Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3)

Abstract Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe3O4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe3O4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe3O4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe3O4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Graphical abstract Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe3O4-ZnO magnetic composite particles (MCPs).


Background
With novel approaches of cancer treatment being extensively researched in order to mitigate prime limitations of current approved therapeutics-nonspecificity and nonselective toxicity-development of targeted and selective cancer therapy has paved its way as a promising tool for next-generation cancer therapeutics by exploiting properties of specific binding like ligand-receptor binding, antigen-antibody mediated immune therapy etc. Targeted nanoparticles (NPs) also provide more therapeutic benefits besides specificity and specific localization like high payload, multidrug conjugation, easy tuning of release kinetics, selective localization, and bypass of multidrug resistance mechanism [1].
Ferrite magnetic NPs (Fe 3 O 4 NPs) are of particular interest in biomedicine from hyperthermia application [2] to targeted therapy and drug delivery [3] because of its superparamagnetic behavior. Unlike strong ferromagnetic substances, they show linear dependence of magnetization with external magnetic field in the same direction with low maximum magnetization, a characteristic property of paramagnetism. But, the magnetic moment is four to ten times more than that of normal paramagnetic substances, hence considered as superparamagnetic [4]. This property allows Fe 3 O 4 NPs to be used as effective vector for targeted drug therapy under the application of external field.
However, it has high aggression and oxidation at physiological pH limiting its direct application which, nonetheless, can be overcome by biocompatible surface coating [3,5]. But, at the same time, coating of Fe 3 O 4 NPs with biocompatible nonmagnetic substance affects magnetization of the particle because of surface spin disorder, formation of dead magnetic zone, etc. which is strongly influenced by thickness and chemical composition of modifier [6]. Hence, tuning of modifier content for maintaining therapeutically significant magnetization value is very important for drug delivery application.
Another important nano-sized particle for cancer therapy is ZnO which has an inherent capacity to induce apoptosis in cancer cells while rendering very less toxic effect towards normal proliferating cells [7,8]. Actual mechanism of this behavior is still not clear, but various arguments have been postulated; most supported being selective uptake of ZnO nanoparticles (ZnO NPs) [9] due to high expression of membrane anionic phospholipids on cancerous cells [10] imparting zinc-related ions disequilibrium inducing reactive oxygen species (ROS) [11,12]. Nonetheless, this selectivity of ZnO NPs acts as promising tool for cancer treatment.
Since ZnO itself is biocompatible [13] and can act as a selective anticancer agent, conjugation of magnetic NPs with zinc oxide NPs could make novel therapeutically important compound. Recent research paper [14] has mentioned the synthesis of core-shelled ZnO-Fe 3 O 4 NPs with molar ratios of Fe 3 O 4 NPS to ZnO at 1:1, 1:10, and 1:20 which shows consecutive reduction of magnetization upon increasing ZnO proportion, being least reduced for 1:1. Although ample study on synthesis of ZnO-ferrite composite can be found as mentioned, there is still dearth on cytotoxicity study of such composite for possible cancer therapy. Hence, we tried to analyze the cytotoxic profile of this nanocomposite while still retaining therapeutically significant magnetization to be used in targeted localization.

Synthesis of Magnetic Fe 3 O 4 NPs
FeSO 4 ·7H 2 0 (0.5 M) solution was mixed to FeCl 3 ·6H 2 O (0.5 M) at 1:1 molar ratio in a conical flask placed on a magnetic stirring hot plate. NH 4 OH (1.5 M) was then added dropwise to iron solution maintained at 70°C with vigorous magnetic agitation. Thus, precipitated black-colored particles were then filtered, washed with deionized water and ethanol, and then oven dried at 60°C overnight. The dried particles were finally collected by magnetic separation.

Synthesis of ZnO NPs
NaOH 0.25 M was added dropwise to 0.05 M zinc acetate maintained in a conical flask under vigorous mechanical agitation at room temperature; the cloudy viscous sediment thus obtained was filtered using vacuum filtration, dried overnight at 50°C, and calcined at 200°C for 2 h in a muffle furnace to obtain white fine powdered ZnO.

Conjugation to Form MCPs
Ex situ conjugation approach as described by Shal and Jafari [14] was used to make the nanocomposite; 0.05 g of Fe 3 O 4 NPs was dispersed in 0.05 M trisodium citrate solution prepared in 10 mL distilled water via shaking overnight at room temperature. Then, trisodium citrate-coated Fe 3 O 4 NPs were washed with distilled water thrice and separated magnetically. Finally, 0.05 g of ZnO NPs was then added in a solution of trisodium citrate-coated Fe 3 O 4 NPs resuspended on distilled water (pH 3.5 acidified by acetic acid) and stirred for 24 h at room temperature. Hence, formed magnetic composites were then separated magnetically, washed several times with distilled water, and dried at room temperature.
Characterization X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM) were carried out at IIT, Roorkee, India. XRD spectra were recorded at 0.154 nm wavelength (λ) of Cu-kα radiation using Rigaku-Geiger diffractometer with a range of 2θ from 5°to 80°. Phase identification and crystallographic planes were determined by comparing peak positions with reference JCPDS (Joint Committee on Powder Diffraction Standards) file. Particle size was computed using Scherrer's equation: D = Kλ/βcosθ, where K = 0.9 is the shape factor, β is the full width at half maxima (FWHM) in radians, and θ is the Bragg's angle [15]. For TEM analysis, required volume of sample was sonicated in acetone (1%, w/v) and applied over carbon-coated copper grid. TEM images were recorded at ×10K magnification and 0.2-μm scale over JEOL 1011 (Tokyo, Japan) at 80 kV. Similarly, Fourier transform infrared (FTIR) spectroscopy was performed on powder samples, and precursors using Shimadzu IR Prestige 21 FT-IR Spectrometer and corresponding spectrum was generated using IR-Solution software. VSM was performed using magnetometer (Princeton EG&G applied research model 155) with maximum current of 30 A, reading number 150, scan time 900 s at room temperature, and corresponding data were generated [15].

Cell Viability Assay
MTT assay as mentioned by Adhikari et al. [16] was performed for cell viability assay. Highly proliferating log phase cells were harvested and seeded in 96-well plates in DMEM at density of 10 4 cells per well. The seeded cells were left for attachment, and after 24 h, the media was replaced with fresh media supplemented with nanoparticles (ZnO NPs and MCPs) at concentration range from 200 μg/mL to eightfold serial dilution up to 1.56 μg/mL. After 24 h of treatment with NPs, cells were washed with DPBS twice and 10 μL of 5 mg/mL MTT dye was added. It was incubated for 4 h, and absorbance reading was taken at 570 nm with background subtraction of 630-nm band-pass filter. Percent cell viability was expressed as percent relative absorbance of sample respective to control.

CV Staining
Cultured cells were seeded in 12-well plate at density of 80,000 cells per well and incubated for 24 h. These cells were treated with ZnO NPs and MCPs at near EC50 concentration of 40 and 30 μg/mL, respectively, and left for 24 h. Nonadherent cells were washed off using DPBS, and remaining cells were fixed with 4% paraformaldehyde for 30 min. Then, 0.05% of crystal violet solution in 20% ethanol was added and left to stain for 30 min. Finally, excess stain was washed off using distilled water and observed on phase contrast microscope at ×10 magnification.

Computation and Statistical Analysis
Cytotoxicity was assessed by measuring cell viability as a function of relative absorbance, with respect to control (untreated cells), of dissolved formazan produced by the conversion of MTT dye by active mitochondrial dehydrogenase enzymes present in viable cells [17]. Dose-response was plotted under inbuilt logistic function on Origin 8 given by Eq. (1) as percent cell death versus logarithmic concentration. EC50 (measure for Potency), hill slope (P), and area under the curve (AUC) were assessed for cytotoxicity profile. Statistical analysis including ANOVA was done using inbuilt add-in of Microsoft Excel 2010.

Results and Discussion
Characterization XRD and TEM were performed for the analysis of size of synthesized particle and study of its morphological characteristics, respectively. FTIR was used to verify the conjugation of NPs on composite particle and to assess its purity. Magnetic properties of particles were studied by VSM, to verify whether assynthesized MCPs retained magnetic property of Fe 3 O 4 NPs.

XRD and TEM Analysis
X-ray diffraction pattern of synthesized particle is shown in Fig. 1. ZnO NPs show sharp diffraction peaks corresponding to hkl values of (100), (002),    [18,19]. Considerable decrease in FTIR peaks to that of trisodium citrate in MCPs further verifies that the sample has almost no impurities. Figure 4 shows the magnetization curve of assynthesized Fe 3 O 4 NPs and MCPs versus applied magnetic field (H) between −10 and +10 kOe. For assynthesized Fe 3 O 4 NPs, the saturation magnetization (M s ) is 47.22 emu/g which is reported to vary as a function of particle size and surface disorder [14,20]. Similarly, the saturation magnetization of MCPs is  [20] and (ii) surface defects and strain in nanocomposite [21].

VSM Analysis
The remanence of as-synthesized Fe 3 O 4 NPs and MCPs nanoparticles are 0.596 and 0.227 emu/g, respectively. Both possess negligible remanence at room temperature showing their typical superparamagnetic behavior. The coercivity of MCPs is obtained to be 62.90 Oe while that in as-synthesized Fe 3 O 4 NPs is 64.17 Oe. This decrease in coercivity is attributed to decrease in inter-particle interactions and magnetoelastic anisotropy due to stress resulting from coating of magnetic core with ZnO shell [21]. All in all, MCPs have retained superparamagnetic behavior as compared to assynthesized Fe 3 O 4 NPs.

Cytotoxicity Study
Percent cell death computed from percent viability was plotted against logarithmic concentration of ZnO NPs and MCPs for MDA-MB-231 under inbuilt doseresponse function of Origin 8. The fitted graph was shown in Fig. 5 with Adj. R 2 0.91 along with percent cell death for NIH 3T3 cell line. EC50 value of ZnO NPs was found to be at 38.44 μg/mL whereas for MCPs, it was 31.29 μg/mL. This shows higher potency of MCPs compared to the ZnO NPs alone. Since only potency is not sufficient to describe efficiency of the drug, other parameters like hill slope and area under the curve were computed and compared to further illustrate advantage of the MCPs over naked ZnO NPs.
Hill slope (p value) for MCPs was 1.21 ± 0.26 and that of ZnO alone was 3.24 ± 1.05. Though the value is reduced, it is still positive and greater than 1 which signifies strong cooperativity between MCPs and cell death. This reduction may be particularly important for clinical application as steep dose-response might pose challenges in cases of low compliance and unfavorable pharmacokinetic profile [22].
The toxicity of ZnO NPs on NIH 3T3 significantly differs from that of MDA-MB-231 (p value < 0.05; twofactor ANOVA at α = 0.05). ZnO NPs show less toxicity on NIH 3T3 above concentration 50 μg/mL than that of MDA-MB-231 whereas below concentration 50 μg/mL, they retain certain toxicity. This, however, has been completely reduced in case of MCPs. Up to 100 μg/mL, the toxicity of MCPs on NIH 3T3 was found to be dose independent (Pearson's r = −0.14) and very low. Given that selective killing of ZnO NPs between cancerous and noncancerous cells has been already quoted [7] and similar findings in our result also support this behavior [23], our synthesized MCPs show preferentiality between cancer and noncancerous cell. However, the further decrease in toxicity in NIH 3T3 (p value <0.05) in comparison to ZnO NPs elevate this effect. This may be because of less ZnO moieties on surface of MCPs Area under the relative viability curve (AUC), a parameter that combines efficacy and potency into single . This inherent selectivity of ZnO NPs, however, was found to be synergistically enhanced in MCPs with high potency (EC50 31.29 μg/mL for MCPs and 38.44 μg/mL for ZnO only) and significant hill slope (P) 1.21 measure, was computed as a sum of measured responses (relative cell death) at all tested dose (concentrations) for ZnO NPs and MCPs on both cell lines [24] where higher AUC value corresponds to higher activity for cell death. AUC provides the robust measure for response when single drug is compared between various cell lines at the same concentration range. Difference in AUC for particular drug between cancer and normal cell is of particular importance for clinical purpose, and our results show that difference in AUC for MCPs between MDA-MB-231 and NIH-3T3 is 253.68 which is more than that of ZnO NPs, 131.45, for concentration range, 25 to 200 μg/mL as represented in Fig. 6. This greater difference in AUC, higher potency, and less toxicity towards normal cell make MCPs clinically significant compound for therapeutic use.
It should be noted that, from our XRD and FTIR results, the as-synthesized MCPs contain independent domain of ZnO and ferrite since no alloying or interaction between these to disturb their individual characteristic peaks have been seen. This imputes that the mechanism by which MCPs exert toxicity must be related with individual activity of NPs at given condition. Since researches in cytotoxicity of ZnO NPs show oxidative stress, ionic imbalance, and induction of apoptosis as major cause for their cytotoxicity [25], ROS imparting toxicity must be the probable route for our synthesized NPs. Ferrite as such is nontoxic [26], but in the presence of excess H 2 O 2 , Fe 3 O 4 NPs can convert H 2 O 2 , a reactive oxygen species to more potent ROS and increase oxidative stress in cell [27]. So, it can be hypothesized that in case of MCPs, ZnO induces ROS including H 2 O 2 and Fe 3 O 4 NPs utilize these H 2 O 2 as a substrate to produce more potent ROS triumphing over nearexhausted scavenging activity of cancer cell [28]. This explains the higher potency of MCPs and similar toxicity profile as of ZnO. The relative low abundance of ROS/ H 2 O 2 in normal cell compared to cancer cell [28,29] and inherent nontoxic nature of ZnO NPs towards noncancerous cells cause MCPs to produce less ROS towards normal cell which can even be defended by antioxidant mechanism of cell itself and thus less toxicity, even 100% cell viability (0% cell death) as seen in Fig. 6.
It is important to note that this cytotoxic result is after 24 h of treatment and might not relate to chronic cytotoxic effect. The result of 100% cell viability of our MCPs in normal 3T3 cell, however, does not guarantee nontoxicity in the long run. But, nonetheless, the huge difference between cytotoxicity of our MCPs in cancer cell and noncancerous cell signifies the preferential cytotoxicity of as-synthesized MCPs.

CV Staining Assay
Crystal violet (CV) staining was done to support the result of cytotoxicity depicted by MTT assay. Crystal violet dye (hexamethylpararosaniline) is a mixture of violet rosanilins that stains nucleus deep blue, aiding in their visualization [30]. Since nonadherent dead cells are removed during the washing process of this assay, cells observed as deep blue, stained by crystal violet, account for viable cells only. Result of CV staining, as depicted by

Conclusions
ZnO-Fe 3 O 4 magnetic conjugated NPs retained inherent selective property of ZnO and magnetic property of Fe 3 O 4 NPs and showed preferential cytotoxicity towards breast cancer cell line MDA-MB-231, with no significant cytotoxicity towards noncancerous NIH 3T3 cell. This finding suggests that MCPs, at a concentration range of 50-100 μg/ml, could be exploited for further research as a putative preferential anticancer agent. Also, the dramatic enhancement of selectivity shown by the conjugation of magnetic Fe 3 O 4 NPs with ZnO NPs holds greater promises for effective clinical application. The magnetic property of MCPs can be used to target it to a particular site of cancerous tissue in our body using external magnetic field, while the selectivity property of MCPs can be used to selectively impart cytotoxicity in cancer cells of the targeted site, causing no significant harm to the normal cell around the site. Thus, ZnO-Fe 3 O 4 MCPs can act as highly selective site-specific advanced NPs for targeted cancer therapy. However, further in-depth research on chronic toxicity profile of such composite including ROS production, antioxidant activity profiling, and study of electronic interaction of NPs is recommended for understanding detail mechanism behind this significant result and for possible in vivo application. c Naked ZnO NPs treated. Cells were treated with nanoparticles for 24 h at concentration of near EC50 value 30 and 40 μg/mL, respectively, for MCPs and ZnO NPs. Cells were fixed by 4% paraformaldehyde for 30 min and stained by 0.05%. Crystal violet for 30 min and then subsequently washed with distilled water followed by microscopic observation at ×10