Cytosolic mCherry protein expression and transfection efficiency
4-4-6 and PEI nanoparticles encapsulating the CAG-mCherry plasmid (Addgene #108685, (Mishra et al., 2020)) were formed as described above and added to HEK or CHO cells diluted for transfection (2 mL culture in 6-well plate). Optimized DNA concentrations were determined based on mean fluorescence over 5 days (580± Ex/630Em) using a BioTek SynergyMX plate reader (BioTek Instruments, Inc.) by reading a 3 × 3 grid distributed across the well surface area. For transfection efficacy studies, HEK or CHO cells were dosed with PBAE or PEI nanoparticles formed as described above, delivering 2 µg/mL (HEK) or 4 µg/mL (CHO) mCherry DNA (n =5). Cells were collected on day 5 and CHO cells were treated with Accutase (Sigma Aldrich), according to manufacturer’s instructions. Cells were then resuspended in phosphate-buffered saline (PBS, pH 7.4) containing 2% fetal bovine serum (FBS) and stained with LIVE/DEAD™ Fixable Green Dead Cell Stain Kit (Invitrogen), according to manufacturer’s instructions. mCherry expression in live cells was assessed using an Attune NxT Flow Cytometer (ThermoFisher Scientific). Experiments were performed a minimum of twice with consistent results.