3.3.1. Determination of Mean Diameter, Polydispersity Index, and Zeta Potential of NCs

Mean particle size, polydispersity, and zeta potential were measured at 25 ◦C using a Zetasizer Nanoseries ZS903600 apparatus (Malvern Instruments, Malvern, UK). Each sample was previously diluted in water (1:500) and each analysis was performed at a scattering angle of 90◦ and a temperature of 25 ◦C for mean particle size and polydispersity measurements. For zeta potential, each sample was placed into the electrophoretic cell where a potential of ± 150 mV was used.

#### 3.3.2. Encapsulation Efficiency

CUR and/or MTX content of NCs was determined by the indirect method. In brief, suspensions of nanocapsules were submitted to a combined ultrafiltration/centrifugation using centrifugal devices (Amicon®® 10.000 MW, Millipore, Bedford, MA, USA) at 2200 g during 30 min in triplicate. Free CUR and/or MTX were determined in ultrafiltrate using an HPLC method in a Merck-Hitachi Lachrom equipment (Tokyo, Japan), interface D-7000, UV detector module L-74000, equipped with pumps L-7100 and an integral degasser, controller software (Chromquest, Thermo Fisher Scientific, Incorporated, Pittsburgh, PA, USA), and manual injector (Rheodyne, Rohnert Park, CA, USA) equipped with a 20 μL injector loop and a 100 μL syringe (Microliter 710, Hamilton, Bonaduz, Switzerland). Chromatographic separation was accomplished using a Inertsil®® ODS3 (GL Sciences, Torrance, CA, USA) reversed-phase analytical column (150 mm <sup>×</sup> 4.6 mm, 5 <sup>μ</sup>m) and a GL Sciences Inertsil®® ODS3 guard cartridge system (10 mm × 4 mm, 5 μm) at room temperature (20 ± 2 ◦C) using UV detection at 261 nm. Gradient elution was carried out using a mobile phase consisted of methanol:water acidified with 0.5% acetic acid at a flow rate of 1.0 mL.min<sup>−</sup>1. The mobile phase gradient program was as follows: it was started at 44% MeOH for 3 min, increased to 90% MeOH in 5 min, held constant until 11 min, then returned to 44% MeOH in 12 min. The encapsulation efficiency (EE, %) was calculated using Equation (1):

$$EE(\%) = \frac{\text{total drug content} - \text{free drug content}}{\text{total drug content}} \times 100\tag{1}$$

#### 3.3.3. Field Emission Scanning Electron Microscopy (FESEM)

The freeze-dried formulations were mounted on aluminum stubs and sputtered with gold (IC-50 Ion Coater, Shimadzu, Kyoto, Japan). Morphological analysis was performed and photomicrographs were prepared using a Mira3 LM FESEM (Tescan, Brno, Czech Republic) at an accelerating voltage of 5 kV.

#### 3.3.4. X-ray Diffraction (XRD)

Polymeric nanocapsules were previously deposited on a glass coverslip and dried at room temperature (25 ± 2 ◦C). XRD data were recorded in an Ultima IV diffractometer (Rigaku, Tokyo, Japan). The 2<sup>θ</sup> value was increased from 4◦ to 50◦ at a scan rate of 0.05◦·min−<sup>1</sup> using a Cu-K<sup>α</sup> source (λ = 1.5418 Å) at 30 kV and 40 mA.
