## 2 Results  
### Basic Characterization of Copolymers, CTPP-Cholesterol, and Polymer-Liposome Complexes  
In this study, mPEG-b-P(MAAc)-NH\({}_{2}\) was first synthesized by free radical copolymerization using mPEG-ABCBA as a macroinitiator, MAAc as a monomer, and AET-HCl as a chain-transfer reagent. Then, mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) was prepared by succinimidyl ester (NHS ester)-amine substitution reaction from MAAc-modified NHS ester with histidine. The formed compound of mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) was characterized by \({}^{1}\)H-NMR and FT-IR. The \({}^{1}\)H-NMR spectrum (Figure 2a,b) showed that the carboxylic acids (at 12.3-12.4 ppm) for the PMAAc segment were fully replaced by histidine molecules (at 6.8-8.0 ppm). There were approximately 30 repeating units of histidine in mPEG-b-P(MAAc-g-His)-NH\({}_{2}\), as calculated from the \({}^{1}\)H-NMR spectrum. In addition, several major peaks of copolymers in the FT-IR spectrum (Figure 2c) were also observed at approximately 2800 cm\({}^{-1}\) (C-H stretching for mPEG-b-P(MAAc)-NH\({}_{2}\)), 1750 cm\({}^{-1}\) (C=O stretching for histidine), 1630 cm\({}^{-1}\) (C=O stretching for conjugated amide bonds), and 1400 cm\({}^{-1}\) (C=C stretching for histidine), all of which confirmed successful preparation of mPEG-b-P(MAAc-g-His)-NH\({}_{2}\). Figure 1: Charge–conversion polymer–liposome complexes for mitochondrial–targeting therapy. (**A**) Polymer–liposome complexes were constructed from pH–sensitive charge–conversion copolymers and cationic liposomes via electrostatic interactions between negative charges of copolymers and positive charges of CTPP–containing liposomes. (**B**) After cancer cells internalized polymer–liposome complexes via endocytosis, charge–conversion copolymers became neutral to desorb from the surface of cationic liposomes and induced endosomal escape for releasing cationic liposomes into cell cytosol. The released cationic liposomes could then target mitochondria for mitochondrial–targeting therapy. CTPP was conjugated with cholesterol by Fisher esterification to form a cationic lipid, CTPP-cholesterol. The chemical structure of CTPP-cholesterol was also confirmed by \({}^{1}\)H-NMR and FT-IR. The \({}^{1}\)H-NMR spectrum (Figure 3a) showed the major peaks of cholesterol and CTPP at 5.4-5.5 ppm (C=C-H) and 7.5-8.0 ppm (aromatic C-H), respectively. In addition, the FT-IR spectrum (Figure 3b) further revealed the stretching of C=C double bonds (at approximately 1400 cm\({}^{-1}\)) and C-H single bonds (at 3000 cm\({}^{-1}\)) from CTPP and the stretching of C-H single bonds (at approximately 2800 cm\({}^{-1}\)) from cholesterol. Moreover, CTPP-cholesterol with a molecular weight of 717.48 Da was determined using ESI-mass spectrometry (Figure 3c), which was consistent with its molecular formula. These experimental results confirm the formation of CTPP-cholesterol molecules. Figure 3: _Cont_. Figure 2: Characterization of synthesized copolymers. \({}^{1}\)H–NMR spectrum of (**a**) mPEG–b–P(MAAc) –NH\({}_{2}\) copolymers and (**b**) mPEG–b–P(MAAc–g–His) –NH\({}_{2}\) copolymers. (**c**) FT–IR spectra of copolymers (\(n=3\), showing one measurement in the data). To prepare polymer-liposome complexes, cationic liposomes containing CTPP-cholesterol, DPPC phospholipids, and ceramide were prepared using a four-step method comprising thin film preparation, hydration, sonication, and extrusion. The particle size and size polydispersity index of cationic liposomes were 152.7 \(\pm\) 24.2 nm and 0.39 \(\pm\) 0.09, respectively, as determined by DLS (Figure 4a). The ceramide loading efficiency was 43.0 \(\pm\) 14%, as determined by HPLC. Next, the mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers were adsorbed on the surface of cationic liposomes via electrostatic interaction between cationic CTPP and negative carboxylic acids of histidine to form polymer-liposome complexes (PCLH).