HeLa cells [a human cervical carcinoma cell line, American Type Culture Collection (ATCC)] and NIH3T3 cells (a mouse embryo fibroblast cell line, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco). MDA-MB-231 cells (a human mammary cancer cell line, ATCC) were cultured in minimum essential medium (MEM, GIBO). The cell culture media contain 10% fetal bovine serum (FBS, Gemini), penicillin (100 μg/ml), and streptomycin (100 μg/ml) at 37°C under 5% CO2.

HeLa cells were seeded in 48-well plates before cytosolic protein delivery. The protein solutions were mixed with dendrimer at various weight ratios. The complex solutions were diluted with 100 μl of serum-free DMEM and incubated for 30 min at room temperature and further diluted with 150 μl of serum-free DMEM. The culture media were removed, and the cells were washed twice with PBS, before adding the protein complex solutions into the wells. After incubation for 4 hours, the culture media containing proteins were removed, and the cells were washed twice with PBS. The fluorescence intensity of the treated cells was determined by flow cytometry (BD FACSCalibur, San Jose). The cells were also observed by laser scanning confocal microscopy (LSCM, Leica SP5, Germany). The commercial protein delivery reagents such as PULSin and TransEx were used as positive controls according to the manufacturers’ protocols. Naked proteins without complexation with the dendrimers were used as negative controls. In a separate study, trypan blue (0.04%) was added to the treated cells before flow cytometry analysis to quench the fluorescence of proteins physically adsorbed on cell membrane. The time-dependent intracellular delivery of dendrimer/protein complexes into HeLa cells was observed by LCSM. The cells were incubated with dendrimer/BSA-FITC complexes for 1, 2, 3, 4, 6, and 12 hours, respectively. The acidic organelles in the treated cells were stained by LysoTracker Red (DND-99, Invitrogen) before confocal imaging. To investigate the internalization mechanism of protein/dendrimer complexes, we used BSA-FITC as the model protein. HeLa cells were preincubated with endocytosis inhibitors such as cytochalasin D (10 μM), chlorpromazine (20 μM), genistein (700 μM), and methyl-β-cyclodextrin (10 mM) for 2 hours before protein transduction experiments, respectively, or incubated at 4°C during cytosolic protein delivery. The cells treated without any inhibitors and transduced at 37°C were tested as a control.

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