectra were recorded on a Perkin-Elmer Lambda-35 (200–1100 nm) spectrophotometer. Powder X-ray diffraction measurements (PXRD) were carried out at room temperature on a PANalytical X'Pert PRO MPD X-ray diffractometer with Cu X-ray tube providing a Kα wavelength of 1.5418 Å. The average crystallite size (D) of the samples was determined using the Williamson–Hall equation βhkl cos θhkl = kλ/D + 4ε sin θhkl, where λ is the wavelength of the CuKα radiation, k a constant equal to 0.9 and βhkl the instrumental corrected broadening measured at the halfmaximum intensity of the (hkl) peak at θhkl Bragg diffraction angle. From the plot of βhklcosθhkl vs. 4sinθhkl one can evaluate the contribution of the microstrain and crystallite size: from the intercept of the linear fit curve with the ordinate the average crystallite size is extracted, and from the slope of the line the microstrain ε is obtained. The HighScore Plus powder diffraction software from PANlaytical using the Rietveld method was utilized for the evaluation of the lattice parameters. Thermal measurements were performed on a Netzsch STA 449 F1 Jupiter Simultaneous Thermal Analyzer in dynamic air (30 mL/min), with a heating rate of 5 ºC min-1. The size and morphology of the particles were evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM measurements were carried out on field emission Hitachi H-4100FE microscope. TEM experiments were performed on a JEOL ARM 200F electron microscope operated at 200 kV. Porosity and surface area of the samples were determined by nitrogen adsorption–desorption analysis at -196o C using a Micrometrics ASAP 2020 analyzer. Specific surface areas (SBET) were calculated according to the Brunauer– Emmett–Teller (BET) equation. The total pore volume (Vtotal) was estimated from the amount adsorbed at the relative pressure equal with 0.99. The average pore diameter and pore size


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distribution curves were obtained using Barrett–Joyner–Halenda (BJH) method from the desorption branch. Photoluminescence analysis (PL) was performed on a JASCO FP 8300 spectrophotometer using 325 nm excitation line of xenon light. X-ray photoelectron spectroscopy (XPS) was carried out on PHI Quantera equipment with a base pressure in the analysis chamber of 10-9 Torr. The X-ray source was monochromatized Al Kα radiation (1486.6 eV) with the overall energy resolution estimated at 0.65 eV by the full width at half-maximum (FWHM) of the Au4f7/2 photoelectron line (84 eV). Although the charging effect was minimized by using a dual beam (low energy electrons and Ar+ ion beam) as neutralizer, the spectra were internally calibrated using C1s line (BE = 284.8 eV) of the adsorbed hydrocarbon on the sample surface (C-C or (CH)n bonding). Electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra were recorded on a JEOL FA 100 spectrometer at room temperature using the following settings: 100 kHz modulation frequency, 0.998 mW microwave power, 4 min sweep time, 1 G modulation amplitude, time constant 0.1 s, amplitude 600. Spin trapping experiments. The samples were prepared as following: 0.010 g of zinc oxide materials was mixed with 0.2 ml fresh solution of 5,5-dimethyl-1-1-pyrroline-n-oxide (DMPO) in water (1M). After one minute, the samples were centrifuged and the solution was transferred to a capillary tube and placed in the spectrometer cavity. This general procedure was applied in different conditions. Thus, samples of ZnO were used as prepared and dried in vacuum in order to eliminate the adsorbed oxygen. DMPO solution was prepared in water that obviously contains atmospheric O2, and, in order to prove the effect in generation of •OH radicals in contact with ZnO samples, the solutions were degassed by bubbling inert gas and then added to vacuum dried ZnO samples. Control experiment with DMPO solution has been also performed proving the absence of EPR signal.


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The presence of oxygen singlet species was proved using a solution of 0.1 M 2,2,6,6-tetramethyl1-piperidinyloxy (TEMP) in water. 2.3. Antimicrobial activity assays. Microbial suspensions of 1.5  108 CFU mL-1 (0.5 McFarland density) obtained from 15 to 18 hours bacterial cultures advanced on solid media have been used. The tested “as received” nanoparticles have been suspended in DMSO to prepare a stock suspension of 10 mg mL-1 concentration. The quantitative assay of the antimicrobial activity was completed by the liquid medium microdilution method. Two-fold serial dilutions of the stock suspensions (ranging from 1000 μg mL-1 – 1.95 μg mL-1) have been performed in a 200 μL volume of sterile nutrient broth / 1:1sterile nutrient broth:mannitol, distributed in ninety-six multi-well plates and then seeded with 50 μL microbial inoculum. Untreated and sterility controls have been used. The plates were incubated for 24 hours at 37o C, and the minimum inhibitory concentration (MIC) values were established as the lowest concentration of the tested compound that inhibited the overnight growth of microbial cultures, compared to the untreated culture. The culture density was measured by reading the absorbance of the wells content at 620 nm. For the evaluation of the influence of the tested nanoparticles on the capacity of microbial strains to colonise the inert substratum, the microplates used in the MIC assay have been emptied and washed three times with phosphate buffered saline. The biofilm formed on the wall of the plastic well was fixed for 5 min with cold methanol, coloured for 15 min by violet crystal solution and re-suspended with a 33% acetic acid solution. The absorbance of the stained suspension was measured at 490 nm and the minimal biofilm eradication concentration (MBEC) was established as the lowest concentration of the tested compound that inhibited the biofilm development on the plastic wells, as compared to the untreated cultures.


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2.4. Biocompatibility tests. The Hep G2 cells were grown in complete DMEM containing 10% fetal bovine serum at 37oC in a humidified atmosphere with 5% CO2. The cells were seeded at a cell density of 2 x 105 cells/ cm2 in 24-well plates and left to adhere overnight. Then, these were incubated for the next 24 hours with ZnO samples (15.6, 31.5, 62.5 and 125 μg mL-1) which were previously sterilized under UV light. Untreated cells were used as control for all in vitro experiments. Cell viability assay. The cellular proliferation was measured using the MTT assay. This colorimetric test measures the succinate dehydrogenase mitochondrial activity in order to quantify the viable cells. After 24 hours of incubation with nanoparticles, the culture medium was removed and the cells were incubated with 1 mg/mL MTT for 2 hours at 37o C and 5% CO2. The purple formazan crystals formed in the viable cells were dissolved with 2-propanol and the absorbance was measured at 595 nm using a FlexStation 3 multi-mode microplate reader. The cytotoxicity level of the tested samples was classified in accordance to the ISO 109935:2009 - Biological evaluation of medical devices ‒ Part 5: Tests for in vitro cytotoxicity recommendations. Nitric oxide (NO) release measurement. The culture medium was collected after 24 hours of cell growth in the presence of particles in order to quantify the NO concentration released in the medium as an indicator of inflammation and cytotoxicity. 80 µL of culture supernatants were mixed with an equal volume of Griess reagent which is represented by a stoichiometric solution (v/v) of 0.1% naphthylethylenediamine dihydrochloride and 1% sulphanilamide in 5% H3PO4. The absorbance was read at 550 nm using a FlexStation 3 microplate reader and the NO concentration was calculated using a NaNO2 standard curve. Intracellular reactive oxygen species (ROS) measurement. ROS production was determined using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), which passively enters within cells where is hydrolyzed by intracellular esterases to 2′,7′-dichlorodihydrofluorescein (H2DCF).


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This product is further oxidized by a range of intracellular ROS to 2′,7′-dichlorofluorescein (DCF), a highly fluorescent compound. After 24 h of exposure to nanoparticles, Hep G2 cells cultured in 24-well plates were washed with PBS and incubated at 37ºC with 10 µM H2DCF-DA for 30 min in dark. After that, cells were washed and resuspended in an appropriate volume of PBS for fluorospectrophotometric detection using 488 nm excitation and 515 nm emission wavelengths. Results were expressed as relative fluorescent units per number of cells and represented as percentage related to control. Statistical analysis. The in vitro assays were performed in triplicates and the results were presented as mean ± standard deviation (SD) of three independent experiments. The statistical significance was analyzed by Student’s t-test and values of P less than 0.05 were considered significant.

RESULTS AND DISCUSSION FTIR and UV-Vis spectroscopy. The FTIR spectra of the two ZnO samples (Figure S1) show the presence of hydroxyl groups (from water and/or traces of diol) and carbonyl groups (resulted from polyol hydrolysis by-products like acetone and ester,21 the thermal analysis disclosing residues content for ZnOHS sample with 25 % higher than in the case of ZnOSS material, see Figures S2, S3 and Table S1) adsorbed on the surface together with the specific Zn-O absorption band attributed to the phonon absorptions of the zinc oxide lattice.

21

A relevant observation

concerning the Zn-O bond is related to the energy and the absence of any splitting of the corresponding absorption that is usually characteristic to spherical-shaped ZnO particles. Also, the decreased intensity and bathocromic shift of the Zn-O absorption observed in the case of ZnOSS (465/450 cm-1 ZnOHS/ZnOSS) could be a result of the differences between the two materials (crystallinity, defects, sizes and/or morphologies).40-42


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The electronic spectra of the two ZnO materials are very similar (see Figure S4) and show a strong and large UV absorption band structured in two shoulders: at 350 nm assigned to electronic transition from the valence band to the conducting band (O2pZn3d),43 and at 250 nm correlated with defects from würtzite-type structure.44 The estimated band gap values are 3.12/3.23 eV (ZnOHS/ZnOSS, see Figure S5). The significant difference between the aggregate sizes for ZnOHS (ca. 250 nm) and ZnOSS (ca. 20 nm) could be at the origin of the of the slight red shift and the enlargement of the exciton band (350 nm) as well as the diminished band gap energy (3.12 eV) in the case of the hollow structures. PXRD analysis. The PXRD patterns revealed the formation of pure hexagonal-ZnO würtzite phase for both ZnO materials (Figure S4). The estimated lattice parameters (a = 0.32546(6)/0.32532(5) nm and c = 0.5212(1)/0.52114(8) nm, ZnOHS/ZnOSS) are almost equal to the standard ones (a = 0.324982 nm and c = 0.520661 nm, JCPDS no. 36-1451), and the mean crystallites sizes values are somewhat lower for the ZnO sample obtained at lower zinc precursor concentration (15.9/17.3 nm for ZnOHS/ZnOSS). The estimated lattice strain of both ZnO samples with a value of 0.1(1). SEM and TEM analysis. The morphology and degree of aggregation of ZnO materials proved to be very sensitive to zinc(II) source concentration as well as to reaction conditions.21 For ZnOHS sample (0.1 M), obtained at the lowest temperature (90o C) and after 4h of reactions, SEM and TEM micrographs show the formation of uniform spheres with sizes of ca. 250 nm, with hollow cores with a diameter of ca. 100 nm, the external shells being assembled from several layers of ZnO nanocrystallites (Figures 1a,b and Figure S7).21 The assembly of ZnO hollow spherical-shaped aggregates through the hydrolysis of zinc (II) precursors represents a rare case.45,46 In the case of zinc(II)-enriched sample, ZnOSS, obtained at higher temperature


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(120o C) after 6h of reaction, the SEM micrographs indicate the formation of smaller and quasispherical aggregates (up to 20 nm), most of them gathered in larger clusters of ca. 100 nm with no well-determined pattern (Figures 1c,d and Figure S8).

Figure 1. SEM (a and c) and TEM (b and d) micrographs for ZnOHS (a, b) and ZnOSS (c, d) materials. TEM analysis show that the small aggregates, with solid cores, mainly consist of quasispherical crystallites. Also, hexagonal-shaped nanocrystallites are rarely observed, the SAED pattern being typical for polycrystalline ZnO würtzite structure (see Figure S8). BET analysis. ZnOHS, has the highest surface area of 91.9 m2 g-1 and a total pores volume of 0.334 cm3 g-1, while ZnOSS sample has a lower surface area of 40.13 m2 g-1 and a total pores volume of 0.265 cm3 g-1. Photoluminescence spectroscopy (PL). A preliminary defect analysis was carried out by photoluminescence measurements (PL). Both PL spectra for the two ZnO samples (Figure 2) are similar and present a strong and sharp UV emission (ca. 378 nm) and, also, visible emissions


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covering violet-blue, green, yellow and orange regions. The UV emission, a common feature of crystalline ZnO materials, is attributed to the free exciton recombination.47 The decreased intensity of the UV band for ZnOHS sample is most likely caused by morpho-structural differences between ZnOHS and ZnOSS materials.48 The emissions from visible region of PL spectra are due to intrinsic and/or extrinsic defects. The violet-blue multi-structured emission bands (410-490 nm) are rarely observed in the PL spectra of zinc oxide and could result from zinc vacancies, interstitial zinc defects49-51 and/or the presence of chemical species attached on ZnO surface.52-55 At first glance, the low-intensity of the green emission (500-550 nm), usually attributed to oxygen deficiency, is quite unexpected considering the reducing action of 1,4-BD that favors the formation of the oxygen vacancies on the ZnO surface (withdrawal of the surface oxygen by polyol).56-58 For our samples, the polylol by-products attached to the ZnO surface21 most likely fill the oxygen vacancies located on or near the surface and determine this unusual behaviour. The quenching of the green emission of zinc oxide was observed in several cases of TOPO- and OD-,59 and PVP-capped60 ZnO nanoparticles [TOPO = trioctylphosphine oxide, OD = 1-octadecene, PVP = poly(vinyl pyrrolidone)]. The weak yellow (595 nm) and orange (623 nm) shoulders are related to interstitial oxygen.61


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Figure 2. Room temperature PL spectra for ZnOHS (a) and ZnOSS (b). XPS spectroscopy. In order to investigate the nature of the ZnO surface defects as well as the chemical species attached on the zinc oxide outer layers, we carried out an XPS study on the ZnOHS and ZnOSS materials. The XPS spectra indicate the presence of zinc, oxygen and carbon elements on the outermost surface layer (< 10 nm). The corresponding numerical data and quantitative assessment for the two ZnO samples are gathered in Table 1, in the as “received stage” and after a gentle argon ion etching, in order to remove the surface contaminants in the first two monolayers (~ 0.5 nm) first two monolayers (~ 0.5 nm) in order to avoid the damage of the surface chemistry of the materials (Figures S9 and S10). For both samples, the XPS spectra of O1s exhibit an asymmetric profile indicating the presence of multicomponent oxygen species on the samples surface region and into the inner layers (Figure S9 and Table 1). The O1s core level could be coherently fitted by three nearly Gaussian subspectral components, centred at ca. 530, 531.5 and 532.8 eV respectively. The main peak from ca. 530 eV is attributed to O2- ions of the würtzite structure of the hexagonal Zn2+ ion array, which is surrounded by zinc atoms with the full supplement of nearest-neighbour O2− ions.59-61 The medium binding energy peak from ca. 531.6 eV could be assigned to multiple


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effects as following: the oxygen ions in the oxygen deficient regions,62-64 the presence of zinccontaining hydroxide species (resulted either from polyol residua or zinc(II) layer double hydroxide intermediates),21,65-67 as well as to C–O and C=O type bonds.68-71 The highest binding energy with the maximum at ca. 532.8 eV is usually attributed to the presence of the oxygencontaining species (bound, adsorbed and/or chemisorbed) like H2O (mainly), O2, CO2 and/or interstitial oxygen atoms, Oi (consistent with the orange emissions from PL spectra, vide supra).63,64,72-74 The intensity of the two higher binding energies remained significant even after the Ar+ etching, denoting that the oxygen vacancies are more abundant on the outermost surface layer, but also that the oxygen species are present beneath the surface. Both assertions are in accordance with the chemical processes of the synthesis and defect localization in the ZnO structure. The lower atomic relative concentrations value of the O/Zn ratio (0.91) for ZnOHS sample proves that the hollow spherical material has, at least at the surface level, a higher oxygen vacancy. The band-like spectrum in the C1s region (Figure S10 a-d and Table 1) displays an asymmetric broad tail, which clearly suggests that this element exhibits also several chemical states.71,74 The fitting of the experimental line profile revealed the presence of three main peaks attributed to the contributions from C-C/C-H (ca. 284 eV, main peak), C-O/C-OH (ca. 286 eV, carbon in alcohol or ester) and O=C-O (ca. 288 eV, carbon in carboxyl or ester groups) bondings. For both samples, the Ar+ etching process reduces by half the carbon content, the ZnOHS sample having the largest amount of carbon on the surface, both in the “as received” and etched forms, and thus confirming the thermal analysis results.


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The Zn2p3/2 photoelectron spectra (with BE at ca. 1021 eV) and the more sensitive to the chemical shifts Zn LMM Auger transition (BE at ca. 498 eV) confirm the formation of the zinc oxide with würtzite structure (Figures 3 a, b).

Figure 3. The superimposed high-resolution photoelectron Zn2p3/2 (a) and Auger ZnLMM (b) spectra in the “as received” stage (solid lines) and after 0.5 min Ar+ ion etching (dotted lines) for the ZnOHS (solid and dotted black lines) and ZnOSS (solid and dotted green lines) samples. The BEs values are shown in Table 1. Table 1. The XPS data: Binding Energies (BEs), oxygen and carbon species relative concentrations (%) and atom relative concentrations (atom %) Binding energies, eV / percentage chemical oxygen species (%) Sample

ZnOHS as received

ZnOHS after 0.5 min Ar+ etching

ZnOSS as received

ZnOSS after 0.5 min Ar+ etching

C1s

O1s

284.8: C-C/CHn (70.2%)

530.1 (65.5%)

286.3: C-O/C-OH (13.8%)

531.6 (28.6%)

288.7: O=C-O (16.1%)

532.8 (5.8)

284.8: C-C/CHn (69.6%)

530.0 - (72.4%)

286.3: C-O/ C-OH (15.5%)

531.5 (23.7%)

288.7: O=C-O (14.9%)

532.9 (3.8%)

284.8: C-C/CHn (74.8%) 286.4: C-O/ C-OH (7.4%) 288.9: O=C-O (16.8%)

Atomic relative concentrations, atom % C O Zn

Zn2p3/2

ZnLMM

1021.3

498.1

18.6

43.0

38.4

1021.3

498.1

9.0

43.6

47.4

1021.2

498.0

16.5

43.4

40.1

1021.3

498.0

7.2

45.0

47.8

530.1 (65.5%) 531.6 (29.0%) 532.8 (5.5)

284.8: C-C/CHn (72.1%)

530.0 (73.9%)

286.3: C-O/ C-OH (10.8%) 288.8: O=C-O (17.1%)

531.5 (22.0%) 532.9 (4.1%)


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EPR study. The electron paramagnetic resonance spectroscopy (EPR) represents a useful tool to investigate the native defects in ZnO materials, such as oxygen and zinc vacancies.77 EPR spectroscopy coupled with various spin trapping agents is also a robust method to detect specific free radicals generated by the metal oxide surfaces, the so-called reactive oxygen species (ROS) that form relatively stable spin adducts.78 ZnOHS and ZnOSS samples exhibit very similar EPR spectra and the discussion will be focused only on ZnOSS material. The zinc(II)-enriched sample, ZnOSS, shows a first-order EPR spectrum only under UV irradiation with Xe lamp (Figure 4). The determined value of g is 2.0094 that is similar to free electron (geff = 2.0023), indicating the presence of unpaired electrons. A large number of studies attributed this signal to oxygen vacancies with a single trapped oxygen (VO+).79,80 The broad and asymmetric profile of the low-field EPR signal (g > 2) could be also resulted from a combination of different deep paramagnetic defects: singly ionized oxygen (VO+), zinc vacancies (VZn-) and/or interstitial zinc, Zni.81,82 These presumptions are consistent with the PL results (green and blue-violet emissions assigned to oxygen and zinc vacancies and/or interstitial zinc defects). Moreover, the EPR spectrum of ZnOSS sample is silent in dark, sustaining the hypothesis that structural defects are at the origin of the paramagnetic signal.83 Nevertheless, the formation of O2- superoxide ion from hydroxyl and/or -C=O groups of the impurities adsorbed on the ZnO outer layers could not be excluded.84


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Figure 4. EPR spectrum of irradiated ZnOSS material. The capacity of zinc oxide to produce oxygen-containing radicals and chemical species is a

key

physico-chemical

parameter

in

estimation

and

assessing

the

ROS

related

biological/environmental effects. Generation of Hydroxyl Radicals. The most reactive oxygen-based radical is hydroxyl radical (•OH) that reacts very quickly with almost every type of molecule found in living cells. Such reactions will probably dominate the recombination of two •OH radicals to form hydrogen peroxide (H2O2).85,86 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) is commonly used for trapping hydroxyl radicals. DMPO is EPR silent, both in air saturated and deaerated water solution and, in reaction with hydroxyl radicals, forms a relatively stable paramagnetic DMPO–OH• adduct.87,88 We investigated the reaction of dried ZnOSS material with DMPO agent in different conditions: (a) dried ZnOSS mixed with deoxygenated DMPO solution, in dark; (b) air exposed ZnOSS mixed with deoxygenated DMPO solution (in natural light) and (c) dried ZnOSS mixed with oxygenated DMPO solution (in natural light). In case (a), the EPR signal attributed to the formation of DMPO-HO• adduct is very weak and can be explained by a minor zinc leaching (Figure 8a, black line). When the dried sample was exposed to air (by inserting a syringe needle into the rubber


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cup of sample tube, case b), the clear four-lines EPR signal (1:2:2:1) was attributed to the formation of DMPO-HO• adduct, with the corresponding hyperfine splitting constants (hfc), aN = aH = 14.95 G (Figure 5a, red line). Also, a secondary product resulted from DMPO degradation with hfc of aN = 16.94 G was observed. In the last case (c), the DMPO solution contains O2 adsorbed from air and the EPR spectrum indicates a possible contribution of a C• centred radical (resulted from the polyol by-products that cover the ZnO surface), with hfc of aN = 15.88 G and aH = 23.24 G. The lower intensity of the EPR signals compared with case (b) shows that the surface carbonaceous residua act as •OH radicals scavenger (Figure 5a, blue line). We investigated further the effect of the UV light on the EPR signal of the ZnOSS-DMPO mixture. The exposure of DMPO aqueous solution to UV light, for 10 minutes, gave no EPR signal. The water dispersion of ZnOSS-DMPO mixture, irradiated with UV light, gives weak and multiple peaks in EPR spectrum corresponding both to DMPO-OH• and C-centered radicals (Figure 5b), having similar values of the hyperfine coupling constants (see above). Generation of singlet oxygen species. 2,2,6,6-Tetramethylpiperidine (TEMP) is a wellknown trapping agent for singlet oxygen species, 1O2.87,88 The reaction of TEMP with singlet oxygen radical affords the free radical 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TEMPO) showing a specific three-lines EPR spectrum. In the absence of ZnO, in water, immediately after adding TEMP, no EPR spectrum was observed. When TEMP is added to ZnOSS aqueous suspension, after centrifugation, it was observed an EPR spectrum with three lines of equal intensity that indicates the formation of TEMPO radical (aN = 17.2 G) resulted from the oxidation of TEMP by the singlet oxygen species (Figure 6a).


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Figure 6. The EPR spectra of DMPO adducts trapped from ZnOSS samples in the following conditions: (a) dried ZnOSS sample mixed deoxygenated DMPO solution in the dark (black line), air saturated sample of ZnOSS mixed with deoxygenated DMPO solution (red line) and dried sample of ZnOSS mixed with oxygenated DMPO solution (blue line); (b) UV light irradiated aqueous solution containing DMPO-ZnO mixture. The EPR signals corresponding to DMPOOH• adduct are marked with green asterisks (*) while those attributed to C• centred radical are indicated with black dots (•). Irradiation with UV light for 10 minutes, of the suspension of ZnOSS in water, followed by an immediate addition of TEMP led to an increase of TEMPO radical signal (Figure S11). Antimicrobial activity. The hierarchical ZnO materials exhibited a wide spectrum of antimicrobial activity, being active against Gram positive and Gram negative bacteria as well as against fungi strains (Table 2). All pathogens included in the ESKAPE group (i.e. Ent. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter sp.)89 are susceptible to the scrutinized materials both in planktonic and, more important, in biofilm growth form. The data show that practically all strains are susceptible to at least one of the two investigated materials.


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ZnOSS exhibited a stronger biocidal effect against planktonic cultures (particularly Gram positive ones), while ZnOHS inhibits better the biofilm development of both Gram positive and negative strains. The Gram-positive bacterial cell wall is composed of a thick peptidoglycan layer, crossed by (lipo)teichoic acid filaments, negatively charged, which could attract nanoparticles. Also, the Gram positive bacterial cell wall is exhibiting abundant pores through which small nanoparticles can easily penetrate, induce cellular lesions, and eventually the cell death. By comparison, the Gram negative bacteria have a more complicated cell wall, represented by the outer membrane composed of lipopolysaccharides, lipoproteins and phospholipids and the periplasmic space containing the peptidoglycan layer, which form a more effective penetration barrier that limits the penetration of nutrients and the elimination of catabolism products at the level of porins (protein channels crossing the bacterial wall).90 In contrast to earlier reports,91,92 the ZnOHS material was more efficient against biofilm-embedded Gram positive and negative cells, as revealed by the obtained MBEC values which were lower than the corresponding MIC ones. It is well known that in biofilm growth state, the pathogenic cells are much more resistant compared to their planktonic counterparts, through different mechanisms including (but not limiting to) poor antibiotic penetration, nutrients limitation, slow growth and selection of persister cells.93


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Table 2. Comparison between MIC and MBEC values for ZnOHS and ZnOSS materials with literature data.

MIC / µg mL-1

Microbial strains ZnOH

ZnOS

P. aeruginosa ATCC 27853 A. baumannii 230

S

S

15.62

250

E. coli ATCC 25922

125

15.62

K. pneumoniae 40

7.81

5

Ent. cloacae 56

6.

MBEC / µg mL-1

Literature data (*, **)

ZnOHS

ZnOSS

Literature data (*, **)

* 2.9,95 5,101, 6.7,96 18,102 30,97 98,156,180,103 50094,98;** 3.23,96 30,96 10-512104 ** 32->256,105 14-16106

62.5

500

* 50020,21

500

125

62.5

500

31.25

* 0.97,20 17.56,96 25,102 50,115 62.5,20 68,100 128-512,112 275,109 512,108 600113,114 * 5,111,117 15.62,20 40,116 50020,98

500

15.62

15.62

1.95

* 50-70,119 520,121 1700-6000120; ** 4-12,121 30-50119

62.5

500

S. aureus ATCC 25925

500

31.25

3.90

125

7.

MSSA ICUB 1

125

1.95

* 2.4,95 4.8,96 46-100,125 50,115 90,124125,94 128-256,123 200-500,102 400114; ** 2,105 0.125-512126

125

500

8.

S. aureus ATCC BAA-1026 S. aureus ATCC 6538

1000

31.25

7.81

62.5

1.95

3.90

7.81

62.5

MRSA ICUB 1

15.62

62.5

125

250

S. saprophyticus ATCC 15305 Ent. faecalis ATCC 29212 Ent. faecium VA-R17 B. subtilis ATCC 6633

7.81

7.81

* 128127

31.25

62.5

1000

31.25

* 25,129 520120; **128128

31.25

250

15.62

15.62

* 20.25,133 125,133 520120

62.5

62.5

1000

1.95

* 6.25,102 31.25,20 62.5,20,135 160-240,103 110136

15.62

500

3.90

500

500

500

31.25

15.62

* 2.5,137 5-10,13710.55,96 25,102 160136; ** 2.77-322,96 >80139 * 10.8138; ** >80139

15.62

500

1 2 3 4

Gram negative strains

9. 10. 11.

Gram positive strains

12. 13. 14. 15. 16.

Fungi strains

C. albicans ATCC 26790 C. neoformans ATCC 204092

250

1.95

* > 50020 ; ** 1281028104 * 250,119 >50020

** 512-248,126 > 1024104

** 64-512,130 128 >512, >512,132 ** 32-256134 * 3.9,20 > ** 16-1024104

128-

50020;

* Inorganic compounds; ** Inorganic compounds-antibiotic mixtures or antibiotics.

ACS Paragon Plus Environment

22

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Both spherical-shaped ZnO nanostructures are active against P. aeruginosa ATCC 27853 reference strain in a different manner: ZnOHS inhibits better the biofilm development, whereas ZnOSS has a stronger effect on its planktonic growth. To our knowledge, the MIC value of 1.95 µg mL-1 (for ZnOSS sample) is lower than previously reported values for bare ZnO,94-102 dopedZnO103 and ZnO materials in combination with antibiotics97 and even for current antibiotics.96,104 Although the low concentrations of the oxides inhibited the biofilm progress (62.5 µg mL-1 for ZnOHS), the obtained MBEC values are slightly higher than those reported for other types of ZnO based-materials20,21 and antiobiotics.104 Also, it is worth to mention the low MIC value (15.62 µg mL-1) obtained for ZnOHS material against A. baumannii, which proves that the antimicrobial effect of the hollow structures is higher than those reported for different classes of large-spectrum antibiotics, such as β-lactams, aminoglycosides, fluoroquinolones and carbapenems.105,106 In case of E. coli ATCC 25922 reference strain, similar to P. aeruginosa, the solid spheres have a higher efficiency against the strain in planktonic growth (MIC for the ZnOSS is ~32 times lower than MBEC) and the hollow ones toward biofilm (MIC value for ZnOHS being two times higher than MBEC). Both registered MICs (125/15.62 µg mL-1) are lower than previously reported ones for other ZnO materials,20,94,96,99,100,102,107-112 ZnO-doped with transition and lanthanide ions112 and Ag-ZnO composites.113,114 The solid spheres of ZnOSS are more active than ZnO-chitosan and graphene-oxide-ZnO composites.115 The ZnOHS hollow spheres activity against biofilm is higher than this corresponding to other types of ZnO materials20 and even of antibiotics.104 The antimicrobial activity of the two materials with spherical morphologies (ZnOHS and ZnOSS) against K. pneumoniae 40 clinical strain in planktonic growth state is also very good (with very low MIC of 7.81/31.25 µg mL-1 for ZnOHS/ZnOSS), better than the majority


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of the reported values,20,98,116 although slightly higher than the best listed one, of 5 µg mL-1.20,111,117 Only ZnOSS oxide shows a good antibiofilm activity (MBEC of 15.62 µg mL-1) lower than the previously reported one,20 but higher than in other experiments using Ag/ZnO nanocomposites.118 Both samples are active against Ent. cloacae 56 clinical strain and the obtained MIC values (15.62/1.96 µg mL-1 ZnOHS/ZnOSS) represent the best result obtained in a series of inorganic antimicrobial agents119 and nanoscaled and bulk ZnO formulation,100,120 and a large spectrum of antibiotics as well as antibiotics-inorganic composites.119,121 ZnOHS sample inhibits vigorously the Ent. cloacae biofilm development, the obtained MBEC value of 62.5 µg mL-1 being the lowest reported for inorganic biocides. All four S. aureus strains (two reference and two clinical) are sensitive to the spherical nanostructures. The lowest MIC values of 1.95 µg mL-1 were attained for S. aureus ATCC 6538 (ZnOHS) and MSSA ICUB 1 (ZnOSS), while, for biofilm, a MBEC value of 3.90 µg mL-1 was reached with ZnOHS for S. aureus ATCC 25925. Although slightly above the lowest values reported for inorganic materials, the MICs and MBECs are much lower comparatively with other ZnO materials,86,94-96,102,111,121-123 Ag-ZnO

114,124,125

and graphene oxide-ZnO composites,115 and

antibiotics.104,126 A potentially bactericidal effect is also manifested towards S. saprophyticus ATCC 15305 clinical strain both in planktonic and biofilm state, the recorded MICs (7.81 µg mL-1 ZnOHS/ZnOSS) and MBECs values (31.25/62.5 µg mL-1 ZnOHS/ZnOSS) being lower than those obtained with alternative ZnO materials.127 Concerning the behaviour against Ent. faecalis ATCC 29212 reference strain, ZnOSS is efficient against the planktonic strain, with MIC a value (31.25 µg mL-1) notably lower than those reported for other ZnO materials120 and antibiotics as kanamycin128 and slightly higher than in ZnO-graphene oxide composites.129 ZnOHS material possesses an excellent inhibitory effect on Ent. faecalis ATCC 29212 biofilm development


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(MBEC = 31.25 µg mL-1) superior to that of a broad range of antibiotics.130-132 The two ZnOHS and ZnOSS spherical structures, exhibit identical MICs and MBECs (15.62/62.5 µg mL-1 MIC/MBEC) against Ent. faecium VA-R17 clinical strain. The inhibitory activity against the planktonic strain is higher compared to previously reported cases of ZnO materials, ZnO- and Ag-graphene oxide composites.133 Also, the antibiofilm activity of the two samples is higher than several combinations of antibiotics.134 The antibacterial activity of the solid spheres toward planktonic B. subtilis ATCC 6633 is superior to that of other ZnO materials, the obtained MIC values (1.95 µg mL-1) being lower than most of the reported values for bare,20,101,102 dopedZnO,103,135 ZnO-based composites136 and some antibiotics.104 Although the hollow structure, ZnOHS, inhibited the biofilm development (15.62 µg mL-1), its anti-biofilm activity is weaker than in the case of other materials.20 The quite different antimicrobial response could be correlated with the morpho-structural peculiarities.137 The ZnOHS aggregates have a larger size (ca. 250 nm), but also a high specific surface area (91.9 m2 g-1) which could probably facilitate a much efficient direct contact with the bacterial strains (especially in biofilm form), the adhesion of the material on the surface of microbial cells31 inducing a significant damage of the cellular membrane and increased possibilities for ROS attack.138 ZnOSS solid spheres are smaller aggregates (ca. 20 nm) gathered in clusters of sizes up to 100 nm, have a lower surface area (40.13 m2g-1) and manifested a higher activity on the planktonic form of pathogens. The smaller assemblies formed by the ZnOSS materials, as well as their quasi-spherical form could facilitate the contact between nanoparticles and the single planktonic microbial cells in suspension. In exchange, in case of biofilm cells, the big size of aggregates could facilitate the formation of an impermeable pellicle covering the biofilm and affecting its exchanges with the environment.


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The ZnO compounds were tested also as antifungal agents toward two reference fungal strains, i.e.: C. albicans ATCC 26790 and C. neoformans ATCC 204092. ZnOHS shows the best antifungal activity, being active against both fungal strains in planktonic growth state (3.90/31.52 µg mL-1 for C. albicans/ C. neoformans) and against C. neoformans biofilm (15.62 µg mL-1), while ZnOSS affects only C. neoformans in planktonic state (15.62 µg mL-1). The obtained MICs are higher but comparable to the ones reported for several zinc oxide-based materials (ZnO,94,96,102,139 Pd-doped ZnO139 and ZnO-chitosan composites136), polycarbonate-based cationic polymer140 and different antibiotics.96,141 The hollow spherical ZnOHS morphology is active toward the biofilm formed by C. neoformans, as proved by the low MBEC value (15.62 µg mL-1). Influence of a hydroxyl-scavenging agent addition on the antimicrobial activity of the tested materials. Taking into account other reports, the wide spectrum of the antimicrobial activity of the two ZnO materials could be partially explained by the oxidative stress induced by ROS generation (see EPR study section). In order to investigate the ROS effect on the antimicrobial activity of the tested materials, the MIC and MBEC values were comparatively assessed for five microbial strains belonging to the most clinically representative species, in culture medium with and without mannitol, a polyalcohol with well-known hydroxyl radicalscavenging properties.142,143 The MIC and MBEC ratio values obtained in the two experimental conditions are gathered in Table 3. The resulted ratios clearly indicate that, in the majority of cases (excepting ZnOHS against E. coli and Ent. faecalis, highlighted in grey in Table 3), the MIC and MBEC are significantly higher in the presence of mannitol, proving that the hydroxyl radical-scavenger action of mannitol reduced the antimicrobial efficiency of the respective materials and led to increased active antimicrobial concentrations. A drastic increase (up to 32


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times) of the MBEC values is observed in case of ZnOHS against P. aeruginosa and of ZnOss against E. coli proving that their antimicrobial activity against these strains is strongly dependent on the ROS release (Table 3), but the interference of other mechanisms are not excluded.

Table 3. MIC and MBEC ratios after cultivation of microbial strains in nutrient broth with mannitol versus in nutrient broth without mannitol (1:1).

MIC

MBEC

E. coli

S. aureus

P. aeruginosa

Ent. faecalis

ZnOHS

1:2

16:1

8:1

1:4

ZnOSS

16:1

8:1

2:1

4:1

ZnOHS

1:1

4:1

32:1

4:1

ZnOSS

32:1

16:1

4:1

8:1

It must be taken into account that the amount of the released ROS depends also on the respiratory of the tested strains, which varies from a strictly aerobic to a facultative aerobic or even anaerobic one, the resulting ROS amounts varying accordingly. Adding mannitol to the culture media could cause an important change in the metabolism of the bacterial strains, as it can be used as a carbon source by the strains possessing the respective enzymatic pathway, either by oxidation (favoring in this case the accumulation of a high amount of intracellular ROS) or by fermentation (this is an anaerobic process in which the amount of intracellular ROS is lower than in the case of the aerobic respiration). This could explain the different ratios obtained for different strains in the two experimental conditions. Regarding the three exceptions, in which the MIC or MBEC values were not changed or were even decreased in the presence of mannitol, two of them occurred for E. coli, and one for Ent. faecalis strain and involved only the ZnOHS material. The two strains have both a facultative aerobic respiratory metabolism. This could


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suggest that the ROS- mediated antimicrobial activity depends both on the amount of the ROS generated by the tested materials and on the detoxifying mechanisms of ROS present in different microbial cells, which, on their turn, are dependent on the respiratory type of the respective bacteria (in our case, for example, E. coli is oxidase negative, catalase positive, while Ent. faecalis is oxidase negative, catalase negative). The results obtained on a wide range of microbial strains highlight that the antimicrobial efficiency of the tested materials is influenced by several factors like: materials features, microbial types (species, strains, growth state, either planktonic or biofilm) and/or culture conditions (composition of the culture medium). As consequence, it is quite difficult to predict a scalable pattern of microbial sensitivity/resistance towards the ZnO samples. Cytocompatibility. The effect of ZnO materials on human liver cells was studied by MTT and ROS assays. Figure 7A shows a dose-dependent decrease of the cell viability after 24 hours of exposure to these samples. These results were in accordance with the levels of NO released in culture medium (Figure 7B), as these indicated an increase of cytotoxicity for the highest tested concentration. No significant differences were noticed between the two samples, regardless to the investigated concentration. At low active concentrations, up to 30 µg/ml, antibacterial and antifungal operating concentrations the ZnO samples exhibited a very good biocompatibility.144 Given that the highest decrease of the cell viability (38%) was registered at a concentration of 62.5 µg mL-1 of the ZnO materials, the measurements of ROS level in mammalian cells was accomplished in the same conditions: the same increase of 21% was obtained for both samples (Figure 7C). Although a higher ROS level was expected for ZnOHS materials due to its smaller band gap (3.12 eV) and higher oxygen vacancies concentration,86,145 the strong scavenging action of the carbonaceous impurities adsorbed on the material surface annihilate this effect. Also, it


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can be assumed that the decrease of viable cell number after the exposure to ZnO materials was mediated by ROS generation, which most probably has induced irreversible oxidative damages.146,147 However, the present study highlighted the good biocompatibility of the tested ZnO samples for antibacterial and antifungal operating concentrations up to 30 µg/ml.

A

B

C Figure 7. Cell viability (A), NO (B) and ROS levels (C) measured after 24 hours of liver cell growth in the presence of ZnO samples (ZnOHS and ZnOSS). Results obtained as mean ± SD (n=3) were presented as percentage of control.

CONCLUSIONS In summary, two ZnO-based spherical aggregates, obtained through the hydrolysis of zinc precursors in 1,4-butanediol, were thoroughly investigated in terms of surface chemistry,


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ROS production, antimicrobial and cytotoxic activity. Thus, a surface defects study was carried out for the two ZnO samples through PL, XPS and EPR spectroscopy revealing the existence of deep and shallow oxygen and zinc-related defects as well as of the carbonaceous impurities (polyol residua) adsorbed on the ZnO outer layers. The ability of the two ZnO materials to generate ROS species was monitored through EPR spectroscopy coupled with spin trapping techniques (using DMPO and TEMP as spin traps) under different environmental conditions (in the absence and presence of the oxygen, in dark, visible and UV light) and revealed that both samples generate hydroxyl, singlet oxygen radicals as well as C-centered radicals (resulted from the carbonaceous impurities attached on the ZnO outer layers). Also, the ROS production was increased for oxygenated samples and exposure to UV light. The ZnO surface reactivity is confirmed by the significant biocide action of the two zinc oxide materials, the ZnOHS and ZnOSS samples exhibiting a wide spectrum of antimicrobial activity against Gram positive, Gram negative as well as fungi strains (including all pathogens of the ESKAPE group). The microbial growth inhibition effect was dependent on microbial strains features (species, different strains of the same species and growth state, either planktonic or biofilm), cultivation conditions (composition of the culture medium) and the morpho-structural features of the employed zinc oxide materials. The reduced antimicrobial efficiency in the presence of mannitol, a ROSscavenging compound, proved that ROS-mediated irreversible oxidation is one of the mechanisms explaining both the antimicrobial activity and the cytotoxicity on human liver cells. ZnOHS proved to be a more potent anti-biofilm agent while ZnOSS exhibited a stronger biocidal effect against planktonic cultures. However, the cytotoxicity of the tested materials on mammalian cells is very good for active antimicrobial concentrations. The different biological responses of ZnOHS and ZnOSS samples are most likely related to the combined effect of the


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structural characteristics (band gap, aggregate sizes and specific surface areas) of the two materials and also to the presence of the polyol residua attached on the ZnO surfaces. These results shed more light in the very complex processes that took place at the surface of hierarchical ZnO nanomaterials, capped with organophilic surfaces that finally direct their biocidal action. Therefore, large perspectives are opened in designing new types of biocompatible nanomaterials showing very good inhibitory effect on bacteria and fungi strains development.

ASSOCIATED CONTENT Supporting Information. FTIR spectra for ZnOHS and ZnOSS materials (Figure S1); TG, DTG and DSC curves for ZnOHS and ZnOSS samples (Figures S2 and S3); UV-Vis spectra ZnOHS and ZnOSS materials (Figure S4); Tauc diagram for ZnOHS and ZnOSS materials (Figure S5); PXRD patterns for ZnOHS and ZnOSS materials (Figure S6); SEM and TEM micrographs and SAED pattern for ZnOHS sample (Figure S7); TEM micrographs and SAED pattern for ZnOSS material (Figure S8); The O1s and C1s deconvoluted photoelectron spectra in the “as received” stage and after 0.5 min Ar+ ion etching for ZnOHS and ZnOSS materials (Figures S9 and S10); The EPR spectra corresponding to TEMPO radical resulted from the reaction of singlet oxygen species at ZnOSS/water interface in the absence and in the presence of UV light (Figure S11); Thermal analysis for ZnOHS and ZnOSS materials (Table S1). AUTHOR INFORMATION Corresponding Authors


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*E-Mail: [email protected] *E-Mail: [email protected] *E-Mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. FUNDING SOURCES Romanian National Authority for Scientific Research (CNCS - UEFISCDI) - research project 234 PED-2017. ACKNOWLEDGEMENTS This work was financially supported by the research project 234 PED-2017 of the Romanian National Authority for Scientific Research (CNCS - UEFISCDI). The authors thank Dr. Gabriela Ionita from the “Ilie Murgulescu” Institute of Physical Chemistry for the EPR measurements.

REFERENCES (1) Fariq, A.; Khan, T.; Yasmin, A. Microbial synthesis of nanoparticles and their potential applications in biomedicine. J. App. J. Appl. Biomed. 2017, 15, 241-248.


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43x23mm (300 x 300 DPI)

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Zinc oxide spherical-shaped nanostructures: investigation of surface

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