3.3.5. Fourier-Transform Infrared Spectroscopy (FTIR)

FTIR analyses of raw materials, freeze-dried polymeric nanocapsules, and physical mixture were carried out from 4000 to 400 cm−<sup>1</sup> using a Prestige-21 IR spectrophotometer (Shimadzu, Kyoto, Japan) in KBr pellets with 32 scans and a resolution of 4 cm<sup>−</sup>1.

#### *3.4. In Vitro Drug Release Study*

The release experiments were performed for free CUR, free MTX, and formulations NCUR-2, NMTX-1, and NCUR/MTX-2 by dialysis diffusion procedure in phosphate-buffered saline (PBS, 20 mmol.L<sup>−</sup>1) containing 0.1% (*w*/*v*) Tween®® 80 at pH 7.4 [21,22] by adding each sample in PBS (1.5 mL) into a dialysis bag (Spectra/Por®® molecular porous membrane tubing, MWCO 10,000, Spectrum Laboratories, Rancho Dominguez, CA, USA), and dialyzed against fresh PBS (13.5 mL) at 37 ◦C and under continuous magnetic stirring of 50 rpm. Aliquots of 500 μL were withdrawn at predetermined time intervals and replaced by the same volume of fresh medium. CUR and MTX concentrations were determined individually in each sample by the previously described HPLC method. The in vitro drug release assay was carried out in triplicate from three different batches.

#### *3.5. In Vitro Cell Culture-Based Assays*

### 3.5.1. Cell Culture

Calu-3 cell line was cultured in RPMI 1640 medium at pH 7.4, containing 10% fetal bovine serum, supplemented with 24 mmol·L−<sup>1</sup> sodium bicarbonate, 2 mmol·L−<sup>1</sup> L-glutamine, 1 mmol·L−<sup>1</sup> sodium pyruvate, 10,000 U·L−<sup>1</sup> penicillin, and 10 mg·L−<sup>1</sup> streptomycin. The cultures were maintained in a humidified oven at 37 ◦C with 5% CO2 atmosphere.
