Optimization of 599 peptide-mediated delivery of siRNAs into oral cancer cells.

<p>(<b>A</b>) Fluorescence microscopy analysis of CAL 27 oral cancer cells incubated for 2 hours with DY547-conjugated siRNA targeting CIP2A (D-siCIP2A; red) alone or in complex with increasing amounts of 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs). Nuclei (blue) were counterstained with DAPI. Scale bar: 50 µm. (<b>B</b>) CAL 27 cells incubated for 2.5 hours with D-siCIP2A alone or in complex with increasing amounts of 599 peptide (ranging from 1 to 100-fold molar excess of siRNAs). For comparison, the cells were also transfected using the commercial transfection reagent, INTERFERin™ (IFN). The amount of siRNA delivered into cells in pmol per mg of protein is reported with each treatment normalized to D-siCIP2A alone. Data are mean ± SEM of four separate experiments, where ***P<0.001, **P<0.01 compared to D-siCIP2A alone treated cells (ANOVA, Dunnett’s Multiple Comparison Test). (<b>C</b>) Assessment of long-term toxicity (as measured by a cell proliferation assay) of CAL 27 cells 48 hours post-treatment with either a non-targeting siRNA (siNT) alone, increasing concentrations of 599 peptide alone, or increasing amounts of 599 peptide (ranging from 1 to 100-fold molar excess of siRNAs) in complex with siNT. For comparison, the cells were also transfected with the commercial transfection reagent, IFN. Untreated cells were defined as 100% viable. Data are mean ± SEM performed in triplicate (n = 3), where ***P<0.001, **P<0.01 compared to untreated cells (ANOVA, Dunnett’s Multiple Comparison Test).