Fig. 5.

Intracellular trafficking and distribution of released Dox from CP-hyd-Dox/CP-BHQ2 mixed micelles and its comparison with free Dox (control) as a function of time. (A) Schematic illustration of internalization and intracellular trafficking of free Dox and Dox delivered by CP-hyd-Dox/CP-BHQ2 mixed micelles (micelle Dox). Free Dox diffuses across the cell membrane to the cytosol and then distributes to endo-lysosomes and nuclei. Micelle Dox is internalized by endocytosis to endo-lysosomes, and after linker cleavage at low pH (black arrow), Dox diffuses out of endo-lysosomes to the cytosol and then enters nuclei. (B) Intracellular localization of released Dox from micelles and free Dox at different time points. In the free Dox group, nuclei show obvious Dox fluorescence at 3 h; afterwards, Dox fluorescence in endo-lysosomes becomes bright and exceeds that in nuclei. In the micelle Dox group, Dox fluorescence first appears in endo-lysosomes, and after 12 h, Dox fluorescence in nuclei is notable. The white scale bar indicates 2 μm. (C) Comparison of Dox accumulation between endolysosomes and nuclei, as reflected by the Average Dox intensity_{(endo-lysosomes)}/Average Dox intensity_(nuclei) ratio as a function of time. The ratio in the free Dox group increases over time while the ratio in the micelle Dox group decreases over time. At 24 h, the ratios in these two groups are similar to each other. (D) The trafficking of Dox between endo-lysosomes and cytosol, as reflected by the Average Dox intensity_{(endo-lysosomes)}/Average Dox intensity_(cytosol) ratio as a function of time.The ratios in both free Dox group and micelle Dox group increase over time and the rates are similar. (E) Distribution of Dox in endo-lysosomes as a function of pH at different time points, as reflected by the Average Dox intensity_{(endo-lysosomes)}/Average Dox intensity_(cytosol) ratio plotted as a function of local pH. Irrespective of different internalization pathways, both free Dox and released Dox from micelles gradually approach the theoretical distribution equilibrium between endo-lysosomes and the cytosol as indicated by the black line and squares. The calculation of the theoretical distribution is described in the text.