Cytosolic delivery of enzymes such as β-Gal into HeLa cells was determined by intracellular β-Gal enzymatic activity measurements. In situ X-Gal staining kit and a quantitative β-Gal enzyme activity assay kit (Beyotime Biotech) were conducted according to the manufacturer’s protocols. Briefly, HeLa cells were treated with dendrimer/β-Gal complexes for 4 hours with a similar procedure as described above. Then, the treated cells were washed twice with PBS and fixed for 10 min at room temperature. The cells were further washed three times with PBS and incubated with the working solution containing 5% X-Gal for 2 hours at 37°C in the absence of CO2. The treated cells were further washed with PBS and observed by an optical microscope (Olympus, Japan). The intracellular β-Gal activity of the treated cells was quantitatively analyzed using a β-Gal enzyme activity assay kit. Generally, the treated cells were lysed with cell lysis buffer. Then, 50 μl of cell lysates was mixed with 50 μl of working solution containing enzyme substrate O-nitrophenyl-β-d-galactopyranoside and incubated for 30 min at 37°C. After incubation, 150 μl of stop reaction solution was added in each well. The enzyme activity was determined by measurement of the absorbance of the solution at 420 nm using a microplate reader (Thermo Fisher Scientific, Germany). The enzyme activity of native β-Gal at the same concentration was measured as control and defined as 100%. For in vitro β-Gal activity assay, the enzyme activity was measured by X-Gal staining assay. Generally, the enzyme/P4 complex solutions were incubated with substrate solution containing X-Gal at 37°C for 1 hour. Dimethyl sulfoxide was added to dissolve the yielding product, and the absorbance of the solutions at 633 nm was recorded by a microplate reader. Native β-Gal activity was measured as control. 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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