*3.5. In Vitro Cell-Based Assays of DPLGA and ADN-DPLGA Nanoparticles* 3.5.1. In Vitro Cell Toxicity (MTT Assay)

A549 (adenocarcinoma human alveolar basal epithelial cells) were selected due to the availability of overexpressed ARs. Cells were obtained from National Centre for Cell Sciences (NCCS, Pune, Maharashtra, India). Cell lines were maintained as prescribed by the ATCC guidelines. For cytotoxicity evaluation, different working dilutions of DTX in sterile phosphate buffer saline were created using a 10 mg/mL stock solution in DMSO. Cytotoxicity of all the formulations and naïve drugs was determined by MTT assay based on reduction of MTT dye (yellow) by the vital mitochondrial enzymes to blue-colored formazan product. A549 cells (1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) were seeded in 96-well plates and were allowed to attach overnight by incubating at 37 ◦C. For assessing the cytotoxicity, cells were exposed to different dilutions of DTX formulation and standard DTX and were incubated for 48 h at 37 ◦C in DMEM supplemented with a 10% FBS medium. After incubation, the media were aspirated, and the cells were washed twice with phosphate buffer saline (pH 7.4). The cells were processed for MTT assay [7]. The mean % of cell viability relative to untreated cells was estimated from data from multiple experiments (n = 6). The IC<sup>50</sup> value was calculated using the curve fitting method.

#### 3.5.2. Receptor Competition Assay

For competitive receptor assay, A549 cells were treated with free ligand before exposing cells with optimized formulations followed by the MTT assay as described earlier in the previous section [38]. Briefly, 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells were co-incubated with an excess of ADN. After 30 min of incubation, cells were washed twice with phosphate buffer saline (pH 7.4), followed by treatment of the cells with nanoparticle formulations and the pristine drug DTX. After incubation (48 h at 37 ◦C), cells were processed for MTT assay as reported earlier [21]. A comparison was completed between the IC<sup>50</sup> values from the receptor competition assay and previously obtained IC<sup>50</sup> values.
