2.2.8. Cytotoxicity Studies

Cytotoxicity studies of PZQ, VHS and SEP were performed by using PZQ veterinary antiparasitic doses. Consequently, the amount of mineral (VHS and SEP) that should be subjected to study was five times higher than that of PZQ. To do so, intermediate dilutions of 100 mM and 10 mM were prepared in dimethyl sulfoxide (DMSO). The same procedure and concentrations were used for cytotoxicity studies of PZQ–VHSet and PZQ–SEPet.

AlamarBlue™ bioassay (Thermo Fisher Scientific, Carlsbad, CA, USA) was used to study cell proliferation. To do so, HCT 116 cells were seeded in 96-well plates (density of 3–6 <sup>×</sup> 104 cells/cm2) in a final DMEM volume of 200 μL. The corresponding solid samples were subsequently added (PZQ, VHS, SEP, IP PZQ–VHSet, or IP PZQ–SEPet). All samples were tested in a wide range of concentrations (100 μM, 20 μM, 4 μM, 800 nM, 160 nM, 32 nM, and 6.4 nM) and three replicates were used for each experience. Then, the well plates were incubated at 37 ◦C, 5% CO2 for 48 h. At the end of contact time, 10 μL of cell viability reagent (PrestoBlue™, Thermo Fisher Scientific) was added to each well. The PrestoBlue™ reagent is a resazurin compound that works as a cellular viability indicator since its color changes from blue to pink when reduced by living cells. After 15 min of incubation in presence of PrestoBlue™ reagent, fluorescence was quantified at 535–90 nm in a microplate reader (Tecan, Zürich, Switzerland).
