## 2 Results
### Stability, pH-Sensitivity, and Endosomal Escape of Polymer-Liposome Complexes  
To understand the stability of polymer-liposome complexes formed via electrostatic interaction, these complexes were treated with PBS at pH 7.4 and 37 \({}^{\circ}\)C under continuous shaking. Any changes in the particle size and size distribution were measured by DLS. As shown in Figure 5a,b, both PCLH and PCLM were stable and maintained their properties even after 72 h of treatment, which indicates that copolymers on polymer-liposome complexes could stabilize their nanostructures. PCLH and PCLM were treated with PBS at pH 6.5 and 5.0 at 37 \({}^{\circ}\)C to understand the pH-responsiveness and behavior of polymer-liposome complexes when they were in early and late endosomes. The pKa value of the imidazole ring for histidine is 6.3 [25]. A portion of histidine on mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers was protonated to obtain positive charges at pH 6.5 and neutralize the negative charges of the copolymers. However, cationic liposomes did not afford any pH-responsiveness. Therefore, the liposomes still maintained their particle sizes and size distributions at pH 6.5, even though the copolymers desorbed from the surface of cationic liposomes (Figure 5c,d). Conversely, most histidine molecules afforded positive charges and caused intramolecular and intermolecular electrostatic interactions with carboxylic acid (pKa value at approximately 1.8) at pH 5.0, thereby causing PCLH aggregation. In contrast, because PMMAc has a pKa of approximately 4.8 [26], mPEG-b-P(MAAc)-NH\({}_{2}\) copolymers tightly coated on cationic liposomes at pH 6.5 and formed intramolecular and intermolecular hydrogen bonds to increase the particle sizes (Figure 5e,f). Figure 4: Characterization of cationic liposomes and polymer–liposome complexes. DLS analysis of (**a**) cationic liposomes, (**b**) PCLH, and (**c**) PCLM. TEM images of (**d**) cationic liposomes, (**e**) PCLH, and (**f**) PCLM (\(n=3\), one measurement from the data is displayed in the figure). Figure 5: _Cont._To further confirm that mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers could detach from cationic liposomes, polymer-liposome complexes were treated at pH 6.5 and pH 5.0 PBS at 37 \({}^{\circ}\)C and analyzed by GPC. For PCLH at both pH 6.5 and pH 5.0 (Figure 6a), the GPC peaks for mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers became stronger on increasing the time to 4 h, indicating the greater desorption of copolymers from cationic liposomes with time. However, the intensities of mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers in the GPC diagram decreased at 6 h because of the occurrence of intramolecular and intermolecular interactions. In contrast, PCLM did not show any copolymer signals at either pH 6.5 or pH 5.0 (Figure 6b), indicating that the surface of cationic liposomes still contained mPEG-b-P(MAAc)-NH\({}_{2}\) copolymers. TEM was also used to observe the interaction of PCLH at 6 h. As shown in Figure 6c,d, the thickness of mPEG-b-P(MAAc-g-His)-NH\({}_{2}\) copolymers on PCLH at pH 5.0 decreased (dark region) and PCLH aggregated to form clusters, which was consistent with the DLS and GPC analysis results. In contrast, the TEM images showed the complete dark aggregates of PCLM at pH 5.0, demonstrating the trapped behavior of cationic liposomes by mPEG-b-P(MAAc)-NH\({}_{2}\) copolymers. Figure 5: Stability and pH-responsiveness of polymer–liposome complexes. Particle size and particle size distribution (PDI) of (**a**) PCLH and (**b**) PCLM at pH 7.4 PBS and 37 \({}^{\circ}\)C. (**c**) Particle size and (**d**) PDI of PCLH at different pH PBS and 37 \({}^{\circ}\)C. (**e**) Particle size and (**f**) PDI of PCLM at different pH PBS and 37 \({}^{\circ}\)C (\(n=4\)). Because PCLH could desorb their copolymers but aggregate at low pH as described above, the endosomal escape ability of PCLH contributed to the protonation of histidine when PCLH was diluted into a culture medium and taken up by cancer cells. In this study, cationic liposomes were labeled with a FITC dye for CLSM observation.