*3.6. Characterization of Dispersed SEDDS*

Both Citrem and Standard SEDDS preconcentrates were clear oily formulations that immediately dispersed into milky emulsions both in DI water and MES-HBSS buffer (pH 6.5). The dispersion in DI water was evaluated in order to assess properties of the formulations and in MES-HBSS to mimic the pH, as well as a more complex environment of in vitro testing media, as shown in Table 6.


**Table 6.** Size and zeta potential of Citrem and Standard SEDDS. The parameters were tested in deionized (DI) water and in 2-(N-morpholino)ethansulfonic acid-Hanks' Balanced Salt Solution (MES-HBSS) (pH 6.5). PdI: polydispersity index.

\* *p* < 0.05 statistical difference relative to nonloaded Standard SEDDS in DI water, \*\*\* *p* < 0.001 statistical difference relative to nonloaded Standard SEDDS in MES-HBSS buffer and ### *p* < 0.001 statistical difference relative to the respective nonloaded SEDDS. Results are presented as mean ± SD (*n* = 3).

> The size (z-average) was found between 180 and 270 nm for both SEDDS in both investigated media, with the PdI ranging from 0.125 to 0.360. In the case of the Citrem SEDDS, there was no significant difference in size across the formulations and media. The Standard SEDDS loaded with DOTAP-OND formed significantly larger nanostructures in the DI water than nonloaded Standard SEDDS (\* *p* < 0.05). In MES-HBSS buffer, a similar phenomenon was observed; in this case, the DDAB-OND-loaded Standard SEDDS created larger structures than the nonloaded formulation (\*\*\* *p* < 0.001).

> The zeta potential was significantly influenced by the composition of SEDDS and the dispersing media. In DI water, both nonloaded SEDDS showed a negative surface charge: −35.5 ± 0.6 and −5.2 ± 2.1 mV for Citrem and Standard SEDDS, respectively. After loading, the surface charge of the Citrem SEDDS increased to about <sup>−</sup>25 mV (### *<sup>p</sup>* < 0.001) and turned positive in the loaded Standard SEDDS to about 13 mV (### *p* < 0.001). Both complexes increased the zeta potential of the nonloaded Citrem and Standard SEDDS to the same extent. The Citrem and Standard SEDDS in MES-HBSS buffer (pH 6.5) showed no significant differences within the same kind of SEDDS among the nonloaded and loaded SEDDS; about −9.5 and 0 mV for Citrem and Standard SEDDS, respectively.
