In vitro cytotoxicity assay of pBAE-CRHR-NB-Zw was evaluated in HeLa cell line and compared with that of the intermediate polymeric complexes of pBAE-CRHR, pBAE-CRHR(EKEKEK)1, and pBAE-CRHR(ERERER)2. At monolayer confluency cells were trypsinized and seeded in a 96-well plate at 1 X 10 4 initial cell density in 100 µl of DMEM supplemented growth medium and allowed to grow up to 80% of confluency prior to polyplex treatment. 24 hours later, 100 µl of MTT solution in medium (0.5 mg/ml) was added to each well and incubated for additional 3 h. The medium was carefully aspirated, leaving purple formazan crystals, which were dissolved in DMSO (100 µl). The absorbance was measured at 540 nm using TECAN microplate reader.