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

Nanocapsules with or without CUR and/or MTX were successfully obtained by the interfacial deposition of the preformed polymer method. Formulations containing no CUR showed a liquid aspect with a slightly bluish-white opalescent coloring. However, CUR-loaded nanocapsules presented a liquid aspect and an intense yellow color.

2.1.1. Determination of Mean Diameter, Polydispersity Index, and Zeta Potential

Results of particle size, polydispersity, and zeta potential are summarized in Table 1. CUR and/ or MTX-loaded and non-loaded NCs revealed mean sizes between 287.83 and 325.16 nm with a polydispersity index (PDI) varying from 0.290 to 0.351, which represent a certain system homogeneity.


**Table 1.** Mean diameter, polydispersity index (PDI), zeta potential, and encapsulation efficiency (EE) of the obtained CUR and/or MTX-loaded and non-loaded nanocapsules.

\* Values are depicted as mean ± standard deviation (SD).

In general, NCs obtained by the interfacial deposition of preformed polymer method have demonstrated mean diameters between 200 and 300 nm and PDI between 0.2 and 0.3, particularly for the works carried out using poly(lactic-co-glycolic acid) and PCL [26]. However, NCs of larger diameter may be related to the presence of PEG in their composition. PEG chains create a more viscous organic phase, which affects its dispersion into the aqueous phase during stirring and leads to higher particle sizes with broader polydispersity [27].

Also, other aspects may directly influence the particle diameter in nanosystems. One of them is the oil used for preparing nanocapsules that influences some of the core structure properties, such as viscosity, hydrophobicity, and surface tension [28]. PDI values close to 0 are considered monodisperse and greater than 0.5 indicate heterogeneous dispersion [29]. Taking all these into account, suitable nanometric-scaled size and adequate polydispersity were recorded for NCs with or without CUR and/or MTX.

Negative zeta potential values were observed for all the colloidal suspensions of nanocapsules (Table 1). Zeta potential analysis allows identifying the electrical charges that occur on the surface of the nanoparticles. Particles are regarded as stable when their zeta potential values are higher than ± 30 mV [30]. Moreover, negative values were achieved for NCs on account of the anionic nature of PCL due to the presence of carboxylic acid functional groups [28]. The statistical analysis showed that mean diameter, PDI and zeta potential were similar for the formulations with or without CUR and/or MTX (*p* > 0.05).
