*2.2. In Vitro Drug Release Study*

NCUR-2, NMTX-1, and NCUR/MTX-2 were initially chosen for in vitro drug release experiments due to their appropriate morphologies and sizes, suitable drug loadings and encapsulation efficiencies, and high amorphization of the drug(s) into NCs. The release profiles for free drugs and these NCs recorded by a dialysis method are summarized in Figure 4.

**Figure 4.** In vitro release profiles for free CUR, free MTX and nanocapsules (NCUR-2, NMTX-1, and NCUR/MTX-2).

Free CUR and free MTX showed drug release patterns with a fast diffusion in the dissolution medium used. Free CUR achieved a mean drug-released value of 80% at 41 min of the experiment. For free MTX, a mean drug release of 80% was obtained at 195 min of the experiment. NCUR-2 released 80% of CUR at 2,640 min (44 h). NMTX-1 released 80% of MTX at 8,160 min (136 h). NCUR/MTX-2 had a mean drug release of 80% at 4,140 min (69 h) and 5,400 min (90 h), respectively for CUR and MTX. Therefore, NCs demonstrated prolonged release profiles with no burst effect in comparison to free drugs. Vakilinezhad et al. [22] described PLGA nanoparticles containing CUR and MTX with an initial burst release that was attributed to the drug dispersion into the superficial layers of these particles. In the present study, a typical core-shell structure was strategically planned to avoid this effect and to achieve a prolonged drug release for circumventing the administration of multiple doses per day of chemotherapy [42].

Considering the release profiles obtained for NCs, the difference in the release rate could be explained by the concentration gradient between each formulation and the dissolution medium. At higher drug concentrations, this process was faster since this gradient was the driving force for drug dissolution. Moreover, at higher drug-loading concentration, CUR and/or MTX could be distributed near to the nanocapsules surface providing a more rapid in vitro release [43]. In that sense, NCUR-2, prepared at the higher theoretical drug concentration of 3.0 mg·mL<sup>−</sup>1, presented a faster dissolution and reached a complete release in almost 2 days. On the other hand, NMTX-1, obtained at the lowest theoretical drug concentration of 0.1 mg·mL<sup>−</sup>1, revealed a very slow dissolution rate in about 7 days. The co-loaded formulation NCUR/MTX-2 presented the best dissolution features for controlling drug release. The theoretical drug concentrations proposed for NCUR/MTX-2, 0.5 mg·mL−<sup>1</sup> of CUR and 0.3 mg·mL−<sup>1</sup> of MTX, achieved a suitable intermediate dissolution rate in about 3 days. In this formulation, the release behavior of these drugs was closer as depicted in Figure 4, when compared to the other formulations tested. This performance is desired as it could ensure a simultaneous effect in vivo to provide both chemotherapeutic activity and inhibition of P-gp.

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