Figure 3.

Cytotoxicity of anti-nucleolin VHHs against nucleolin-overexpressing cancer cells. Different VHH constructs, grafted with a nucleolin-binding peptide sequence either onto CDR1 or CDR3, with (αNCL-CDR1-L or αNCL-CDR3-L, respectively) or without (αNCL-CDR1 or αNCL-CDR3, respectively) flanking linkers at the end of the grafted CDR, were incubated with (a) MDA-MB-435S or (b) 4T1 cancer cells, at concentrations up to 8 µM, for 72 h at 37 °C. The parental VHH, without the targeting component to nucleolin, was included as control. In the end of the incubation, cytotoxicity was assessed by the MTT assay. Data represent the mean ± SD of at least three independent experiments, performed in duplicate. Differences in cytotoxicity among the tested VHHs were evaluated by one-way ANOVA followed by Tukey’s test (*p < 0.05, **p < 0.01, ***p < 0.001).