Complexation and protection of siRNA from RNase. (A) Agarose gel electrophoresis of polyplex with different N/P w/w ratios. Uncomplexed siRNA is shown as white bands on the gel. Complete complexation took place at N/P w/w ratios of 1/4 and 1/6 w/w. (B) siRNA degradation assay showed no increase in absorbance (a measurement of free siRNA) in 1/4 P(SiDAAr)₅PEG₃ polyplex, indicating polymer protecting siRNA from RNase degradation. Data are the mean ± S.E.M.; n = 2 to 3. P < 0.01 between siRNA, siRNA-polyplex + nuclease vs. siRNA + nuclease.

Stability of polyplex in the presence of polyanion. Polyanion competition assay revealed siRNA polyplex stability in the presence of varying concentrations of heparin sodium. (A) Gel image. (B) Quantification of band intensity. Partial siRNA release took place at 2500 µg/ml of heparin sodium, whereas at 5000 µg/ml of heparin sodium, nearly all of the siRNA was released from the polyplex.

Polyplex uptake in metastatic and resistant breast cancer cells. Uptake in MDA-MB-231-Luc-D3H2LN (A) and MCF-7/Adr cell lines (B) by flow cytometry and the respective mean fluorescence intensity following incubation of cells with polyplex for 4 hours. Luc-siRNA PP, cells that were treated with polyplex prepared with TRITC-conjugated polymer; No treatment, cells that were not treated with polyplex. y-Axis represents the data normalized to the maximum number of cells counted, whereas x-axis represents the channel used for measuring fluorescence signal. Untreated n = 1, treated n = 8 to 9 repeats of the same sample measurements.

Cytotoxicity and transfection efficiency of siRNA/P(SiDAAr)₅PEG₃ polyplex in the MDA-MB-231-Luc-D3H2LN cell line. (A) Polyplex toxicity. *P < 0.05 compared with the control group, n = 6. (B) Luc-siRNA transfection with polyplex at varying N/P w/w ratios. The polymer composition of the polyplex increases and becomes highly effective at N/P ratio of 1/4 w/w, where there is a complete complexation of siRNA. *P < 0.01 compared with the control group, n = 2. RLU, relative light unit.

Knockdown ABCB1 with anti-ABCB1 siRNA polyplex and its effect on DOX accumulation and cytotoxicity in MCF-7/Adr cells. (Aa) Flow cytometric analysis of suppression of ABCB1 protein expression following incubation with anti-ABCB1 siRNA polyplex. (Ab) The suppression was seen to increase with incubation time. (Ba) Cotreatment of cells with anti-ABCB1 siRNA polyplex significantly enhanced DOX uptake. (Bb) Change in mean fluorescence intensity of cells cotreated with anti-ABCB1 siRNA polyplex and DOX. Cells were treated with anti-ABCB1 siRNA polyplex and DOX solution at 2500 ng/ml for 4.5 hours prior to flow cytometric analysis of cells for DOX uptake. (C) Cell viability following treating cells with DOX solution alone or cotreatment with anti-ABCB1 siRNA polyplex at different doses DOX (numbers indicate nanograms per milliliter dose) for 72 hours prior to measuring cell viability using MTT assay. n = 6. *P < 0.05; **P = 0.053 compared with DOX alone. PP, polyplex. Flow cytometry: y-axis represents the data normalized to the maximum number of cells counted, whereas x-axis represents the channel used for measuring fluorescence signal.