With bioconjugates 5-7 in hand, we evaluated their cytotoxicity by assaying cell viability on an sstr2-positive rat pancreatic cancer Ar42J cells, using free amatoxins as controls. The MTS cell viability assay showed that all three PDCs were effective at reducing the viability of Ar42J cells in the low nM range, providing calculated EC50 values of 4.2, and 2.3 nM for conjugates 6 and 7, respectively; these displayed up to 1000-fold enhancement in apparent cytotoxicity compared to free amatoxins 3 and 4, both of which gave calculated EC50 values of &tide;2 μM. This points to successful targeting and intracellular accumulation of toxin inside cells through an sstr2-mediated uptake inside the cells. Further evidence supporting target-mediated uptake is the micromolar toxicity of bioconjugates to the sstr2-negative CHO cell line that is otherwise sensitive to non-targeted amanitin. In comparison to -amanitin and synthetic amatoxins 3 and 4, bioconjugates 5-7 are 7- to 14-fold less toxic to CHO cells (control cell line), likely owing to impaired uptake of octreotate-amanitin conjugates that must enter via diffusion mediated processes or other non-specific import mechanisms that are currently unknown. In addition, a blocking study was run in the presence of free TATE-N32, which led to the expected reduction in apparent toxicity.

With bioconjugates 5-7 in hand, we evaluated their cytotoxicity by assaying cell viability on an sstr2-positive rat pancreatic cancer Ar42J cells, using free amatoxins as controls. The MTS cell viability assay showed that all three PDCs were effective at reducing the viability of Ar42J cells in the low nM range, providing calculated EC50 values of 4.2, and 2.3 nM for conjugates 6 and 7, respectively; these displayed up to 1000-fold enhancement in apparent cytotoxicity compared to free amatoxins 3 and 4, both of which gave calculated EC50 values of &tide;2 μM. This points to successful targeting and intracellular accumulation of toxin inside cells through an sstr2-mediated uptake inside the cells. Further evidence supporting target-mediated uptake is the micromolar toxicity of bioconjugates to the sstr2-negative CHO cell line that is otherwise sensitive to non-targeted amanitin. In comparison to -amanitin and synthetic amatoxins 3 and 4, bioconjugates 5-7 are 7- to 14-fold less toxic to CHO cells (control cell line), likely owing to impaired uptake of octreotate-amanitin conjugates that must enter via diffusion mediated processes or other non-specific import mechanisms that are currently unknown. In addition, a blocking study was run in the presence of free TATE-N32, which led to the expected reduction in apparent toxicity.