Figure 2

A. Effect of the Ni-NTA liposome concentration in the CLIA assay. SKBR3 cells were co-incubated with (squares) or without (circles) 20 μg/mL of the anti-ErbB2-scFv-F5-(His)6 and varying concentrations of fluorescently labeled Ni-NTA-liposomes (Ni-NTA = 2% of phospholipid content). Internalization of liposomes was measured in a microfluorimeter. B. Effect of antibody concentration in the CLIA assay. SKBR3 cells were coincubated with 500 μM of fluorescently labeled Ni-NTA-liposomes containing 2 % of phospholipid content and varying concentrations of either the anti-ErbB2-scFv-F5-(His)6 (solid line-circles), or an irrelevant control antibody (anti-VEGRF2-scFv-4G7-(His)6)(squares), or with no antibody (dotted line – circles). C. Effect of the anti-ErbB2-scFv-F5-(His)6 concentration on the uptake of fluorescently labeled, minimally PEGylated Ni-NTA-liposomes (Ni-NTA = 5% of phospholipid content; 0.5 mol.% PEG(M.w. 2,000)-DSPE) by cells with high (SKBR-3 cells) or low (MCF-7 cells) expression of ErbB2 receptor. (-EDTA), cells were washed with Hanks' BSS without EDTA; (+EDTA), cells were washed with Hanks' BSS + 1 mM EDTA. Experiments were done in duplicate. Error bars, SD. For all the experiments, immunoliposomes were allowed to internalize for four hours. The cells were lysed in base before reading the fluorescence in a microfluorimeter. Incubation time was 4 hours.