4T1 cells (105 U) were seeded into a confocal dish and incubated in 1 ml of culture media at 37°C under an atmospheric CO2 level of 5%. When the cell confluence reached 70%, the culture media were changed to fresh ones containing TCPS (blank control), ACC@FITC-CaSi-PEG, ACC@FITC-CaSi-PAMAM-FA/mPEG, and MMP-2–pretreated ACC@FITC-CaSi-PAMAM-FA/mPEG (MMP-2 concentration, 400 ng/ml) while maintaining the nanoparticle concentration at 34 μg/ml. The incubation would continue for 12 hours. The cytosolic fluorescence was then quenched by incubation with trypan blue (200 μg/ml), and then, the cell samples were washed with nonserum-containing culture media. The lysosomes were stained red by incubating with lyso-tracker red solubilized in nonserum-containing culture media (60 nM) at 37°C for 60 min. The supernatant was removed when the incubation was complete, and the cell samples were washed thrice with PBS. The cells were fixed with paraformaldehyde at 4°C for 30 min and washed again with PBS for three times. The cell nuclei were then stained using H33258 (10 μg/ml) for 5 min. The processed cell samples were washed three more times with PBS and mounted using glycerin. The mounted cells were observed on a Leica TCS SP8 confocal laser microscope.

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