4.3.2. Effect of Lipid and Surfactant Ratios on PDI, ZP, and EE

The PDI is an indicative of the particle size distribution. According to the literature, a monodisperse sample has PDI values close to 0, while values between 0.1 and 0.3 indicate a narrow size distribution, between 0.1 and 0.4 indicate a moderate size distribution, and greater than 0.4 represent a wide size distribution [32,63]. From the analysis of the Pareto chart (Supplementary Data, Section 3.1, Figure S4D), the surfactants ratio (Tw/Ph) and lipids ratio (SL/LL) were not statistically significant for PDI, although the ratio of lipids showed a more significant effect than the surfactants ratio through the length of the bar. The lipids ratio (SL/LL) showed a linear and positive correlation with PDI, while the surfactants ratio had a negative effect, which means that PDI values are directly related to the lipids ratio. The contour plot (Supplementary Data, Section 3.1, Figure S5D) showed that the values of PDI were higher than 0.24 with the medium lipids ratio (7.94:1.94) and the high surfactants ratio (4.5:0.5), demonstrating a narrow size distribution for the NLC formulation.

ZP reflects the electric potential and the surface charge of nanoparticles in a suspension and is a predicting factor of the long-term stability. When ZP values are higher than |30| mV, the electrostatic repulsion of the attractive Van der Waals forces stops nanoparticle aggregation from occurring [63,92–94]. Lipids arrangements on the surface of the nanoparticle, surfactant surface absorption, and the charge of the encapsulated drug interfere with ZP [32,92]. The Pareto chart showed that the lipids ratio had statistical significance (*p* = 0.05), showing a positive effect on ZP when a quadratic model was used, while the surfactant ratio had a significant negative effect on ZP for the quadratic model (Supplementary Data, Section 3.1, Figure S4E). Thus, ZP was more influenced by the lipids ratio than by the surfactants ratio. Regarding the ZP contour plot (Supplementary Data, Section 3.1, Figure S5E), optimum values were observed in the range of −32 mv and −34 mv, which were obtained with the medium lipids ratio (7.94:1.94) and high surfactants ratio (4.5:0.5).

The high negative ZP values were probably related to the co-surfactant and the drug. Phospholipon® 90G is an amphoteric molecule that acquires a negative charge at the pH level of the NLC formulation, while rivastigmine is a Bronsted base, which when in aqueous dispersion forms a highly negatively charged hydroxyl group [78,95–97].

Regarding EE (Supplementary Data, Section 3.1, Figure S4F), only the lipids ratio (SL/LL) was statically significant (*p* = 0.05), although it negatively decreased as the lipids ratio increased. The contour plot (Supplementary Data, Section 3.1, Figure S5F) revealed that EE was higher than 95% when lipid and surfactant ratios were at medium levels (7.94:1.94 and 4.00:1.00, respectively). This can be explained by the lipophilic nature of rivastigmine, which becomes more solubilized as the lipid concentration increases. In addition, this can also be related to the surfactants, which create more available space between lipids to accommodate rivastigmine molecules [32,63].

From Table 4, it can be concluded that the CCD fitted to the selected CQAs, while the analysis of the Pareto charts (Supplementary Data, Section 3.1, Figure S4) and contour plots (Supplementary Data, Section 3.1, Figure S5) showed that the best lipids ratio was 7.94:1.94 (%, *w*/*w*) and the best surfactants ratio was 4.5:0.5 (%, *w*/*w*). Accordingly, this rivastigmine-loaded NLC formulation was selected for optimization of the instrumental parameters.

**Table 4.** Results of particle size (Z-Ave (mean particle size); volume distribution (50% of particles with size equal or lower to the given value of D50 and 90% of particles with diameter equal or lower to the given value of D90), polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE) tests for the rivastigmine-loaded nanostructured lipid carriers (NLC) selected following the optimization of formulation variables.


<sup>1</sup> DoE (design of experiment); <sup>2</sup> LD (laser diffraction); <sup>3</sup> SD (standard deviation); <sup>4</sup>n (number of runs); <sup>5</sup> DLS (dynamic light scattering); <sup>6</sup> SL/LL (solid lipid: liquid lipid); <sup>7</sup> Tw/Ph (Tween® 80: Phospholipon® 90G); <sup>8</sup> D50 (50% of particles with a diameter size equal or lower to the given values); <sup>9</sup> D90 (90% of particles with a diameter size equal or lower to the given values); <sup>10</sup> Z-Ave (mean particle size); <sup>11</sup> PDI (polydispersity index); <sup>12</sup> ZP (zeta potential); <sup>13</sup> EE (encapsulation efficiency).
