## 3 Results and Discussion
### Morphological Changes
reported  
Figure 7: Optical microscopy images of cells treated with NPs for 48 h. (**A**) untreated Michigan Cancer Foundation-7 (MCF7) cells; (**B**) MCF7 cells treated with 100 \(\upmu\)g/mL of CoNiZn NPs; (**C**) MCF7 cells treated with 100 \(\upmu\)g/mL of CoNiFe NPs; (**D**) untreated human umbilical vascular endothelial cells (HUVECs); (**E**) HUVEC cells treated with 100 \(\upmu\)g/mL of CoNiZn NPs; (**F**) HUVEC cells treated with 100 \(\upmu\)g/mL of CoNiFe NPs. that single Au, Ag, and Au/Ag alloy NPs have considerable advantages for NP imaging in the cellular environment, a highly diffusing medium [40]. In a similar study, [PERSON] and coworkers studied the cytostatic and antimicrobial effects of newly prepared Ag, Au, and Ag/Au alloy NPs. The biosynthesized NPs had spherical shape, demonstrated good catalytic activity and exerted desirable cytotoxic effects against Hela cervical cancer cells. In their study, the IC\({}_{50}\) of Ag/Au alloy NPs were found to be 24.83 uM, being the most active at concentration of 100 uM as cell viability reaches 47.50% [41]. These findings highlight the benefits of preparing metallic alloy NPs as cheap sources to establish new and safe anti-proliferative agents. Our in vitro findings of CoNiZn and CoNiFe NPs indicated that the CoNiZn NPs exerted more desirable cytotoxicity against breast cancer cells, confirmed by lower IC\({}_{50}\)s. On the other hand, NPs exhibiting magnetic properties are highly appealing for cancer treatment. Magnetic NPs possess unique physicochemical properties, which attract many researchers to perform in vitro investigations about their beneficial applications in medicine [42]. In this regard, cancer therapy and hyperthermia treatment are two of the most promising applications of magnetic NPs that have recently shown promising outcomes [43]. Furthermore, these NPs have shown great advantages in cancer nanoterenostics, as they can play an important role as MRI probes [44]. Still, the synthesized magnetic NPs must be safe when applied to normal human tissues. Based on MTT results, we found that exposure of normal human cells to 12.5 ug/mL of CoNiZn and CoNiFe NPs did not induce any significant cytotoxicity. Furthermore, no evident morphological alteration and membrane damage was noticed when HUVEC cells were treated with the given dose of the NPs for 48 h. Therefore, we believe that it would be appropriate to consider 12.5 ug/mL as the safe dose for both NPs for further assessments and future applications. [PERSON] et al. examined the cytotoxic effects of Co\({}_{0.5}\)Ni\({}_{0.5}\)Pr\({}_{0.1}\)Fe\({}_{1.9}\)O\({}_{4}\) magnetic NPs on the Hela cell line. They found that these NPs did not exert cytotoxic any significant effects in human lung cancer cells at concentrations of up to 500 ug/mL after 24 h exposure [45]. In contrast with these findings, we observed potent cell-killing effects on MCF7 cells after 24 h and 48 h exposure to prepared NPs at concentrations higher than 12.5 ug/mL. The observed difference between our findings and [PERSON] et al. 's results might be due to the composition of the studied NPs (presence of Yb\({}^{3+}\)- and Pr\({}^{3+}\)-substituted cobalt-nickel ferrite NPs in the latter) and the type of the studied cell lines. Still, our NPs had negligible or non-cytotoxic effects at 12.5 ug/mL. By performing both MTT and trypan blue exclusion assays, [PERSON] and coworkers assessed the in vitro toxicity of zinc and nickel ferrite NPs in human erythrocytes and peripheral blood mononuclear (PBM) cells. After treatment with 50 or 200 ug/mL of ferrite NPs for 24 h, the number of alive PBM cells was not decreased, and, thus, nickel, zinc, nickel-zinc ferrite magnetic NPs did not significantly induce cell death in normal human cells [46].