*Article* **Oligonucleotide Delivery across the Caco-2 Monolayer: The Design and Evaluation of Self-Emulsifying Drug Delivery Systems (SEDDS)**

**Jana Kubackova, Ondrej Holas, Jarmila Zbytovska, Barbora Vranikova, Guanghong Zeng, Petr Pavek and Anette Mullertz**


**Abstract:** Oligonucleotides (OND) represent a promising therapeutic approach. However, their instability and low intestinal permeability hamper oral bioavailability. Well-established for oral delivery, self-emulsifying drug delivery systems (SEDDS) can overcome the weakness of other delivery systems such as long-term instability of nanoparticles or complicated formulation processes. Therefore, the present study aims to prepare SEDDS for delivery of a nonspecific fluorescently labeled OND across the intestinal Caco-2 monolayer. The hydrophobic ion pairing of an OND and a cationic lipid served as an effective hydrophobization method using either dimethyldioctadecylammonium bromide (DDAB) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). This strategy allowed a successful loading of OND-cationic lipid complexes into both negatively charged and neutral SEDDS. Subjecting both complex-loaded SEDDS to a nuclease, the negatively charged SEDDS protected about 16% of the complexed OND in contrast to 58% protected by its neutral counterpart. Furthermore, both SEDDS containing permeation-enhancing excipients facilitated delivery of OND across the intestinal Caco-2 cell monolayer. The negatively charged SEDDS showed a more stable permeability profile over 120 min, with a permeability of about 2 <sup>×</sup> <sup>10</sup>−<sup>7</sup> cm/s, unlike neutral SEDDS, which displayed an increasing permeability reaching up to 7 <sup>×</sup> <sup>10</sup>−<sup>7</sup> cm/s. In conclusion, these novel SEDDS-based formulations provide a promising tool for OND protection and delivery across the Caco-2 cell monolayer.

**Keywords:** oligonucleotide; self-emulsifying drug delivery systems; hydrophobic ion pairing; intestinal permeation enhancers; Caco-2 monolayer
