4.3.1. Effect of Lipids and Surfactants Ratio on Particle Size (Z-Ave, D50, and D90)

Table 3 shows that the predicted and observed values were very close, indicating the suitability of the design for the selected CQAs.

Predicted vs. observed plots showing the suitability of the model for the applied design, a Pareto chart of standardized effects studying the interaction effects of CMAs for each dependent response, and contour plots representing the interactions between CMAs and dependent responses were used for further analysis.

From the analysis of the Pareto chart (Supplementary Data, Section 3.1, Figure S4A), the surfactant ratio (Tw/Ph) and lipids ratio (SL/LL) were significant factors for particle size (Z-Ave) (*p* = 0.005), showing a negative effect, which means that particle size increased with increasing ratios of SL/LL and Tw/Ph. In contrast, for the dependent response D50 (Supplementary data, Figure S4B), Tw/Ph had a positive effect and the most statistically significant effect (*p* = 0.05) when a linear model was used, while SL/LL did not show a statistically significant effect. Regarding D90 (Supplementary data, Section 3.1, Figure S4C), it was observed that all independent variables had statistical significance (*p* = 0.05), showing positive effects on size when a linear model was applied, indicating direct relationships.



<sup>1</sup> Z-Ave (mean particle size); <sup>2</sup> volume distribution (50% of particles with size equal or lower to the given value of D50) and 90% of particles with size equal or lower to the given value of D90)); <sup>3</sup> PDI (polydispersity index); <sup>4</sup> ZP (zeta potential); <sup>5</sup> EE (encapsulation efficiency) <sup>6</sup> O (observed results); <sup>7</sup> P (predicted results).

From the observation of the contour plots (Supplementary data, Section 3.1, Figure S5A–C), the smallest NLCs (Z-Ave = 148–128 nm, D50 = 46–56 nm, D90 = 110–160 nm) were found with the medium lipids ratio (7.94:1.94) and high surfactants ratio (4.5:0.5). These results can be explained by the fact that smaller particle sizes are generally obtained when higher concentrations of surfactants are used [46]. It has been described that higher surfactant concentrations ensure NLC stability, decreasing the surface tension and preventing aggregation and crystallization of lipids during storage, in turn avoiding the increased nanoparticle size [91].
