The cytosolic HRP delivery was determined by intracellular HRP enzymatic activity assay. Intracellularly delivered HRP catalyzed a nonfluorescent substrate, Amplex Red, to a fluorescent product, resorufin, in the presence of hydrogen peroxide. Briefly, HeLa cells were treated with dendrimer/HRP complexes for 4 hours with a similar procedure. Then, the cells were washed three times with PBS and incubated in PBS containing Amplex Red (50 μM) and hydrogen peroxide (500 μM). After 30 min of incubation at room temperature, the treated cells were washed three times and observed by LSCM. In another typical intracellular enzyme activity assay, TMB was used as a colorless substrate and turned into a blue product in the presence of HRP. Generally, cytosolic HRP delivery was performed with a similar procedure, and the treated cells were washed five times with PBS. TMB substrate (0.5 ml; 10 μg/ml) dissolved in acetate buffer (pH 5) with 3 mM hydrogen peroxide was added into each well. After 10 min of incubation at room temperature, the mixture solution in each well was imaged by an optical camera. Cytosolic enzyme activity of native HRP without dendrimer complexion was measured as a control. The in vitro HRP enzymatic activity was measured by TMB staining assay. Generally, the native HRP and HRP/P4 complex solutions were centrifuged at 13,000 rpm for 30 min. The supernatants were diluted with distilled water and incubated with TMB solution. The enzyme activity of HRP was determined by measurement of the absorbance of the solution at 630 nm by a microplate reader at 30-s intervals for 11 min. 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.

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