## 2 Results
### Mitochondrial-Targeting and Cytotoxicityicity of Polymer-Liposome Complexes  
CLSM was used to monitor the mitochondrial-targeting ability of PCLH and PCLM. Cancer cell mitochondria and cationic liposomes were labeled with MitoTracker and FITC, respectively. For PCLH (Figure 8a), the intensities of FITC and MitoTracker were merged together after 3 h, indicating successful mitochondrial-targeting of cationic liposomes after desorption from mPEG-b-P(MAAC-g-His)-NH\({}_{2}\) copolymers and evading from endosomes. For PCLM (Figure 8b), although FITC intensity of cationic liposomes was also overlapped with mitochondria (MitoTracker) at 3, 6, and 9 h because anionic copolymers could improve mitochondrial targeting [27], the FITC intensity of PLCM at mitochondrial was lower than that of PCLH, demonstrating that the mitochondrial-targeting ability of PCLM was weak. Mitochondria were isolated from MCF-7 cells to monitor the mitochondrial-targeting ability of PCLH and PCLM after treating with FITC-labeled polymer-liposome complexes for 24 h. The ratio of fluorescent intensity of the isolated mitochondria showed that the accumulation of PCLH was higher than that of PCLM (Figure 9a). PCLH could enhance 1.1-fold liposomal accumulation compared to PCLM. In addition, most PCLM stayed in the cytoplasm of MCF-7 cells. Based on the experimental results, PCLH had an excellent ability of pH-responsiveness for leading the cation liposomes to escape from endosomes and target mitochondria. Figure 7: Endosomal escape of polymer–liposome complexes. Confocal images of (**a**) PCLH and (**b**) PCLM after co-incubation with MCF-7 cells for 3, 6, and 9 h. Scale bar: 20 \(\upmu\)m (\(n=3\), showing one of the measurements from the data). Figure 8: Mitochondrial-targeting ability of polymer–liposome complexes. Confocal images of (**a**) PCLH and (**b**) PCLM after co-incubation with MCF-7 cells for 3, 6, and 9 h. Scale bar: 20 μm (\(n=3\), showing one of the measurements from the data). Because ceramide in the mitochondria is known to increase the production of reactive oxygen species (ROS) and cause mitochondrial damage [28], the levels of ROS in MCF-7 cancer cells were investigated to understand the targeting effect after the cells were treated with PCLH and PCLM for 24 h. As shown in Figure 9b, PCLH was expected to produce higher levels of ROS (around 1.1-fold) in the mitochondria of cancer cells than PCLM because of the efficient mitochondrial-targeting ability of PCLH. On the other hand, since PCLH showed endosomal escape and mitochondrial-targeting ability, both PCLH and PCLM were incubated with MCF-7 breast cancer cells to understand whether PCLH could cause higher toxicity than PCLM. After co-incubation for 24 h, both PCLH and PCLM showed similar cell viability at low concentrations of ceramide. However, PCLH showed higher toxicity than free ceramide at high concentrations of ceramide (Figure 9c), indicating a therapeutic benefit by concentrating ceramide in the mitochondria. In contrast, the cytotoxicity of PCLM was lower than that of PCLH owning to the lack of endosomal escape and mitochondrial-targeting for PCLM. The results of cytotoxicity demonstrate that the half maximal inhibitory concentration (IC\({}_{50}\)) value of PCLH was about 20 \(\upmu\)M, while the values for free ceramide and PCLM were above 40 \(\upmu\)M. Finally, cell-cycle analysis was evaluated using MCF-7 breast cancer cells after treating with PCLH and PCLM. The experimental results show that both PCLM and free ceramide caused 40% of MCF-7 cells to be arrested at the G1 phase (Figure 9d,e). In contrast, PCLH induced more than 46% of  
Figure 9: Function of polymer–liposome complexes on MCF-7 cells.