## 3 Discussion  
Because mitochondria play many important roles in the cell, nanomedicines that can induce mitochondrial dysfunction have become a potential therapeutic strategy in cancer therapy [29]. Several drug carriers have been designed and investigated to deliver payloads to mitochondria for mitochondrial-targeting therapy, including cationic nanoparticles [30,31], mitochondrial-targeting peptide-containing nanoparticles [32], and cell-penetrating mitochondria transit peptide-containing nanoparticles [33]. Among these, the most commonly used structure for targeting mitochondria is CTPP because it may undergo an electrostatic interaction with mitochondrial membranes [19]. However, the targeting effectiveness of CTPP in mitochondria is low because the positively charged CTPP-containing nanoparticles tend to accumulate in normal tissues, making it difficult to deliver payloads into the cell and mitochondria [32]. To overcome the positive charge-exposed limitation caused by cationic nanoparticles, including CTPP-containing liposomes, on mitochondria drug delivery, a charge-conversion copolymer that showed a pH-sensitive charge-conversion segment in addition to a hydrophilic mPEG was designed in this study. The charge-conversion copolymer was based on the fact that histidine molecules on copolymers could possess negative charges that would allow their adsorption on cationic liposomes and then generate positive charges to neutralize negative charges for departing from liposomes in the early and late endosomes (pH 6.5-5.0) and induce endosomal escape via the proton sponge effect for releasing cationic liposomes into the cell cytosol to target mitochondria. Because of the pH-responsiveness of histidine, this charge-conversion copolymer coated on the surface of cationic liposomes could screen the positive CTPP to possess negative zeta-potential of polymer-liposome complexes at pH 7.4 and efficiently separate from cationic liposomes to expose positive CTPP on acidic endosomes/secondary lysosomes. Mitochondrial-targeting drug carriers should not only shield the positive charge of CTPP but should also successfully escape from the trapping of endosomes/secondary lysosomes. Recent reports have revealed that the positively charged nanoparticles could be rapidly internalized via caveolae-mediated endocytosis and then by vesicle-mediated transcytosis [34,35]. This transcytosis process might explain why CTPP-containing nanoparticles have low mitochondrial-targeting efficiency. One study has also demonstrated that approximately 4 h is needed to form secondary lysosomes after nanoparticles were internalized by cancer cells [36]. As endocytosed nanoparticles accumulate in secondary lysosomes, acid hydrolase enzymes could digest the payloads and then cause payload inactivation. Because histidine molecules could absorb protons and exhibit the proton sponge effect at low pH [37,38], in this study, charge-conversion copolymers after desorption from cationic liposomes could rapidly induce endosomal escape and release of cationic liposomes before 3 h without internalizing into secondary lysosomes. Hence, it is clear that cationic liposomes have the ability to target mitochondria. On the other hand, a recent study has showed that anionic copolymers could improve mitochondrial targeting when compared to cationic copolymers and charge-neutral copolymers [27]. According to the literature described above, both copolymers synthesized in this study indeed caused mitochondrial targeting ability of cationic liposomes. However, both copolymers were distributed whole though the cell cytoplasm. The mitochondrial-targeting ability for polymer-liposome complexes was contributed from the electrostatic interactions between cationic liposomes and mitochondria. Ceramide is known as a regulator that modulates cellular proliferation, apoptosis, and autophagy [22]. Several reports have suggested that ceramide-induced cell death is greatly related to mitochondrial dysfunction [22,23], especially for breast cancer cells [39].