*4.3. In Vitro Performance of the Formulations*

The ability of the Citrem and Standard SEDDS to enhance the permeation of OND was investigated in a transport study across the Caco-2 cell monolayer. A significant increase in OND in the basolateral compartment of the SEDDS-treated cells correlated with a decrease in TEER. No changes in TEER after 30 min in the Citrem SEDDS treated samples led to a stable Papp. The TJs opening by the Standard SEDDS plateaued after 60 min, and the final TEER was reached (Figure 8C,D). This resulted in a significantly enhanced permeation for the Standard SEDDS formulations compared to the respective Citrem SEDDS formulations. Finally, all SEDDS formulations enabled a significantly higher amount of transported OND to the basolateral compartment relative to the OND solution (Figure 9). For the Citrem SEDDS, there was no significant difference among the formulations, while the DDAB, DOTAP-loaded and nonloaded Standard SEDDS dispersed in OND solution showed a superior permeation of OND compared to the complex-loaded Standard SEDDS. Due to the complexation, OND is associated within the dispersed SEDDS and, thus, is not readily available for permeation, unlike the case of the noncomplexed OND dissolved in the dispersion medium. Nevertheless, loading OND complexed with a cationic lipid into SEDDS enables the crucial codelivery with PEs into the site of the action [64]. In addition, specific delivery through the intestinal monolayer provided by the Citrem and Standard SEDDS was confirmed, as no fluorescently labeled OND was localized inside the Caco-2 cells after the two-hour-long uptake study (Figure 10). This observation is in accordance with Li et al., who reported only intercellular localization of a macromolecular loading when it was delivered in MCFA-based formulations. In contrast, the loading was observed perinuclearly when employing a formulation based on long-chain fatty acids [65].

The performed experiments did not offer a conclusive answer regarding the choice of a cationic lipid for OND complexation. Although differing in the minimal amount of lipid needed for the complexation (Table 3), as well as the size of the complexes (Figure 3), no differences in the ability to protect OND (Table 7) and in the in vitro performance were observed (Figure 9).

Utilized SEDDS excipients are generally recognized as safe (GRAS) and/or approved by the European and US Pharmacopeia [37,66,67]. All SEDDS disperse into nanoparticles of a comparable size of about 200 nm, but they differ in zeta potential. The addition of Citrem into SEDDS decreases the surface charge of the formed colloidal nanostructures. The negative surface charge diminishes the interactions between SEDDS droplets and the negatively charged cell membranes of the Caco-2 cell monolayer. Due to the lack of this kind of repulsion between the cell surface and Standard SEDDS, there will be more frequent interactions between the Standard SEDDS and the cell membrane, resulting in membrane disturbance and LDH release (Figure 8B). Simultaneously, the opening of TJs by Citrem SEDDS occurs in a less intense manner. Nevertheless, it is noteworthy that McCartney et al. showed the reversible nature of Labrasol-induced reduction in TEER. The same study also reported that a static system such as the Caco-2 monolayer has a limited repair capacity compared to rat colonic mucosae [62].

Medium-chain triglyceride-based SEDDS confirmed their potential in vivo as they outperformed SEDDS based on long-chain triglycerides for the delivery of insulin [63]. However, generally, the SEDDS in vitro–in vivo correlation is known to not always be satisfactory [19], mainly due to the dynamic in vivo environment of complex intestinal fluids of both endogenous and dietary origin and the challenging crossing of the mucus layer [28]. Pathophysiological inflammatory changes, such as the overexpression of positively charged proteins and highly active phagocytes, create conditions for passive targeting. These changes specific to the inflammatory site might favor the negatively charged SEDDS in terms of enhanced effectivity at the target tissue. On the other hand, the permeation enhancement effect of the neutral Standard SEDDS was shown to be more pronounced in the Caco-2 monolayer. The preferential uptake of negatively charged particles by phagocytic cells [68] might limit the ability of the neutral SEDDS to deliver OND into targeted phagocytes.
