The in vitro cell-based experiments of formulations. (A) In vitro cellular uptake of BC-DOX-NPs, CS-DOX-NPs and free DOX in 4T1 cells and B16F10 cells after 1 h, 2 h and 4 h incubation, analyzed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Flowcytometry histogram showing changes in cell uptake of BC-DOX-NPs and CS-DOX-NPs in 4T1 cells and B16F10 cells after preincubation with CS solution (10 mg/ml). (C) Relative uptake of BC-DOX-NPs and CS-DOX-NPs in 4T1 cells after preincubation with various cell uptake inhibitors, compared with control group. *P < 0.05, **P < 0.01. (D) CLSM images of 4T1 cells incubated with BC-DOX-NPs, CS-DOX-NPs and free DOX for 1 h. The cell nuclei were stained by DAPI (blue). The CD44 receptors on 4T1 cells were stained by Rhodamine-labeled secondary antibodies (red). The fluorescence of DOX was shown in green and co-localization was shown in yellow. The bar is 10 µm. (E) Cytotoxicity of BC-DOX-NPs, CS-DOX-NPs, free DOX and blank carries in 4T1 cells after 24 h and 48 h incubation at different concentrations. All data represent mean ± SD (n = 3). **P < 0.01 and ****P < 0.0001 compared with free DOX; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001 compared with CS-DOX-NPs. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)