*4.5. Cytotoxicity Assay*

A549 cells were plated into normal 96-well plates at a density of 5 × 10<sup>3</sup> cells (100 μL/well) to obtain attached cells. The cells were plated into polyHEMA-coated 96-well plates at a density of 1 × 10<sup>4</sup> cells (100 μL/well) to obtain floating cells. After 24 h incubation, the cells were exposed to different concentrations of paclitaxel or paclitaxel-PLHNPs (25 μL/well) for 72 h. A modified MTT assay for non-adherent culture was employed to determine cell viability at the end of treatment [34]. A 25 μL of fresh culture medium containing MTT was added to each well to reach a final concentration of 0.5 mg/mL and incubated for 4 h, followed by adding 100 μL of lysis solution (20% SDS in 10 mM HCl) to solubilize formazan crystals produced by viable cells, then the plates were kept in the dark for 48 h. After that, absorbance was measured at 550 nm and subtracted from a reference wavelength at 650 nm, using a microplate reader. The cell survival rate was expressed as percentage compared with control. Fold resistance was calculated from ratio of the IC50 value in floating cells to the IC50 value in attached cells.
