3.5.2. Cell Treatment

Calu-3 cells were seeded in 96-well plates at a density of 1.5 <sup>×</sup> 104 cells.well−<sup>1</sup> and incubated for 24 h in the culture medium. For assessment of cell viability and cytotoxicity, Calu-3 cell line was incubated with the samples NCUR-2 (200.00–25.00 <sup>μ</sup>mol·L−1), NMTX-1 (100.00–12.50 <sup>μ</sup>mol·L−<sup>1</sup> and 4.00–0.50 <sup>μ</sup>mol·L<sup>−</sup>1) and NCUR/MTX-2 (247.00–61.75 <sup>μ</sup>mol·L−<sup>1</sup> and 9.88–2.47 <sup>μ</sup>mol·L−<sup>1</sup> to CUR; 100.00–25.00 <sup>μ</sup>mol·L−<sup>1</sup> and 4.00–1.00 <sup>μ</sup>mol·L−<sup>1</sup> to MTX) for 72 h at 37 ◦C with 5% CO2 atmosphere. For co-loaded formulation, final concentrations were standardized using MTX due to its lower content in NCs and EE compensation was also performed for CUR content, resulting in fractional values. Tests were obtained using serial dilution procedure and were performed as four independent experiments containing *n* = 4 samples per assay.

#### 3.5.3. Cell Viability by Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) Test

After 72 h of the treatments, 200 <sup>μ</sup>L of a solution of MTT at 0.5 mg·mL–1 was added to the wells following a standard method [56]. The cultures were then incubated at 37 ◦C for 2 h, protected from light, until the presence of formazan crystals. The supernatant was then removed. For the solubilization of these crystals, 200 μL of dimethyl sulfoxide was added. The spectrophotometric absorbance reading was performed at a wavelength of 550 nm in a μQuant microplate reader (BioTek, Winooski, VT, USA). To calculate cell viability (%), Equation (2) was used. The concentration that inhibited 50% of cell growth (IC50) was then calculated by Probit regression [57]:

$$\text{Cell viability } (\%) = \frac{\text{absolute of test}}{\text{absorbance of control}} \times 100 \tag{2}$$

#### 3.5.4. Combination Index

To define the drug-drug interaction potential from the use of NCs containing CUR and MTX against Calu-3 cells, the combination index (CI) was calculated by Equation (3) [58] considering the results obtained by the MTT method:

$$\text{Combination index (CI)} = \frac{(D)1}{(Dx)1} + \frac{(D)2}{(Dx)2} \tag{3}$$

where (Dx)1 and (Dx)2 are the concentration of the tested substance 1 (CUR) and the tested substance 2 (MTX) used in the single treatment that was required to decrease the cell viability by 50% and (D)1 and (D)2 are the concentration of the tested substance 1 (CUR) in combination with the concentration of the tested substance 2 (MTX) that together decreased the cell viability by 50%.
