4.5. Cell Viability/Cytotoxicity Assay

The effect of TiO₂ PEG NPs and EGF-TiO₂ PEG NPs on the A431 cell viability was investigated using a LIVE/DEAD^® Viability/Cytotoxicity kit (Invitrogen, Ltd., Cambridge, UK) according to the manufacturer’s instructions using a fluorescence microplate protocol. Briefly, 1 × 10⁴ cells/well were seeded in a Costar 96-well plate and incubated at 37 °C and 5% CO₂. After 24 h, the cells were exposed to 10 μg/mL of TiO₂ PEG NPs and EGF-TiO₂ PEG NPs for 24 h. The cells were then stained with 1 μM calcein AM to stain live cells and 2 μM ethidium homodimer-1 (EthD-1) to stain dead cells, then the fluorescence intensity was measured using a Spark^TM 10 M multimode microplate reader (Tecan Ltd., Männedorf, Switzerland).