Fig. 5

Attributes of effective αCD33-mAB:protamine conjugation ratios. A Concentrations tested and resulting molar ratios of αCD33 antibody (αCD33-mAB) to SMCC-protamine for the effective conjugation of both components. B Coomassie-stained SDS–PAGE showing uncoupled αCD33-mAB compared to the conjugation products that were coupled as depicted in A. The formation of a protamine-conjugated heavy chain (HC-P) and light chain (LC-P) showed an optimum at a 1:32 conjugation ratio with no further increase at higher ratios. C–H Left: Band-shift assays exhibiting siRNA binding capacity. Right: αCD33-mAB-P/P stored at 4 °C for several days shows precipitation at the bottom of the tube only at ratio 1:50 and 1:120 (blue triangles). I-N Internalization of Alexa488-control-siRNA complexed αCD33-mAB-P/P into OCI-AML2 cells. Complexes of αCD33-mAB-P/P transport Alexa488-siRNA into cells (left panel rectangles), with detailed magnifications (right panels). At conjugation ratios of 1:1–1:10 (I-K) only diffuse greenish background can be detected. O-T Left: Colony formation assays of the different conjugations in OCI-AML2 cells. Significance: *, p < 0.05, 2-tailed T-test. Means plus SD of three independent experiments. D3A, DNMT3A siRNA. S-P, SMCC-protamine. Right: αCD33-mAB-P/P-Alexa488-siRNA complexes form vesicles with different size and efficacy in presence of rising amounts of free SMCC-protamine. No vesicle formation at ratios of 1:1–10 (right: O-Q), apart from some unspecific aggregates (right side in P). α, anti