High Surface Reactivity and Biocompatibility of Y2O3 NPs in Human MCF-7 Epithelial and HT-1080 FibroBlast Cells.

This study aimed to generate a comparative data on biological response of yttrium oxide nanoparticles (Y2O3 NPs) with the antioxidant CeO2 NPs and pro-oxidant ZnO NPs. Sizes of Y2O3 NPs were found to be in the range of 35±10 nm as measured by TEM and were larger from its hydrodynamic sizes in water (1004 ± 134 nm), PBS (3373 ± 249 nm), serum free culture media (1735 ± 305 nm) and complete culture media (542 ± 108 nm). Surface reactivity of Y2O3 NPs with bovine serum albumin (BSA) was found significantly higher than for CeO2 and ZnO NPs. The displacement studies clearly suggested that adsorption to either BSA, filtered serum or serum free media was quite stable, and was dependent on whichever component interacted first with the Y2O3 NPs. Enzyme mimetic activity, like that of CeO2 NPs, was not detected for the NPs of Y2O3 or ZnO. Cell viability measured by MTT and neutral red uptake (NRU) assays suggested Y2O3 NPs were not toxic in human breast carcinoma MCF-7 and fibroblast HT-1080 cells up to the concentration of 200 μg/mL for a 24 h treatment period. Oxidative stress markers suggested Y2O3 NPs to be tolerably non-oxidative and biocompatible. Moreover, mitochondrial potential determined by JC-1 as well as lysosomal activity determined by lysotracker (LTR) remained un-affected and intact due to Y2O3 and CeO2 NPs whereas, as expected, were significantly induced by ZnO NPs. Hoechst-PI dual staining clearly suggested apoptotic potential of only ZnO NPs. With high surface reactivity and biocompatibility, NPs of Y2O3 could be a promising agent in the field of nanomedicine.


Introduction
Screening of nanoparticles (NPs; defined as particles having, at least, one dimension below 100 nm) to be exploited in nanomedicine has been unprecedented in recent years since they provide multiple avenues in diagnosis and therapy of diseases. NPs could be engineered to target cancerous cells and release anticancer drugs therein, thus, avoiding normal cell toxicity that is a bigger obstacle in conventional chemotherapy [1,2]. NPs originating from lanthanide group exhibit remarkable properties owing to their high optical activity, surface reactivity and superior photo stability [3][4][5]. High refractory properties, good thermal conductivity, superior stability and excellent mechanical property make yttrium oxide (Y 2 O 3 ) NPs a fascinating material [6]. Engineering NPs for active targeting to cancer cells while sparing normal cells, however, requires NPs with certain inherent properties. Obviously, biocompatibility and surface reactivity are some of the important criteria. Due to their intrinsic physical properties, inorganic NPs, especially of lanthanide group, offer highly tunable surface properties and stability over polymeric NPs [7,8]. NPs of antioxidant CeO 2 and quite a different pro-oxidant (toxic) NPs of ZnO, for example, have received much attention for their potential usefulness in medicine though both NPs exhibit different biological effects [9]. Some investigators have reported that chemically related NPs of CeO 2 and Y 2 O 3 possess redox potential that enables them to mimic the activity of antioxidant enzymes such as superoxide dismutases (SODs) [10]. Moreover, as a novel treatment strategy, Y 2 O 3 NPs could be used in the therapy of fulminant hepatic failure and oxidative stress-related diseases [11].
The present study was designed seeking a higher surface reactivity of NPs that could be exploited in nanomedicine. Since surface reactivity gives information about the extent to which NPs surface can be tuned, therefore, determining surface reactivity is considered a pre-requisite in the field of nanomedicine. Understanding the surface reactivity of protein molecules such as bovine serum albumin (BSA) is crucial towards predictability of drug interaction with a NP [12,13]. Quantification of BSA reactivity towards NPs have been one of the reliable methods in the evaluation of the degree of NPs activity towards biological models and molecules [14]. In this study, surface reactivity of BSA with Y 2 O 3 NPs was studied and compared with the surface reactivity of CeO 2 NPs and ZnO NPs. To the best of our knowledge, we could not find any report on BSA reactivity with that of Y 2 O 3 NPs. There are, however, reports on BSA reactivity with the NPs of europium-doped Y 2 O 3 (Y 2 O 3 :Eu 3+ ) and ZnO NPs [14]. As will be discussed later, BSA affinity with Y 2 O 3 NPs was found to be highest among all of the three NPs chosen in this study. Strong BSA reactivity suggested Y 2 O 3 NPs could be a potential candidate in drug loading and delivery. In addition to BSA powder suspended in aqueous solution, surface reactivity of Y 2 O 3 NPs towards culture media ingredient was also compared with that of CeO 2 and ZnO NPs since surface modification can significantly reduce the toxicity of NPs [15].
Due to aforementioned properties, NPs of Y 2 O 3 , if found to be biocompatible, could serve the purpose of nano-carrier for various drugs. Biocompatibility of Y 2 O 3 NP was examined in the two human cell lines-human breast (MCF-7) cells and fibroblast (HT-1080). These cells are a proven model for bio-medical testing [9,16,17]. This study, therefore, reports a comparative finding in terms of surface reactivity and biocompatibility of Y 2 O 3 NPs with the chemically related NPs of CeO 2 and chemically un-related NPs of ZnO. It is notable that NPs of Y 2 O 3 , like that of CeO 2 NPs, have been reported to be antioxidant and protective in cells of neural [18,19] and retinal [20], whereas some investigators have reported this NP as oxidative and toxic [21,22]. In this study, we have provided a comparative data on surface reactivity with BSA and media components which is not known. Moreover, detailed mechanisms of potential toxicity carried out in the two human cell lines could further shed light on the discrepancy in toxicity level that exists for Y 2 O 3 NP [18][19][20][21][22].

Y 2 O 3 NPs TEM Sizes, Hydrodynamic Sizes and Zeta Potential
NPs size calculated by TEM imaging at different resolution came to be 35 ± 10 nm while shapes appeared mostly cuboidal ( Figure 1A,B). The matte texture observed in high-resolution (HR) TEM images ( Figure 1C,D) confirms the crystallinity of Y 2 O 3 NPs.
Sizes and zeta potential of NPs in distilled water, phosphate buffer saline, serum free culture medium and complete culture media were measured to see the potential differences caused by serum and media components. Dynamic light scattering (DLS) measurements taken at 200 µg/mL show a differential dispersion of Y 2 O 3 NPs in distilled water, phosphate buffer saline, serum free culture media and complete culture media as given in Table 1.  Surface reactivity of Y 2 O 3 NPs with BSA was examined and compared with surface reactivity of two other NPs-CeO 2 and ZnO-known to exert opposite responses in a wide variety of biological models. Among these NPs, Y 2 O 3 NPs showed significantly higher BSA adsorption (Figure 2A). Control optical density of BSA (500 µg/mL in water) was taken as a reference point. BSA adsorption study with all three NPs was performed in water. BSA adsorbed, at 6 h of incubation, by NPs of Y 2 O 3 , CeO 2 , ZnO was found to be 330 ± 8, 145 ± 6 and 17 ± 4 µg, respectively. Similar is the trend at 24 h of incubation, though, with a slight decrease in BSA adsorption. To see the alteration in protein adsorption, if any, caused by proteins from other source (fetal bovine serum for example), or components of culture media, the Y 2 O 3 NPs were suspended first in the liquid containing the respective adsorbent and incubated for 6 h or 24 h. After the respective period of incubation, NPs were centrifuged and in BSA containing water. Results clearly indicate that serum or media component caused significant shielding effect to BSA adsorption onto NPs surface ( Figure 2B). Time dependent study shows that shielding effect due to serum or media component is quite stable. Data shows that Y 2 O 3 NPs bind with serum proteins at rate of 30 ± 4 µg/mg NP when incubated for 6 h. BSA pre-treated NPs, however, did not show any noticeable amount of serum adsorption. Moreover, reactivity towards either BSA or media component is stable; neither BSA adsorption occurred to media treated Y 2 O 3 NPs nor serum free media caused BSA displacement from Y 2 O 3 NPs pre-treated with BSA (see summary in Table 2). . Adsorption to BSA was highest for Y 2 O 3 NPs while least for ZnO NPs. Y 2 O 3 NPs resulted in highest surface reactivity, therefore, displacement experiment was further extended to Y 2 O 3 NPs to understand the relative stability of adsorption of BSA, serum albumin and media ingredients with respect to each other (B). Triplicates of each treatment group were used in each independent experiment. * Denotes a significant difference from the control (p < 0.05). Redox active NPs such as CeO 2 are known to possess reactive oxygen species (ROS) scavenging capability. This ROS scavenging capacity plays critical role in the antioxidant activity reported of these NPs, though other factors might also exist [23,24]. In this study, SOD-like and CAT-like activity of Y 2 O 3 NPs was measured along with NPs of CeO 2 and ZnO. Table 3 shows that no significant enzyme-like activity of Y 2 O 3 NPs was detected except for the NPs of CeO 2 .

Y 2 O 3 Nps Did Not Cause Significant Decrease in Cell Viability
There was no difference found in the cell viability data due to BSA treated or non-treated Y 2 O 3 NPs in MCF-7 and HT-1080 cells (see Supplementary Figure S1). The rest of the biological parameters were, therefore, conducted using naïve NPs of Y 2 O 3 , CeO 2 and ZnO. As could be observed in Figure 3A-C, NPs of Y 2 O 3 (and chemically similar CeO 2 NPs at 100 µg/mL) did not cause inhibition to cell viability whereas ZnO caused 50% inhibition to cell viability (defined as IC50) at 41 ± 4 µg/mL to MCF-7 cells and 33 ± 4 µg/mL to HT-1080 cells. and ZnO NPs (IC50) have been used in all of the biological experiments since these two NPs have been taken as primarily for comparison purposes with that of Y 2 O 3 NPs. Phase-contrast images (20×, Leica DMi8, Germany) of MCF-7 cells and HT-1080 cells treated with highest concentration (i.e., 200 µg/mL) of Y 2 O 3 NPs, IC50 of ZnO and CeO 2 (100 µg/mL) have been given (C). For IC50 calculation, a scatter plot in Microsoft Excel was inserted followed by setting the Y-axis to logarithmic. Then a trend line was selected and 'exponential' picked. Then 'display equation' was used in calculating ICs. IC50s calculations were further verified and confirmed from the online IC50 calculator (https://www.aatbio.com/tools/ic50-calculator) provided by AAT Bioquest, Inc. (CA 94085, USA). Scale-bar represents 40 µm (micron) in each image. Triplicates (n = 3) of each treatment group were used in each independent experiment. * Denotes a significant difference from the control (p < 0.05).

Y 2 O 3 NPs Did Not Induce Oxidative Stress
Potential oxidative damage induced by NPs was assessed by cellular integrity directly affected by ROS. Lactate dehydrogenase (LDH) release, a marker of cell membrane damage, was measured in cells at concentrations of 50, 100 and 200 µg/mL exposed for 24 h ( Figure 4A). Y 2 O 3 NPs did not cause any LDH release in MCF-7 and HT-1080 cells at concentrations of 50, 100 and 200 µg/mL. Another marker of membrane damage, lipid peroxidation (LPO), followed similar trend in both cells ( Figure 4B). It should be noted that statistically significance membrane damage caused by NPs of ZnO at IC50. As expected, antioxidant CeO 2 NPs did not elevate markers of membrane damage. Similarly, Y 2 O 3 NPs did not induce ROS that was measured by DCF probe and H 2 O 2 specific sensor ( Figure 4C,D). A standard oxidant, H 2 O 2 , was always included as a positive control of ROS during 2 , 7 -dichlorofluorescin diacetate (DCFH-DA) measurement (see Figure S2).

Y 2 O 3 NPs Did Not Cause Mitochondrial Membrane Potential Induction
To test further biocompatibility of Y 2 O 3 NPs, mitochondrial membrane potential (MMP) was determined qualitatively and quantitatively using combined advantage of a fluorescence microscope (Leica DMi8, Wetzlar, Germany) and ImageJ software. Qualitative and quantitative results clearly suggest that Y 2 O 3 NPs exposure at 200 µg/mL, highest concentration of NPs taken in this study, to both cells did not exert negative impact ( Figure 5A,B). Punctate red fluorescence of JC-1 aggregate coming from intact mitochondria is a qualitative evidence of non-toxicity of Y 2 O 3 NPs. In essence, there was no appreciable change in MMP after 24 h of incubation with any concentration of Y 2 O 3 NPs relative to control cells.

Y 2 O 3 NPs Did Not Cause Appreciable Change in Autophagy Activity
Finally, a comparative potential of autophagy modulation due to the three NPs was evaluated to understand the mechanism of biocompatibility in greater depth because many biocompatible NPs established by cytotoxicity and oxidative stress later turned to be inducer of autophagy. Above mitochondrial data provide sufficient set of evidence that suggests Y 2 O 3 NPs are biocompatible in MCF-7 and HT-1080 cells. Similar to the role of mitochondria in cell survival and death, lysosomes are thought to play critical part in determining the fate of the cells. The two related NPs-Y 2 O 3 and CeO 2 , did not induce changes in lysosomal activity in MCF-7 ( Figure 6A) and HT-1080 ( Figure 6B) cells. LTR fluorescence, however, suggested a definite increase in lysosomal or acidic autolysosomes that occurred in ZnO NPs treated cells. A higher LTR fluorescence, quantified for both cells in Figure 6C, is equivalent to extensive acidic vesicle formation within cells.

Y 2 O 3 NPs Did Not Exhibit Apoptotic-Necrotic Potential
Hoechst and PI dual staining clearly shows NPs of Y 2 O 3 and CeO 2 to be non-cytotoxic (Figure 7). However, ZnO NP treated cell depicts IC50 toxicity. Moreover, an apoptotic mode of cells death in the case of ZnO NPs is evident by the fragmented and clumped pattern of chromatin condensation (see Figure S3).  Figure S3). Triplicates (n = 3) of each treatment group were used in each independent experiment.

Discussion
A variety of potential interacting component in relevant aqueous fluids may affect NPs native property prior to biological interaction [25,26]. Zeta potential and hydrodynamic sizes of NPs play crucial role in their secondary surface formation or coronization [27,28]. In this study, as determined by DLS, zeta potential of Y 2 O 3 NPs suspension in complete culture media (−27.0 ± 1.2 mV) was much better than that in water (−16.0 ± 4.2 mV), PBS (−6.0 ± 2.4mV) and serum free culture media (−10.0 ± 4.0 mV). Interestingly, in a previous study, zeta potential of all aqueous (in deionized water) Y 2 O 3 NPs preparations were highly negative with spherical Y 2 O 3 NPs having an average zeta potential of −13 mV, platelets shaped NPs having an average zeta potential of −27 mV and the rod shaped NPs having an average zeta potential of −28 mV [29]. The respective mean hydrodynamic sizes for the spherical NPs (TEM size; 3 nm) was 700 nm, the platelet-shaped NPs (TEM size; 4 nm) was 700 nm and the rod-shaped NPs (TEM size; 11 nm) was 800 nm [29]. It should be recalled that in the present study the shape of the Y 2 O 3 NP (TEM size; 35 ± 10 nm) was cubic in shape. In the present study, hydrodynamic size of Y 2 O 3 NPs in water came out to be 1004 ± 134 nm while in complete culture media it was 542 ± 108 nm. The more negative zeta potential and lesser agglomeration observed in complete culture media for Y 2 O 3 NPs is partially attributable to high surface reactivity of Y 2 O 3 NPs with protein molecules as is evident by next experiment on Y 2 O 3 NPs interaction with BSA proteins. In addition, BSA-treated Y 2 O 3 NPs exhibited better dispersion in all of the tested aqueous media than native NPs (see Table 1).
Protein adsorption to NPs is a dynamic process in fluids be it culture media, blood or other bio fluids independent of nature of the NPs either polymeric [28] or metallic [30]. One of the most notable alterations when NP is under biological fluids is the formation of NP-protein corona [28]. In addition to protein coating, there are reports about non-protein coating on Y 2 O 3 NPs to achieve desirable outcome. Y 2 O 3 NPs coating with glycerol citrate polymer, for example, caused increased antibacterial activity [31]. Realizing the importance of surface reactivity, in this study protein adsorption on to NPs in its native form was quantified using bovine serum albumin (BSA) in a time-dependent manner (see Figure 2). From the results, it can be safely concluded that the cellular response of Y 2 O 3 NPs is actually the response of complete-media-component (that include serum proteins as well media ingredients) coated-Y 2 O 3 NPs rather than only serum protein coated NPs. Thus, living systems mostly interact with the surface modified NPs rather than with bare NPs only when the NPs are surface reactive [32,33]. Bio-molecular corona formed on surface reactive NPs can provide stealth effect during NP uptake by immune cells [34]. Corona formation can also aid in biocompatibility [25]. Of the three NPs tested for surface reactivity, NPs of Y 2 O 3 emerge with highest surface reactivity. The ROS scavenging capacity plays critical role in the antioxidant activity reported for CeO 2 NPs [23,24]. Several reports have shown NPs of Y 2 O 3 and CeO 2 with similar characteristics to offer protection against induced toxicity by pro-oxidants [9,22]. Antioxidant enzyme-like activity exhibited by certain NPs such as that of CeO 2 has been implicated in its potential protective effect against induced toxicity. Only CeO 2 NPs, however, were found to exhibit SOD-like and catalase-like activity in this report (Table 3).
Cell viability in cells treated with the NPs of Y 2 O 3 (35 ± 10 nm) and CeO 2 up to the concentration of 200 µg/mL was find equal to that of control in the MCF-7 and HT-1080 cells. Some investigators, however, have found NPs of Y 2 O 3 (41 ± 5 nm) to inhibit 50% cell growth (i.e., IC50) at the concentration of 108 µg/mL in human embryonic kidney (HEK293) cells [35]. Other researchers have examined NPs of Y 2 O 3 and yttrium ions simultaneously in yeast and have concluded the yttrium ions derived from NPs responsible for the toxicity observed in yeast cells [36]. In mouse normal hepatocyte cell line, Y 2 O 3 NPs (7-8 nm) coated with polymer of acrylic acid have been reported to cause toxicity at the concentration of 10 µg/mL [21]. On the other hand, NPs of Y 2 O 3 have been reported to be antioxidant and protective in cells of neural [18,19] and retinal [20] origin. In this study, no significant difference in cell viability was observed for BSA-coated and native NPs of Y 2 O 3 (See Figure S1). ROS such as H 2 O 2 and O 2•− , can damage cellular components when produced in large amounts but can initiate a diverse array of signaling pathways leading to control cell division, differentiation and migration [37].
Depending on its intracellular concentration and localization, H 2 O 2 initiates either pro-apoptotic or anti-apoptotic response [38]. Levels of H 2 O 2 in the range of 20-50 µM seem to have limited cytotoxicity in many cell types [39]. NPs of Y 2 O 3 and CeO 2 did not cause significant ROS induction in the two cell types. NPs of ZnO, however, induced H 2 O 2 levels over 2-folds in both cells. MMP is the result of ROS that may contribute in inducing death activating pathways [38]. Therefore, NPs are reported to be strong modulator of MMP that can bring the onset of apoptosis, necrosis and autophagy [40,41]. Data suggested that there was no appreciable change in MMP after 24 h of incubation with any concentration of Y 2 O 3 NPs relative to control cells.
The tendency of autophagy modulation is considered as a survival strategy under short-term nutrient stress. However, if the depletion of antioxidants and nutrients is prolonged that might happen under toxicant exposure; the same autophagy may lead to course of cell death. Interestingly, NPs composed of rare earth oxide have been reported as a new class of autophagy inducers [42,43]. Certain NPs, as those of silica, have been widely used in nanomedicine and other applications due to their biocompatibility [44,45]. However, when examined on the parameter of autophagy, these NPs came to cause impairment in lysosomal activity and cellular protein clearance [46,47]. Given the importance of autophagy and lysosomal protein degradation in maintaining cellular homeostasis, therefore, in this report biocompatibility test was extended to analyzing the potential impact Y 2 O 3 NPs might have on the activity of lysosomes. Recall that lysosomes are the major cellular organelle involved in the regulation of autophagy. In our study, the two related NPs-Y 2 O 3 and CeO 2 , did not induce changes in lysosomal activity in both MCF-7 and HT-1080 cells. NPs of ZnO, used as toxic form of NPs, however, increased the number of cellular acidic vesicles and apoptotic mode of cell death in both cell types (see Figure S3 for the apoptotic mode of cell death induced by ZnO NPs).

Yttrium Oxide NPs
NPs of Y 2 O 3 were commercially obtained from Sigma-Aldrich, USA. As per the information provided by supplier, TEM size of Y 2 O 3 was below 50 nm. Color of nanopowder was white.

Transmission Electron Microscopy of Y 2 O 3 NPs
Size of Y 2 O 3 NPs was determined by field emission transmission electron microscopy (FETEM) (JEM-2100F, JEOL, Inc., Tokyo, Japan) using an accelerated voltage of 200 kV [48]. Normal and high resolution (HR) TEM images was also taken to observe the crystallinity of NPs as it happens in HR-TEM texture of typical crystals. Suspension of ultra-sonicated Y 2 O 3 NPs was placed onto a carbon-coated copper grid, air dried and observed with FETEM. Purity of Y 2 O 3 NPs was determined by energy dispersive spectrum (EDS) analysis.

Agglomeration and Zeta-Potential of Y 2 O 3 NPs
Agglomeration behavior and zeta potential of Y 2 O 3 NPs in water, phosphate buffer saline (PBS) and complete cell culture medium was determined by dynamic light scattering (DLS) system (Nano-ZetaSizer-HT, Malvern Instruments, Malvern, UK) as described by Murdock et al. [49]. NPs were freshly suspended in respective aqueous medium (200 µg/mL) and ultrasonicated for 10 min (Ultrasonic Cleaner-8891, Cole-Parmer, 625 Bunker Court Vernon Hills, IL USA). DLS measurement was carried out in cuvettes supplied with DLS system. For DLS measurement, a phenol red-free medium was used.

Determination of Surface Reactivity of NPs
To evaluate surface reactivity, NPs were treated with relevant chemicals such as bovine serum albumin (BSA), serum and media components. To determine the degree of protein adsorption, NPs and BSA were mixed in a 15 mL sterile tube. Final concentrations of BSA and NP were kept at 0.5 mg/mL and 1 mg/mL, respectively in a total volume of 10 mL. Then, a mixture of BSA and NP was put on a gentle shaker at room temperature after ultra-sonication for 20 min. From this NP-BSA mixture, 2 mL was taken in tubes and centrifuged for 10 min at 12,000× g at every 6 h and 24 h to measure protein adsorption. From the supernatant obtained, 100 µL each was taken from either NP-BSA mixture or BSA only solution, and added in cuvette containing 2.0 mL of Bradford reagent. A decrease in absorbance at 595 nm in NP-BSA mixture from BSA only solution was indicative of protein adsorption on NPs. Amount of BSA adsorbed on to NP surface was calculated using BSA standards at 595 nm and expressed as µg BSA adsorbed/mg of NP. Calculation method is similar to that of Patil et al. [50]. Protein adsorption to NPs is a dynamic process in liquids be it culture media, blood or other bio fluids. To see the alteration in protein adsorption, if any, caused by components of culture media, mixture of Y 2 O 3 NPs was suspended in serum free (i.e., protein free) culture medium and left for 24 h. Then, BSA was added as above and any shielding effect caused by media ingredients in protein adsorption was measured at 6 h and 24 h as described above. Surface reactivity of Y 2 O 3 NPs was also determined towards serum, a fluid containing diverse molecules such as albumin and other proteins, hormones, lipids, carbohydrates, etc.

Enzyme-Like Activity of NPs
Enzyme like activity of NPs was determined by the protocol of Marklund and Marklund [51] using autoxidation of pyrogallol. The principle of this method is based on the competition between the pyrogallol autoxidation by O 2 •− and the dismutation of this radical by enzyme SOD in a 75 mM Tris/cacodylic acid buffer, pH 8.2. For uninhibited reaction, a 0.02 OD/min for 3 min at 420 nm was utilized. Amount of enzyme required to inhibit this reaction by 50% was defined as one-unit of SOD enzyme. Catalase was determined by recording the rate of H 2 O 2 disappearance at 240 nm for 3 min as described by Aebi [52]. For this, a 100 mL phosphate buffer, pH 7.0 was mixed with H 2 O 2 to give an absorbance of 0.54 at 240 nm. For calculation purposes, 0.436 mM extinction co-efficient for H 2 O 2 at 240 nm was taken. Reaction was initiated by 50 µL of 1 mg/mL NPs suspension in one mL of phosphate buffer with H 2 O 2 absorbance of 0.54 at 240 nm. A suitable NP control was used for subtracting absorbance decrease due to NPs settling in reaction cuvette from the absorbance caused by potential enzyme-like activity of NPs.

Viability Studies
Cell viability was carried out by MTT, NRU and LDH assay as briefly described below. MTT assay was carried out according to the protocol described by Mosmann [53] with minor modifications. Briefly, around 20,000 MCF-7 cells per well were seeded in 96-well plates in a 100 µL of culture medium. Blue formazon formed in viable cells were solubilized and absorbance taken at 570 nm using a plate reader (Synergy HT, Bio-Tek, Winooski, VT, USA). Cell viability of treated and control cells is given as cell viability in percentage of control. To minimize the interference in absorption that may be potentially caused by the NPs, plates were centrifuged to suspend the NPs, and 100 µL supernatant was carefully transferred (without disturbing NPs at the plate bottom in the original experimental plate) to another fresh well in a 96-well plate as previously reported [54]. The neutral red uptake (NRU) assay is based on the method described by Repetto et al. [55]. Absorbance was taken at 540 nm using a plate reader (Synergy HT, Bio-Tek, Winooski, Vermont, US ) and data is given as cell viability in percentage of control. The activity of cytoplasmic LDH released into the culture media was determined with the method described [56]. A 100 µL sample from the centrifuged culture media was collected after the cells were treated for 24 h. The rate of NADH oxidation was determined by following the decrease in absorbance at 340 nm for 3 min at 30-s interval at 25 • C using a spectrophotometer (Genesys 10 Bio, Thermo Fisher Scientific, Madison, WI, USA). The amount of LDH released is represented as LDH activity (IU/L) in culture media.

Determination of Lipid Peroxidation
LPO was assessed by the thiobarbituric acid reactive substances (TBARS) assay, which detects mainly malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters. MDA was measured by slight modification of the method of Ohkawa et al. [57]. The absorbance of the supernatants was read at 532 nm. Results were calculated as nmol TBARS/mg of cellular protein using 1.56 × 10 5 M −1 cm −1 as molar extinction of MDA-TBA.

Measurement of Total Reactive Oxygen Species by DCFH-DA and H 2 O 2 by Specific Sensor
The generation of intracellular ROS was measured using 2 , 7 -dichlorofluorescin diacetate (DCFH-DA) probe [58]. DCF fluorescent intensity was measured at emission from 528 nm band pass of plate reader (Synergy HT, Bio-Tek, Winooski, Vermont, USA  Mitochondrial outer membrane potential (MOMP) in control and treated cells were determined by JC-1. In healthy cells with high mitochondrial membrane potential, JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in apoptotic or unhealthy cells with low membrane potential, JC-1 remains in the monomeric form showing green fluorescence [59]. Cells were seeded at appropriate densities in 12-well plate. When treatment period was over, media was aspirated off from each well and labeled with 5 µM JC-1 in Hepes buffered HBSS for 20 min. Fluorescence pictures of JC-1 monomer and aggregate in cells were captured by a fluorescence microscope (DMi8, Leica Microsystems, Wetzlar, Germany, using objective 20×, Leica DFC450 C camera and its LAS software version 4.13).

Measurement of Lysosomal Activity by LTR
Lysosomal activity predominantly refers to intracellular acidic vesicles (lysosomes or fusion product of autophagosomes with lysosomes) during autophagy [60]. Lysosomal activity in cells can be tracked by LysoTracker (LTR) dyes. LTR probes freely permeate cell membranes and label live cells. One such LTR dyes is red fluorescent LysoTracker™ Red DND-99 (Thermo Fisher Scientific, Eugene, Oregon, US) as was used in our previous report [61]. After the specific treatment period, cells were labeled at the final concentration of 1 µM LTR within ongoing culture media. After 30 min of labeling in 12-well culture plates, cells were carefully washed with cold HBSS with three times to remove excess LTR. CalceinAM, a green fluorescence emitting live cell dye was labeled at 0.5 µM simultaneously in LTR imaging to corroborate live cells from dead ones. An increase in punctate red fluorescence of LTR in treated cells than that in control cells indicates an increase in lysosomes or intact acidic vesicles (autolysosomes) [62].

Apoptotic-Necrotic Potential Detection by Hoechst/PI Staining
A dual staining based on concurrent use of Hoechst and propidium iodide (PI) was carried out again to confirm the mechanism of toxicity if occurred by the NPs of Y 2 O 3 . This approach was based on the ability of Hoechst to label every cell including live as well as dead and ability of PI to penetrate only dead cells. Further, if there is toxicity, a fair idea about potential mechanism of cell death induced by either apoptosis or necrosis can be deduced by applying logical conventions set by many cytologists [63]. In apoptotic cells, most nuclei show clumped, compact and discrete fluorescence due the pattern of nuclear DNA breakdown whereas in necrotic cell, most nuclei show increased in perimeter, diffused and peripheral fluorescence. In control dead cells, nucleus is neither increased in perimeter (as in necrosis), nor show compactness and discrete fluorescence (as in apoptotic cells) but rather a smooth and centrally intense fluorescence [64]. This is an inexpensive and more dependable method which was successfully applied in our previous publication deciphering mode of cell death caused by apoptosis to that from apoptosis-independent [61].

Protein Estimation
The total protein concentration was measured by using a ready-to-use Bradford reagent with bovine serum albumin as protein standard [65].

Statistics
One way ANOVA (analysis of variance) followed by Dunnett's multiple comparison tests were employed for statistical analysis of data. Statistical significance was attributed at p < 0.05. The experiment was repeated three times (n = 3) carried in triplicates. For a particular set of the experiment, a burst of images was captured at the constant exposure of time, gain, saturation and gamma. Images representing three independent experiments were used in this report. Calculation of corrected total cellular fluorescence (CTCF) was conducted in ImageJ software (NIH, Bethesda, MD, USA). Using the region of interest (ROI) manager command, the constant area selection was obtained to each image opened in ImageJ. CTCF was calculated by subtracting the mean of background (without cell) fluorescence from the mean of cellular fluorescence (i.e., mean integrated density). Scale bar in images was set using ImageJ after calibrating the scale in terms of pixels/micron according to the respective objectives used during imaging.

Conclusions
NPs of Y 2 O 3 could be desired agent in drug loading and targeted delivery as shown by their high surface reactivity, biocompatibility and their inability to affect cellular autophagy functioning. Higher surface reactivity also provides opportunity to test drugs loading, and in the further fine tuning of the NPs surfaces with diverse array of available biocompatible surfactants [27].
Supplementary Materials: Figure S1: Native (or pristine) Y 2 O 3 NPs were first treated with different relevant adsorbents such as BSA, filtered serum or media ingredients without added serum (i.e., incomplete culture media) to confirm their surface activity. Now differently surface modified NPs were adjusted at the concentration of 200 µg/mL in complete culture media and were exposed to MCF-7 cells and HT-1080 cells for 24 h. Cell viability was not significantly different with that of control or native (unmodified) NPs. Triplicates (n = 3) of each treatment group were used in each independent experiment. Figure S2: ROS was measured using DCFH-DA along with H 2 O 2 that was used as positive control. H 2 O 2 (0.65 mM) was exposed to MCF-7 cells and HT-1080 cells that was sufficient to induce cytotoxicity almost 50% in cell population. Figure S3: An apoptotic mode of cells death in the case of ZnO NPs is evident by the fragmented and clumped pattern of chromatin condensation. All yellow circles meant for zooming the part of images are equal in area.