2.3.6. In Vitro Cytotoxicity Studies

HeLa (human cervix adenocarcinoma cells) (Cellonex) were cultured in Dulbecco's Modified Eagle Medium (DMEM)-(Lonza) supplemented with 10% w/w fetal calf serum and antibiotics (penicillin/streptomycin/amphotericin B) at 37 ◦C in a 5% CO2 incubator. HeLa cells were transferred to 96-well plates at a cell density of 1 × 10<sup>4</sup> cells per well in 150 μL culture medium and grown overnight. A single concentration of 50 μM of the test compound was incubated with the cells for an additional 48 h, and cell viability in the wells assessed by adding 20 μL 0.54 mM resazurin in PBS for an additional 2–4 h. The numbers of cells surviving drug treatment were determined by reading resorufin fluorescence (excitation 560 nm, emission 590 nm) with a SpectraMax ® M3 plate reader (Molecular Devices, San Jose, CA, USA). Fluorescence readings for the individual wells were converted to percent (%) cell viability relative to the average readings from untreated control wells. Plots of % cell viability vs. log(compound) were used to determine IC50 values by non-linear regression using version 5.02 GraphPad Prism (GraphPad Holdings LLC, La Jolla, CA, USA).

The compounds investigated were the optimized NCC, the individual API and a physical mixture of the API in stoichiometric ratios identical to those used to produce the NCC.
