Influence of surfactant-tailored Mn-doped ZnO nanoparticles on ROS production and DNA damage induced in murine fibroblast cells

The present study concerns the in vitro oxidative stress responses of non-malignant murine cells exposed to surfactant-tailored ZnO nanoparticles (NPs) with distinct morphologies and different levels of manganese doping. Two series of Mn-doped ZnO NPs were obtained by coprecipitation synthesis method, in the presence of either polyvinylpyrrolidone (PVP) or sodium hexametaphosphate (SHMTP). The samples were investigated by powder X-ray Diffraction, Transmission Electron Microscopy, Fourier-Transform Infrared and Electron Paramagnetic Resonance spectroscopic methods, and N2 adsorption–desorption analysis. The observed surfactant-dependent effects concerned: i) particle size and morphology; ii) Mn-doping level; iii) specific surface area and porosity. The relationship between the surfactant dependent characteristics of the Mn-doped ZnO NPs and their in vitro toxicity was assessed by studying the cell viability, intracellular reactive oxygen species (ROS) generation, and DNA fragmentation in NIH3T3 fibroblast cells. The results indicated a positive correlation between the specific surface area and the magnitude of the induced toxicological effects and suggested that Mn-doping exerted a protective effect on cells by diminishing the pro-oxidative action associated with the increase in the specific BET area. The obtained results support the possibility to modulate the in vitro toxicity of ZnO nanomaterials by surfactant-controlled Mn-doping.

ZnO nanoparticles (NPs) have been shown to exhibit promising in vitro antitumor and antimicrobial activity based on the action of released Zn 2+ ions and generated intracellular reactive oxygen species (ROS), leading in some cases to the activation of apoptotic signaling pathways in mammalian cells [7][8][9] . The antimicrobial effect of ZnO NPs was used as an active principle in the design of cosmetic and personal care products 10 and functional textile fabrics 11,12 . In vivo studies regarding the use of ZnO NPs as zinc sources or drug delivery systems in diabetes [13][14][15] and cancer therapies 16 have revealed their potential to interfere with zinc homeostasis and promote the bio-availability of therapeutic drugs or biomolecules (e.g., enhancing the efficiency of cancer therapy in animal models 17,18 , decreasing blood glucose by increasing the level of insulin in the blood of diabetic rats 13  The resulting suspension was allowed to age at a constant 60 °C temperature for several hours. The precipitates were separated by centrifugation, washed several times with double-distilled water, and let to dry overnight in an oven at 60 °C. The samples prepared using the above-mentioned procedure are further labeled in the text as follows: • Experimental techniques. Powder X-ray Diffraction (XRD) measurements were performed using a D8 ADVANCE diffractometer (BRUKER-AXS GmbH, Germany) with Ni filtered Cu radiation (λ = 1.54184 Å). The lattice parameters and the average crystallite size were determined by Rietveld refinement, using the Topas v.3 software (Bruker) 27 .
Fourier-Transform InfraRed (FTIR) spectroscopy measurements were performed using a Spectrum BX II (Perkin Elmer) spectrometer in the 4000-350 cm −1 spectral range, by accumulating 64 scans at a resolution of 4 cm −1 . The samples were finely crushed with KBr in a 1:50 mass ratio and pressed into thin pellets 27 .
Transmission Electron Microscopy (TEM) studies were performed using a JEOL 2100 system equipped with an Energy Dispersive X-Ray Spectroscopy (EDS) detector and ASTAR crystallographic analysis unit. All the samples were prepared using the conventional powder method.
Electron Paramagnetic Resonance (EPR) spectroscopy investigations in the X (9.86 GHz) frequency band were carried out at room temperature on a Bruker ELEXSYS E-580 spectrometer equipped with the calibrated Super High QE (SHQE) cylindrical cavity resonator (ER 4123SHQE). Reference-free determinations of the Mn 2+ ions concentration in weighted amounts of powder samples inserted into pure fused silica tubes were performed using the absolute spin quantitation routine included in the XEPR software from Bruker 28 .
BET surface area & Porosity measurements Nitrogen sorption isotherms were recorded at 77 K using a Micromeritics ASAP 2020 apparatus. Each sample was degassed at 100 °C for 12 h under vacuum before analysis. The BET surface area was calculated according to the Brunauer-Emmett-Teller (BET) equation, using adsorption data in the relative pressure range between 0.05 and 0.30. The total pore volume was estimated from the amount adsorbed at the relative pressure of 0.99. The pore size distribution curves were obtained using the Barrett-Joyner-Halenda (BJH) method from the desorption branch [46][47][48] . Figure 1 presents the X-ray diffractograms of the two sets of ZnO samples doped with variable Mn 2+ nominal concentrations in the presence of either PVP or SHMTP. The two sets of samples exhibit similar XRD patterns, indexed as single-phase ZnO with a hexagonal structure, space group P63mc (JCPDS 89-1397). In the case of the ZnO samples prepared in the presence of PVP (Fig. 1a), the relative intensities of the XRD peaks are typical for nanocrystals with polyhedral morphology, the average crystallite size being 38 ± 3 nm.   38 . Therefore, as expected, a low doping level does not affect the lattice parameters. TEM investigations. TEM analysis revealed morphological and structural aspects of the investigated samples. Two types of systems have been identified, defined by the influence of the used surfactants: i) a system of quasi-spherical, monodispersed nanoparticles with a size of ~ 30 nm and ii) a system consisting of both single nanoparticles and broad distribution of aggregates with complex morphologies, with a resulting size distribution spreading from ~ 30 nm to ~ 150 nm, in the PVP and SHMTP samples, respectively.

XRD investigations.
The CTEM images in Fig. 2 (a)-(c) (top left) show the simple morphological system of the PVP samples whereas in Fig. 2 (d)-(f) (top left) the complex morphologies present in the SHMTP samples are pictured.
All the SAED patterns in Fig. 2 (bottom left) display lattice planes of hexagonal ZnO (space group 186). All the samples are well crystallized.
A quantitative description of the two types of morphologies of ZOM2000P and ZOM2000S, as observed by conventional imaging, is shown in the size distribution in Fig. 2 (right). The bi-modal behavior of the two distributions, with maxima at S1 = 30 nm and S2 = 60 nm as well as their corresponding FWHM, i.e. relatively narrow for S1 for both samples but much broader for S2 of ZOM2000S, suggests that in the presence of the PVP surfactant the NPs system tends to be much more uniform (mainly with small crystal sizes), whereas in the presence of SHMTP surfactant it exhibits a much more complex morphology (mainly with large crystal sizes). It is www.nature.com/scientificreports/ worth mentioning that both types of nanoparticles as they are quantitatively described by S1 and S2 are present in both ZOM2000S and ZOM2000P, but their prevalence changes dramatically.
EPR spectroscopy. The EPR spectra of the two sets of ZnO:Mn samples, normalized for differences in mass and spectrometer parameters (i.e. resonator quality factor Q, number of scans), are displayed in Fig. 3 a-b. As expected, the intensity of the spectra increases with the increase in the Mn 2+ doping levels. The spectra are quite alike for the two sets of samples prepared with different surfactants, except in the case of the ZOM50P sample for which the intensity of the spectrum is markedly higher than for the corresponding ZOM50S sample. The dominant features in the EPR spectra are two groups of six lines with different intensities and linewidths (~ 0.3 mT and ~ 1.7 mT, respectively), characteristic to the hyperfine structure of isolated Mn 2+ ions (S = 5/2, I = 5/2). The similar line separation of ~ 7.8 mT shows that both sets of lines belong to Mn 2+ ions localized in the ZnO lattice, namely in ZnO nanocrystals (the broad lines) and in a disordered ZnO phase (the narrow lines) 39 . The actual concentrations of the Mn 2+ ions in weighted amounts of the investigated ZnO:Mn samples were determined by the quantitation of the recorded X-band EPR spectra. The error in these determinations was estimated to decrease from 30% for the 50 ppm nominal concentration to 25% for 2000 ppm. According to the results presented in Fig. 3c, the actual Mn 2+ concentration is one to two orders of magnitude lower than the nominal doping concentration. Moreover, the actual concentration of the Mn 2+ ions in the PVP samples is lower than in the SHMTP samples with the same nominal concentration, except in the case of the ZOM50P sample. Another oddity of the ZOM50P sample is that the actual Mn 2+ concentration is very close to the one determined for the ZOM500P sample.  BET & porosity. The recorded N 2 adsorption-desorption isotherms are shown in Fig. 5. All the samples display typical type IV isotherms accompanied by H3 type hysteresis loops, according to the IUPAC classification 46 . The hysteresis loops appear at high relative pressures (> 0.8), which suggests the presence of non-rigid aggregates with irregular and large slit-shaped pores 47,48 . The pore size distribution curves (Fig. 5, right side) confirm these observations and the data are in good agreement with the microscopic investigations that reveal irregular pores randomly distributed, mainly consisting of interparticle voids. The textural parameters (Table 1) present low values, as expected when taking into account the morphology of the samples. It can be noticed that the S BET and total pore volume values are slightly higher for the sample obtained using PVP as a surface agent (surfactant) in comparison with the samples prepared in the presence of SHMTP. The values are directly proportional to the dopant concentration, regardless of the surface agent (surfactant) used. This can be attributed to the formation of larger aggregates of nanoparticles in the case of the samples prepared in the presence of SHMTP, as TEM investigations revealed. These results are in agreement with the size distributions obtained from TEM determinations, the ZOMP samples with small particle size and narrow size distribution being associated to larger surface areas and larger pore volumes compared to the ZOMS samples partially composed of large size aggregates with broad size distribution, characterized by reduced surface areas and smaller pore volumes. Cellular studies results. Cell viability tests. For NPs concentrations below 4 µg/mL, the cell viability is not significantly affected (see Fig. 6); for nominal doping concentrations of 500 ppm and 2000 ppm of Mn 2+ , a slight growing stimulation effect (viabilities above 100%, more pronounced in the case of ZOM2000S) can be observed.
At these NPs concentrations, in the case of the 50 ppm Mn 2+ nominal concentration (black lines in Fig. 6), the viability slightly decreases with NPs concentration (even if not statistically significant). At higher NPs concentrations, the viability drops, going down to zero for concentrations above 16 µg/mL. Intracellular ROS measurement. The ROS production increases with the concentration of NPs in both PVP and SHMTP formulations (Fig. 7). The higher the nominal doping concentration, the less pronounced is the dependency of the ROS production on the NPs concentration: the doping concentration flattens the dependency of ROS production on NPs concentration. This behavior was observed to be more pronounced in the case of ZOMS samples compared to ZOMP. For ZOM2000S, the dependence of the ROS production on the NPs concentration becomes insignificant.
Cellular DNA fragmentation. In order to assess the DNA fragmentation, comet assay images were acquired for untreated cells and cells treated with ZOMP and ZOMS NPs, respectively, as exemplified in Fig. 8 a-c. In the case of the cells exposed to NPs, the fragmentation of DNA was revealed by the presence of the comet-shaped fluorescent halo shown in Fig. 8b and c. No such halo was observed in the case of control cells.
The tail lengths of the comets are presented in Fig. 8d and e, for the PVP and SHMTP formulations, respectively.     www.nature.com/scientificreports/ For all NPs concentrations DNA fragmentation was observed (tail lengths from exposed cells are significantly longer compared to the negative control). The DNA fragmentation induced by exposing cells to NPs was not as thorough as in the case of the positive control (cells treated with H 2 O 2 ).
In the case of the PVP formulation (Fig. 8d), increasing either the NPs or Mn nominal doping concentrations does not produce a further augmentation of the DNA fragmentation.
In the case of the SHMTP formulation (Fig. 8e)

Discussion
Two series of Mn-doped ZnO NPs, each with a nominal dopant concentration of 50, 500, and 2000 ppm, were obtained following a surfactant-assisted co-precipitation synthesis method, in the presence of either PVP or SHMTP. In order to assess the influence of the used surfactants/SDAs on the physicochemical properties of the produced nanomaterials, the synthesized samples were characterized with respect to their crystal structure, particle size and morphology, textural properties (specific surface area and porosity and surface chemistry) and Mn-doping efficiency. The relationship between the observed surfactant-controlled properties of the synthesized NMs and their in vitro oxidative stress and cytotoxic effects was investigated by evaluating the cellular viability, intracellular ROS generation, and DNA fragmentation induced in non-malignant murine fibroblast NIH3T3 cells.
In the case of the ZOMP samples, the shape-directing action of PVP suppressed the c-direction of the ZnO crystals and led to the growth of single phase quasi-spherical monodisperse nanoparticles (≈ 38 nm), with lattice parameters unaffected by the doping process. The difference in the particles morphology suggested by the XRD results is confirmed by TEM investigations. The constancy of the lattice parameters with respect to the changing nominal concentration of Mn 2+ is not surprising considering the low real concentration of Mn 2+ ions incorporated in the ZOMP samples (12-20 ppm as indicated by the EPR results illustrated in Fig. 3c) and the small difference between the ionic radii of Zn 2+ and Mn 2+ . It thus appears that the nucleation and growth of the ZOMP nanocrystals were mainly dictated by the action of PVP which hindered the incorporation of Mn into the samples.
The ZOMP nanoparticles exhibit type IV N 2 adsorption-desorption isotherms (Fig. 5 (left)) which are characteristic for mesoporous materials. Moreover, the pore size distribution (Fig. 5 (right)) and the fact that the hysteresis loops appear at high relative pressures indicate the presence of non-rigid particle aggregates, in agreement to the TEM results. The values of BET specific surface area and total pore volume (Table 1) are directly proportional to the dopant concentration. Considering the low level of doping, it appears that these surface properties are very sensitive to the presence of Mn 2+ ions. The low measured values of the BET surface area are not surprising considering the low synthesis temperature and the lack of thermal treatment after co-precipitation.
In comparison to ZOMP, the ZOMS samples exhibit significant differences regarding particle size distribution, morphology, and doping efficiency. The samples prepared in the presence of SHMTP consist of anisotropic ZnO NPs and aggregates with complex morphologies, leading to a broad size distribution ranging from ~ 40 nm to ~ 150 nm. The notable (002)-shape anisotropy, with a crystallite size of 49 ± 4 nm along (002) and 23 ± 2 nm for other crystallographic directions, indicates that the particle extension is promoted in the c-direction. SHMTP is thus less effective in constraining the shape of the ZnO nanocrystals in comparison to PVP. The anisotropic growth appears to facilitate the incorporation of Mn 2+ ions into the ZOMS samples, a concentration of 42 ppm of Mn 2+ being found by EPR determinations in the ZOM2000S sample (Fig. 3c). The increased doping efficiency in the ZOMS samples compared to the ZOMP samples could be due to their particular morphology, which exposes crystal faces with favorable binding energy for the Mn 2+ ions 49 . The surprisingly low amount of Mn detected by EPR in both ZOMP and ZOMS series could be due to either inefficient doping due to the synthesis conditions or the presence of larger amounts of Mn in the samples in the form of the EPR silent Mn 3+ ions. Concerning the textural properties of ZOMS, the measured BET specific areas and total pore volume are directly proportional to dopant concentration but the S BET values are smaller compared to those of the ZOMP samples (Table 1). Similar to the case of ZOMP, the type IV N 2 adsorption-desorption isotherms of ZOMS (Fig. 5 (left)) indicate the mesoporous nature of the samples. The pore size distribution (Fig. 5 (right)) together with the high values of the relative pressure where hysteresis loops occur suggest the presence of non-rigid aggregates, larger than in the case of the ZOMP series, with large slit-shaped irregular pores.
The FTIR spectroscopy investigations (Fig. 4) confirmed that no trace of the two surfactants remained in any of the studied ZnO:Mn samples after the washing and drying processes, any direct involvement of the surfactants in the in vitro action of the tested nanomaterials being thus excluded.
To summarize, the two series of samples, ZOMP and ZOMS, are distinct with respect to three aspects: i) particle size and morphology; ii) Mn-doping level; and, iii) specific surface area and porosity. While PVP induced the formation of quasi-spherical monodisperse nanoparticles, SHMTP allowed anisotropic growth on the c-direction leading to complex aggregates with a large size distribution. The Mn-doping of the synthesized ZnO nanocrystals was hindered by PVP and facilitated by SHMTP. Also, PVP led to higher specific surface areas and larger pore volumes compared to SHMTP. These results suggest that PVP could be helpful in producing mesoporous ZnO nanomaterials with high surface area and large pore volume for photocatalysis or gas sensing applications while SHMTP could be employed when Mn-doped anisotropic ZnO nanoparticles are desired. www.nature.com/scientificreports/ Regarding the in vitro toxicity effects of the studied Mn-doped ZnO NPs, the MTS viability tests allowed the determination of the NPs concentrations for which it was justified to perform the other types of studies (ROS production and DNA fragmentation). NPs concentrations of up to 4 µg/mL did not significantly impact cellular growth. At these low NPs concentrations, the MTS assay showed that increasing the Mn 2+ doping level diminishes the cytotoxic effect of NPs for both surfactant formulations.
The ROS production and DNA fragmentation revealed the oxidative stress induced in the cells exposed to NPs. The results of both tests led to the conclusion that oxidative stress is lower when using higher Mn-doping, this tendency being more pronounced in the case of the SHMTP formulation. For instance, the protective effect of doping is better observed in the case of ZOM2000S, where the ROS production does not depend on the NPs concentration anymore and the DNA fragmentation was found to be the smallest.
As it was shown by Laurent et al., 2005, treating NIH3T3 fibroblasts with low amounts of H 2 O 2 increased their proliferative rate, while further increased amounts of H 2 O 2 resulted in growth arrest and cell death 50 . In our case, the ROS production due to small concentrations of NPs seems to play a similar role: at low NPs concentrations, there is a slight increase in the cell viability compared to controls, while at high NPs concentrations the viability drops. At higher NPs concentrations, the higher amount of ROS production exceeds the defense capability of the cells. Doping with Mn 2+ has an antioxidant protective effect which is more pronounced in the case of the SHMTP series, probably due to the higher Mn 2+ nominal concentration (see Fig. 6) in samples with the same NPs amount.
It is established that one of the main mechanisms responsible for the in vitro toxic effects of the ZnO nanomaterials involves the action of the Zn 2+ ions released due to ZnO dissolution in alkaline phosphate media 19 . Moreover, it has been shown by several groups that the solubility of the ZnO nanoparticles in such media is diminished by TMIs doping 32,51 . Based on these considerations, we argue that the observed negative correlation between the cytotoxic and oxidative stress responses, on the one hand, and the doping level of the tested Mndoped ZnO NMs from each series (ZOMP or ZOMS), on the other hand, is caused by the reduction of the ZnO dissolution induced by Mn-doping. This argument is also supported by the observed weakening of the dependence of the magnitude of the oxidative stress indicators on the concentration of the tested NPs with increasing dopant concentration in either ZOMP or ZOMS.
A comparison between the two series with respect to the dopant-dependent dissolution aspect would be forced since the dissolution of ZnO (and other types of nanoparticles) is also known to be size-and shape-dependent 52,53 .
Previous ZnO toxicology studies considered that the specific surface area is one of the key factors involved in the toxicity of ZnO nanomaterials 54 . In the case of ZOMP samples, the interaction of their larger surfaces with the cells and culture environment led to enhanced intracellular ROS production and DNA damage in comparison with the ZOMS series which was associated to smaller surface areas. This result indicates a positive correlation between the specific surface area and the magnitude of the studied in vitro toxicological effects. Such a relationship is expected considering the higher cellular stress experienced by cells exposed to larger nanomaterial surfaces 55 . This finding can also be described in terms of particle size distributions, emphasizing that samples with small particle size and narrow size distributions (e.g. ZOM2000P in Fig. 2), associated with bigger S BET values, induce more intense cell responses in comparison to samples with larger particles/aggregate size and broad size distribution (e.g. ZOM2000S in Fig. 2) which are characterized by smaller surface areas. However, in each of the studied sample series, ZOMP and ZOMS, there is a negative correlation between the values of S BET (which are directly proportional to the nominal doping concentration) and the cell oxidative responses. Based on these considerations, one can thus argue that Mn-doping exerts a protective effect on cells by diminishing the pro-oxidative action of the increased specific surface area.
The present results encourage the conduction of further studies on the use of surfactants/shape-directing agents to control the Mn-doping of ZnO and the mechanisms of in vitro toxicity of Mn-doped ZnO nanomaterials.

Conclusions
Two surfactants with distinct shape-directing properties, polyvinylpyrrolidone (PVP) and sodium hexametaphosphate (SHMTP), were used in a low temperature co-precipitation synthesis method to produce quasi-spherical or anisotropic Mn-doped ZnO nanocrystals, respectively. The two types of nanomaterials showed differences with respect to size distribution, Mn-doping level and surface properties. The PVP-based synthesis led to ZnO samples consisting of nanoparticles with a mean size of ~ 38 nm, quasi-spherical morphology and monomodal size distribution. The ZnO samples obtained in the presence of SHMTP were composed of larger nanoparticles with broad size distribution, ranging from ~ 40 to 150 nm, and with complex morphologies.
Comparing the samples in terms of textural properties, the ZOMP samples present larger surface areas and larger pores than the ZOMS samples. The values of both textural parameters increased with Mn concentration in all cases.
In what regards the NPs doping, PVP was shown to hinder Mn incorporation into ZnO while SHMTP allowed for a more efficient Mn-doping.
The relation between relevant material properties, Mn-doping level, specific surface area and porosity, and the main in vitro oxidative stress toxic effects was assessed using murine fibroblast cells. The cell viability, intracellular ROS production, and DNA fragmentation were shown to depend on the morpho-structural properties of the tested ZnO nanoparticles as well as on the Mn-doping characteristics dictated by the used surfactants. The ZnO samples prepared in the presence of PVP were shown to be more cytotoxic than the ones prepared in the presence of SHMTP.
The ZnO PVP samples (ZOMP) display a stronger cytotoxic effect compared to SHMTP samples (ZOMS) due to morpho-structural characteristics that make them more reactive in the in vitro environment like smaller particle size, narrow size distribution, larger surface areas and pore volumes, and Mn incorporated inefficiently.

Scientific Reports
| (2020) 10:18062 | https://doi.org/10.1038/s41598-020-74816-0 www.nature.com/scientificreports/ The cytotoxic action is diminished with increasing the Mn concentration as follows: the cell viability is affected more pronouncedly in the case of ZOMP samples but the effect weakens at higher doping levels; the cells exposed to ZOMP samples produce a higher quantity of ROS, which also decreases when the Mn concentration is increased; the DNA fragmentation is enhanced in the cells treated with ZOMP samples but it does not depend on the NPs concentration or Mn concentration. A peculiar effect was observed in the case of the ZOM2000S sample, for which the DNA fragmentation was reduced with increasing the NPs concentration.
The reduction of the cytotoxic effects following the increase of Mn-doping level suggests the possibility of using Mn-doping to improve the cytocompatibility of ZnO nanomaterials, even if they have characteristics, such as small particle size and large surface area, generally known to correlate to high cytotoxic effects.
Why are these findings important? When considering ZnO nanoparticles for antimicrobial applications, cosmetics or topical ointments for dermatological use, it is of prime importance to ensure their biocompatibility while maintaining the desired functional properties. Our study indicates that the use of convenient shapedirecting agents and surfactants during the synthesis process and manganese doping may constitute a valid approach for this purpose.
Aside from gaining new insight regarding the ZnO in vitro toxicity modulation by surfactant-tailored morpho-structural properties and Mn-doping, our results suggest that structure-directing agents may represent a non-expensive and facile way to engineer doping processes and modulate the biocompatibility of nanomaterials. Cell viability test. CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega G3581, USA) (MTS) was used to measure the cell viability. Viable cells convert the tetrazolium reagent to the soluble formazan product, the concentration of which can be photometrically measured (maximum absorption at 490 nm). The formazan concentration is linearly correlated to the number of viable cells.

Methods
Sterile ZOMP/ZOMS NP suspensions were added in each well at final concentrations of 0, 1, 2, 4, 8, 16, 32, and 64 µg/mL (for each concentration four identical wells were used) and the cells were further incubated for 24 h; the medium was then removed and the cells were washed with 0.9% NaCl solution. 300 µL of Dulbecco's Modified Eagle Media without Phenol-red (Gibco 21063-029, UK) containing MTS (volume ratio 5:1) was added to each well and incubated 2 h at 37 °C. The clear supernatant was transferred to new plates and the absorbance at 490 nm was recorded using a plate reader (Awareness Technology Inc., Taiwan). After subtracting the blank value, the absorbance values were normalized by dividing the average absorbances of identical samples by the average absorbance of the controls (samples with 0 µg/mL NPs).

Intracellular ROS measurement.
The Image-iT Live Green Reactive Oxygen Species Detection Kit (Ter-moFisher Scientific I36007, USA) (H 2 DCFDA) was used to measure the production of ROS induced by ZOMP/ ZOMS. H 2 DCFDA is a membrane-permeant nonfluorescent compound. After entering the cell, it is deacetylated by cellular esterases and later oxidized by ROS into a highly fluorescent compound (DCF) which can be quantified by fluorescence (λ ex = 495 nm, λ em = 529 nm).
Sterile ZnOMn NPs suspensions were added in each Petri dish at final NP concentrations of 0, 1, 2, 4, and 8 µg/mL and the cells were further incubated for 24 h. The medium with NPs was then removed and the cells were washed with a sterile buffer (1.26 mM CaCl 2 , 5.4 mM KCl, 0.44 mM KH 2 PO 4 , 0.34 mM Na 2 HPO 4 , 3.31 mM NaHCO 3 , 0.5 mM MgCl 2 , 0.41 mM MgSO 4 , 137 mM NaCl, 5.56 mM D-glucose, pH 7.4). H 2 DCFDA was added to the cells (final concentration 2 µM) and further incubated for 40 min. The cells were washed and the fluorescence intensity was measured (Zeiss Observer D2 inverted microscope, Germany, equipped with RatioMaster D-104C Horiba system, USA, Felix Gx acquisition software). The signal was collected from one single cell during 10 s, and the average value was normalized to the cell surface area (Zeiss AxioVision rel 5.9 and Image J). For each NPs concentration, 30 cells were randomly chosen from 3 different Petri dishes. Cellular DNA fragmentation. The comet assay is a single cell electrophoresis technique used to measure DNA fragmentation. The cells are incorporated in an Agarose gel, deposited on a microscope slide, lysed, and subjected to electrophoresis. The genetic material from each cell will migrate depending on its degree of fragmentation: smaller DNA fragments will migrate faster 56 . Samples were stained with Propidium Iodide and fluorescence images were recorded (λ ex = 490 nm, λ em = 510 nm) showing a "comet" shaped pattern. The fragmentation degree was quantified by the tail length of the comet (defined as the distance between the geometrical centers of the head and of the comet).
Between 150 and 300 randomly captured images of comets were acquired for each NPs concentration and their tail length was measured.
Statistical analysis for cellular studies. All data were analyzed using a one-way analysis of variance (ANOVA) with Tukey's multiple comparison tests to determine statistical significance among treatments using Origin 8.6 software. Differences were considered significant for p < 0.05.