Fig. 6

The αCD33-mAB-P/P nanocarriers only transport siRNA in presence of free SMCC-protamine (SMCC-P). A Coomassie-stained SDS–PAGE showing αCD33-mAB, αCD33-mAB coupled with SMCC-P and HPLC-fractions 29–30 of αCD33-mAB-P/P upon effective depletion of unbound SMCC-P; HC = heavy chain, LC = light chain, -P = SMCC-protamine. B Antibody–protamine conjugates with fluorescent Alexa488-siRNA in cell-free incubation overnight on chamber slides. αCD33-mAB-P/P with free SMCC-P forms visible vesicular structures (left panel), while αCD33-mAB-P after depletion of free SMCC-P (fraction 30, see A) do not form visible structures (right panel). C. DLS and zeta-potential measurement of αCD33-mAB-P/free protamine-scr-siRNA carriers. D. αCD33-mAB-P/P-scr-siRNA nanoparticles were left to form for 2 h and subjected to electron microscopy on copper grids by phosphotungstate negative staining. E–H. Immunostaining of nanocarriers with an anti-human IgG antibody to illustrate the accessibility and location of the αCD33-mAB in the outside rim and the siRNA in the lumen of the αCD33-mAB-P/P-nanocarriers. I. Protamine was chemically coupled to Cy3 and then incubated with αCD33-mAB-P that was depleted from free protamine and with non-fluorescent control-siRNA to form nanocarriers. This complexation was performed for 2 h at RT and nanocarriers were then immobilized o/n on slides for immunostaining as in G. J. αCD33-mAB-P/P-Cy3-control-siRNA show homogeneous Cy3 (blue) micelles. K. The same vesicles as in L show anti-human IgG-Alexa647 (red) fluorescence in ring-like structure around each vesicle (staining as depicted in E). L. Overlay of panels J and K. α, anti