*4.2. Loading the Complexes into Citrem and Standard SEDDS and Evaluation of the Formulations*

Both nonloaded and complex-loaded SEDDS were characterized in terms of size and zeta potential in DI water and MES-HBSS buffer (pH 6.5). The Citrem SEDDS created more uniform nanostructures in terms of size, irrespective of the investigated loading and media. Dispersion of the Standard SEDDS seemed to be more influenced by a loaded substance, as a significant difference in size among the Standard SEDDS formulations was observed (Table 6). However, all SEDDS remained in the range suitable to trigger phagocytosis by macrophages, which are highly active in inflamed intestinal tissue [11,58]. Moreover, in the affected area, positively charged proteins are overexpressed [9]. Thus, negatively charged formulations such as Citrem SEDDS present an attractive approach for local targeting to the diseased tissue.

In DI water, a significant difference in zeta potential between nonloaded and complexloaded SEDDS was observed for both the Citrem and Standard SEDDS. After complex loading, the zeta potential becomes more positive, suggesting the interference of both cationic lipids from the complexes with the surface charge. Given that OND was complexed with cationic lipids in excess, the shift of zeta potential towards more positive values can be expected. Nevertheless, these differences were not observed in the MES-HBSS buffer (pH 6.5). An interplay between ions present in the buffer and decreased pH seemed to mitigate the differences in zeta potential for individual SEDDS.

The Citrem and Standard SEDDS dispersed in FaSSIF were imaged by cryo-TEM (Figure 7). Previous studies imaging dispersed SEDDS by Tran et al. [59] and Fatouros et al. [60] showed oil droplets after SEDDS dispersion, unlike the Citrem and Standard SEDDS, for which vesicular nanostructures prevailed over oil droplets. In comparison to SEDDS characterized in the previous cryo-TEM studies, Citrem and Standard SEDDS contain a higher percentage of lipophilic surfactants. High concentration of lipophilic surface-active molecules can lead to the formation of vesicles, as well as higher complexity and diversity of the vesicular structures.

The majority of the observed nanostructures were between 100 and 300 nm. This range correlates with the size measured by dynamic light scattering (Table 6).

As the impact of the SEDDS excipients on the complex stability was evaluated, a slight but significant decrease in complex stability induced by anionic Citrem was observed (Table 4). However, this slight decrease seems to have translated into a low ability of the Citrem SEDDS to protect OND. In the presence of the S1 nuclease, only 16% of the loaded OND amount was protected against hydrolysis (Table 7). The Standard SEDDS excipients showed no interference with the complex stability, which improved the protection of the loaded OND 3.5-fold (up to 58% of the loaded OND amount). This suggests that there are also other factors in addition to the negatively charged Citrem that lead to complex destabilization.

Both the Citrem and Standard SEDDS disperse into nanodroplets and vesicular structures (Figure 7). Complexed OND is likely to remain inside the nanodroplets; the vesicle formation, however, could impair the complex stability. It would require further examination to track the complex inside the vesicles and to evaluate the impact of vesicular structures on the stability of the complexes. Thus, OND protection could be further improved by optimization of the SEDDS composition.

Upon oral administration, SEDDS excipients undergo digestion by lipases present in the gastrointestinal tract. The impact of gastric lipases, together with low gastric pH, can be circumvented by administering SEDDS preconcentrates in enteric-coated capsules. In the small intestine, the majority of triglycerides are digested by lipases [61]. In order to maintain the dispersed colloidal structures intact and, thus, to prolong the protection of OND, a lipase inhibitor, orlistat, was added to the SEDDS. Only 0.25% (*w*/*w*) of orlistat sufficed to decrease the level of lipolysis in the tested SEDDS to about 10% (Figure 6). This indicates that orlistat incorporation is a potentially useful approach to inhibit SEDDS digestion and to enable the delivery of well-protected OND. Previous studies have shown that the inhibition of lipolysis of a MCFA-based delivery system did not interfere with its permeation-enhancing effect [62,63].
