Fig. 4

Evaluation of the BCSC-targeted and penetration effects of DOX/HA-b-PCDA NPs. A The uptake of DOX/HA-b-PCDA NPs and free DOX by BCSC-enriched 4T1 mammosphere cells under fluorescence microscope, scale bar = 100 μm. B Intracellular distribution of DOX/HA-b-PCDA NPs in BCSC-enriched 4T1 mammosphere cells under CLSM, for observation, the nuclei were stained with DAPI (blue), lysosome were stained with lysosome tracker-green. To observe the DOX fluorescence, the excitation wavelength was 553 nm and the detection wavelength was 578–630 nm; to observe the lysotracker-green fluorescence, the excitation wavelength was 493 nm and the detection wavelength was 528–547 nm. scale bar = 20 μm. C Typical flow cytometry profiles of the cellular uptake of DOX/HA-b-PCDA NPs and free DOX by BCSC-enriched 4T1 mammosphere cells. D The mean fluorescence intensity of DOX in BCSC-enriched 4T1 mammosphere cells after incubation with DOX solution or DOX/HA-b-PCDA NPs solution with or without pre-incubation with 1 mM of HA solution. E The DOX fluorescence images observed by CLSM images at different lays of the mammospheres from bottom to top, after incubation with different drug formulation solutions for 4 h, scale bar = 100 μm. “***”, p < 0.001