Fig. 1

The αCD33-monoclonal antibody (mAB) gemtuzumab-protamine (αCD33-mAB-P/P) conjugates bind and transport siRNA only into CD33-positive cells. A We cloned a plasmid expressing the heavy (HC) and light chain (LC) of the full IgG1 monoclonal antibody gemtuzumab. After transfection of the production cell line CHO-S, the mAB was purified via FPLC. Shown here is a representative elution profile (right panel). IRES, internal ribosomal entry side; GFP, green fluorescent protein. B First, protamine was conjugated chemically to the linker sulfo-SMCC, then this linker was coupled to the αCD33-mAB. The naturally anionic siRNA could then complex by electrostatic binding to the protamine moieties. In complex with siRNA, the αCD33-mAB-P and free SMCC-protamine spontaneously form vesicular structures, which we call αCD33-mAB-P/P-siRNA nanocarrier. C Coomassie-stained SDS–PAGE showing uncoupled αCD33-mAB and αCD33-mAB coupled with SMCC-protamine (αCD33-mAB-P/P). The shift of molecular weight after protamine conjugation via sulfo-SMCC to the αCD33-mAB can be seen for the heavy chain (HC-P) and for the light chain (LC-P). SMCC-P, unbound SMCC-protamine; M, molecular weight marker. D Band-shift assay. Agarose gel-electrophoretic analysis of the binding capacity of siRNA to the αCD33-mAB-P/P complex. Up to 16 mol siRNA per mol of mAB-P can bind to the αCD33-mAB-P/P complex. E–K Fluorescence microscopy of nanocarriers. Vesicles were formed by self-assembly upon incubation of 60 nM αCD33-mAB-P/P with 600 nM Alexa488-control-siRNA for 2 h at RT and documented by fluorescence microscopy after immobilization of the nanocarriers on coated slides without cells o/n at 37 °C. L–S Fluorescence microscopy analysis of siRNA internalization mediated by αCD33-mAB-P/P-siRNA complex into CD33-positive cells (L-N and P-R) but not into negative control cells (O and S). Upper panels (L-O): Green fluorescence depicting Alexa488-control siRNA in vesicular structures within the cells. Lower panels (P-S): Overlay of blue Hoechst staining and green fluorescence from upper panels. α, anti