EpCAM-targeted delivery of nanocomplexed siRNA to tumor cells with designed ankyrin repeat proteins

A, SDS-PAGE of purification fractions of fusion protein C9-P (22.6 kDa). CL, cleared lysate; I, insoluble fraction; FT, IMAC flow through; W1-3, three wash fractions; E1-3, three elution fractions; M, molecular weight marker. After protein expression and cell lysis in denaturing buffer (8 mol/L urea), the protein solution was purified by Ni-NTA in a batch purification, and subsequently loaded on a polypropylene column. Non–his-tagged impurities and nucleic acids were washed from the column using denaturing buffer. Subsequently, the his-tagged DARPin was refolded on the column by gradually lowering the urea concentration followed by elution in buffer containing 250 mmol/L imidazol. B, binding of C9-P to the 21mer bcl-2 siRNA. Samples were run on a native 15% polyacrylamide gel and stained with Coomassie blue. C, binding of FITC-labeled siRNA to MCF-7 cells in the presence of C9 (black filled peak) or as a complex with C9-P (open grey peak). Cells were incubated on ice with a 4:1 mixture of FITC-labeled siRNA and C9 or C9-P for 1 h. After repeated washing, cell binding was determined by flow cytometry analysis.

A, binding of EpCAM-targeted DARPin C9 and the fusion protein C9-P to EpCAM-positive MCF-7 and HT-29, and to EpCAM-negative HEK293T cells. Black filled peak, secondary antibody alone; open grey peak, C9 (without protamine fusion); open black peak, C9-P. Cells were trypsinized, resuspended in ice-cold PBS containing 1% FCS, and stained with the indicated proteins (1 μmol/L). FACS analysis was done following visualization with Alexa 488-conjugated anti-his-tag antibodies. B, cell binding activity of monomeric and dimeric C9 to EpCAM-positive MCF-7 and negative HEK293T control cells. Varying concentrations of DARPin C9 or the dimers C9D or C9LZ were added to the cells for 1 h before adding Alexa 488-conjugated anti-his-tag antibodies to determine the maximum binding concentrations by flow cytometry analysis.

Internalization of FITC-conjugated bcl-2 siRNA in MCF-7 cells. The siRNA was complexed to C9-P in a molar ratio of 4:1 (A), to lipofectamine (B), or mixed with C9 lacking an oligonucleotide binding domain as negative control (C) and added to cells grown on coverslips. After incubation at 37°C for 4 h, cells were fixed and visualized by confocal microscopy. Z-stacks were examined to ensure the signal is inside the cells. Scale bar, 10 μm.

Down-regulation of bcl-2 mRNA (A) and bcl-2 protein (B) in MCF-7 cells upon treatment with bcl-2-targeted siRNA in the presence of C9 or complexed to the various C9 fusion proteins in a ratio of 4:1 or with LipofectAMINE. Cells were treated for a total of 48 h, then lysed and subjected to mRNA or protein analysis by qPCR or Western blotting, respectively. Values were standardized to ribosomal RNA or actin. Following lysis, bcl-2 protein levels were determined by densitometric quantification of bcl-2 bands and normalization to actin. As negative controls, an irrelevant nonfunctional siRNA sequence delivered with C9-P and a mixture of anti–bcl-2 siRNA with C9 (without protamine) were used. Error bars, mean + SD, n = 3. *, P < 0.01, compared with untreated control; †, P < 0.05 compared with C9-P plus siRNA. C, representative Western blot of bcl-2 protein expression in MCF-7 cells upon treatment with bcl-2-targeted siRNA delivered with the indicated fusion proteins.