Poly(
ethylene glycol)-conjugated
polyethylenimine (
PEG-PEI) is a widely studied cationic
polymer used to develop non-viral vectors for
siRNA therapy of
genetic disorders including
cancer. Cell lines stably expressing
luciferase reporter
protein typically evaluate the transfection efficacy of
siRNA/
PEG-PEI complexes, however recent findings revealed that
PEG-PEI can reduce
luciferase expression independent of
siRNA. This study elucidates a cause of the false positive effect in
luciferase assays by using
polymer nanoassemblies (PNAs) made from
PEG, PEI, poly-(
l-lysine) (PLL),
palmitate (PAL), and
deoxycholate (DOC):
PEG-PEI (2P),
PEG-PEI-PAL (3P), PEG-PLL (2P'), PEG-PLL-PAL (3P'), and
PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of
luciferase using a
colorectal cancer cell line expressing
luciferase (HT29/LUC) concluded that 2P and 2P' caused no
luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P'<2PD<3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased
ATP and cellular
protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of
luciferase in the cells. These results indicate that hydrophobically modified
PEG-PEI induces cellular distress causing over-ubiquitination of the
luciferase protein, producing false positive
siRNA transfection in the
luciferase assay.