*3.1. Materials*

Curcumin (CUR, ≥94% curcuminoid content, Sigma-Aldrich, St. Louis, MO, USA), methotrexate (MTX, >99% pure, Fermion, Espoo, Finland), poly(ε-caprolactone) (PCL, Mw 10,000–14,000 g.mol<sup>−</sup>1, Sigma-Aldrich), poly(ethylene glycol) 6000 (PEG, Mw 5,400–6,600 g.mol<sup>−</sup>1, Cromato Produtos Químicos, Diadema, Brazil), sorbitan monooleate (Span 80, Oxiteno, Mauá, Brazil), polysorbate 80 (Tween 80, Delaware, Porto Alegre, Brazil), medium chain triglycerides (MCT, 99% pure, Focus Química, São Paulo, Brazil), acridine orange base (AO, Sigma-Aldrich), ethidium bromide (EB, Sigma-Aldrich), methylthiazolyldiphenyltetrazolium bromide (MTT, Sigma-Aldrich), sulpho-rhodamine B (SRB, Sigma-Aldrich), penicillin-streptomycin (Sigma-Aldrich), and acetone (≥99.9% pure, Vetec Química, Rio de Janeiro, Brazil) were used as received. HPLC-grade methanol was purchased from Tedia (Rio de Janeiro, Brazil). RPMI 1640 medium and fetal bovine serum were obtained from Vitrocell (Campinas, Brazil). Water was purified in a Milli-Q Plus water purification system (Millipore, Bedford, MA, USA). All other solvents and reagents were analytical grade. The Calu-3 cell line was obtained from the Bank of Cells of Rio de Janeiro (BCRJ, Brazil) and was kindly provided by Dr. Katia Sabrina Paludo.

#### *3.2. Preparation of Polymeric Nanocapsules (NCs) Containing Curcumin (CUR) and*/*or Methotrexate (MTX)*

The interfacial deposition of the preformed polymer method was used for preparing NCs containing CUR and/or MTX [20]. Six different formulations (Table 3) were obtained depending on the amount of CUR and/or MTX in their composition. Briefly, PCL and PEG 6000 were solvated in the organic phase containing span 80, CUR and/or MTX, MCT, and acetone. This phase was carefully added to the aqueous phase containing tween 80 and purified water under vigorous magnetic stirring at 45 ◦C. The obtained colloidal emulsion was kept under magnetic stirring for 10 min. The organic solvent was then quickly removed by evaporation under reduced pressure at 40 ◦C. Non-loaded nanocapsules were also prepared as the negative control. All formulations were obtained in triplicate from three different batches. For providing a comparative analysis, a physical mixture of polymers and drugs (PM PCL/PEG/CUR/MTX) at the same molar ratio was prepared by mortar and pestle mixing before characterization procedures.


**Table 3.** Composition of polymeric nanocapsules containing curcumin (CUR) and/or methotrexate (MTX).
