The cytosolic delivery of toxic proteins was tested on MDA-MB-231 cells (saporin and RNase A) and HeLa cells (trypsin). The cells were cultured in 96-well plates overnight. Saporin, RNase A, and trypsin complexes with P4 were prepared as described above. The dendrimer/protein complexes were diluted with 100 μl of serum-free MEM and incubated for 30 min. Then, the mixtures were further diluted with 500 μl of serum-free MEM. The cell culture media were removed, and the cells were washed with PBS. Protein complexes (100 μl) were added into each well. After 6 hours of incubation, the media were replaced with 100 μl of fresh MEM containing 10% FBS, and the cells were further incubated for 42 hours. The cytotoxicity of cells treated with saporin, RNase A, and trypsin complexes was evaluated by MTT assay. Five repeats were tested for each sample. Proteins were tested at various concentrations. Naked saporin, RNase A, trypsin, and dendrimers at equal concentrations were tested as controls. For in vitro RNase A activity assay, the enzyme activity was measured by an RNaseAlert Kit according to the manufacturer’s protocol. Briefly, RNaseAlert substrate dissolved in assay buffer was incubated with native enzyme or the RNase A/P4 complex in RNase-free buffer. The recovery of enzyme activity was conducted by the addition of BSA (0.1 mg/ml) or heparin sodium (0.1 mg/ml) into the complex solutions. The fluorescence intensity was recorded at 5-min intervals for 30 min. The excitation and emission wavelengths were λex = 490 nm and λem = 520 nm, respectively.

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