*3.1. Characterization of Nanoparticles and Their E*ff*ects on Cell Viability, Iron Content, and ROS*

Two kinds of iron nanoparticles were used in this study: one was PBNPs, and the other was FeNPs. The former was in blue color, and the latter was in brown color (Figure 1A,B). The PBNPs were at sizes of 152 nm and had potentials of −1.94, while the FeNPs were at sizes of 20 nm and had potentials of −20.9 (Figure 1A,B). The CCK8 assay showed that only the cell viability of HL60 was significantly reduced by FeNP treatment (Figure 1C). The cell viability of KG1a was not significantly reduced by all treatments (Figure 1C), indicating that KG1a was more resistant to iron nanoparticles. The intracellular iron content measurement revealed that the treatment of both iron nanoparticles significantly increased the intracellular iron content of two cell lines; however, the FeNP treatment more significantly increased the intracellular iron content of two cell lines (Figure 1D). Additionally, treatment of both iron nanoparticles more significantly increased the intracellular iron content of HL60 than KG1a (Figure 1D). The qPCR detection of the expressions of two leukemia stem cell (LSC) marker genes, CD34 and CD38, indicated that CD34 was only highly expressed in KG1a and that CD38 was expressed in two cells at low levels (Figure 1E). This demonstrated that KG1a had higher stemness than HL60. When treated with two kinds of nanoparticles, CD34 expression in KG1a was slightly downregulated by FeNPs but upregulated by PBNPs, whereas CD38 was significantly downregulated by PBNPs in two cells and by FeNPs in HL60 (Figure 1E). The ROS measurement indicated that the FeNP treatment increased the ROS level in both cell lines but that the PBNP treatment decreased the ROS level in the two cell lines (Figure 1F). Altogether, these data indicated the difference between two leukemia and two kinds of iron nanoparticles.

**Figure 1.** The characterization of two kinds of iron nanoparticles and their effects on KG1a and HL60: (**A**,**B**) hydrated particle size and zeta potential of Prussian blue nanoparticles (PBNPs) (**A**) and Fe3O4 nanoparticles (FeNPs) (**B**), (**C**) cell viability of the PBNP- and FeNP-treated HL60 and KG1a cells, (**D**) cellular iron contents of the PBNP- and FeNP-treated HL60 and KG1a cells, and (**E**) QPCR detection of CD34 and CD38 expression in two cells. HF, FeNP-treated HL60; HP, PBNP-treated HL60; KF, FeNP-treated KG1a; KP, PBNP-treated KG1a. ns, no significance; \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001. (**F**) ROS levels of the PBNP- and FeNP-treated HL60 and KG1a cells.
