For the cellular uptake evaluations, the 4T1 or A375 cells were seeded into a six-well plate at a density of 105 U per well. When the cell confluence reached around 70%, fresh media containing 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) were used to replace the exhausted culture media, where TCPS was used as control. The nanoparticle concentration was maintained at 34 μg/ml, and the incubation periods were set to 12 and 24 hours. When the incubation was complete, the media were drained and the cells were washed three times with PBS. Non–EDTA-Na–containing trypsin was then added to detach the cells, which were further washed twice with PBS and concentrated by centrifugation. The intracellular FITC intensity of the purified cells was monitored by a CytoFLEX system (Beckman Coulter).

For the CLSM study, 4T1 or A375 cells were first seeded into the confocal dish at a density of 1 × 105 and incubated in 1 ml of culture media overnight at 37°C under an atmospheric CO2 level of 5%. The exhausted culture media were replaced with fresh ones containing TCPS (blank control), ACC@FITC-CaSi-PEG, ACC@FITC-CaSi-PAMAM-FA/mPEG, and MMP-2–pretreated nanoparticle concentration was maintained at 34 μg/ml and the incubation would continue for 12 or 24 hours. Fresh culture media containing trypan blue (200 μg/ml) was subsequently added to replace the old ones to quench the cytoplasmic fluorescence. The cell samples were then washed with nonserum-containing culture media, and the cell membrane was stained with rhodamine-labeled wheat germ agglutinin solubilized in nonserum-containing culture media (10 μg/ml). When the cell membrane staining was complete, the cells were washed three times with PBS and fixed by paraformaldehyde at 4°C for 30 min. The fixed cells were again washed three times with PBS, and the cell nuclei were stained using H33258 (10 μg/ml) for 5 min. The double-stained cells 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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