*3.12. Uptake Study*

The uptake of the fluorescently labeled OND formulated into SEDDS was examined in confluent Caco-2 cells using fluorescent microscopy. Figure 10 shows merged images: blue Hoechst staining cell nuclei, red PI staining dead cells and green FAM-labeled OND. Images depicting staining with a single dye can be found in the Supplementary Materials (Figure S1).

signal indicating that OND was localized only inside dead Caco-2 cells.

**Figure 10.** Fluorescence microscope images of fluorescently labeled oligonucleotide (OND) (**A**) MES-HBSS buffer, (**B**) OND solution in MES-HBSS buffer, (**C**,**D**) nonloaded Citrem and Standard SEDDS dispersed in OND solution, respectively, (**E**,**F**) DDAB-OND loaded in Citrem and Standard SEDDS, respectively, and (**G**,**H**) DOTAP-OND loaded in Citrem and Standard SEDDS, respectively. The size bar represents 100 µm. Blue represents the cell nuclei of living cells stained by Hoechst dye, red represents dead cells stained by propidium iodide and green represents fluorescently labeled OND. **Figure 10.** Fluorescence microscope images of fluorescently labeled oligonucleotide (OND) (**A**) MES-HBSS buffer, (**B**) OND solution in MES-HBSS buffer, (**C**,**D**) nonloaded Citrem and Standard SEDDS dispersed in OND solution, respectively, (**E**,**F**) DDAB-OND loaded in Citrem and Standard SEDDS, respectively, and (**G**,**H**) DOTAP-OND loaded in Citrem and Standard SEDDS, respectively. The size bar represents 100 µm. Blue represents the cell nuclei of living cells stained by Hoechst dye, red represents dead cells stained by propidium iodide and green represents fluorescently labeled OND.

> **4. Discussion**  *4.1. Preparation of Cationic Lipid-OND Complexes*  Cationic lipid-DNA complexes have been shown to be a less immunogenic but, also, a less efficient alternative to viral vectors for the delivery of nucleic acids [4]. These macromolecules are often formulated as lipoplexes with preformed liposomes to enhance the cell transfection of large DNA molecules (several kb) [51], as well as shorter siRNA (tenths Similar to the OND solution (Figure 10B), no OND was observed in living Caco-2 cells upon incubation with the Citrem or Standard SEDDS or when OND was administrated in the aqueous phase (nonloaded SEDDS; Figure 10C,D) or complexed inside the SEDDS (DDAB-OND and DOTAP-OND; Figure 10E,F and Figure 10G,H, respectively). The green signal of FAM-labeled OND was observed only in combination with the red signal indicating that OND was localized only inside dead Caco-2 cells.

The uptake of the fluorescently labeled OND formulated into SEDDS was examined in confluent Caco-2 cells using fluorescent microscopy. Figure 10 shows merged images: blue Hoechst staining cell nuclei, red PI staining dead cells and green FAM-labeled OND. Images depicting staining with a single dye can be found in the Supplementary Materials

Similar to the OND solution (Figure 10B), no OND was observed in living Caco-2 cells upon incubation with the Citrem or Standard SEDDS or when OND was administrated in the aqueous phase (nonloaded SEDDS; Figure 10C,D) or complexed inside the SEDDS (DDAB-OND and DOTAP-OND; Figure 10E,F and Figure 10G,H, respectively). The green signal of FAM-labeled OND was observed only in combination with the red

#### of bp) [52], for parenteral delivery to the systemic circulation. In this study, we prepared **4. Discussion**

(Figure S1).

#### hydrophobic complexes consisting of cationic lipids and OND in order to load them into *4.1. Preparation of Cationic Lipid-OND Complexes*

SEDDS, a well-established lipid-based oral delivery system. The presented experiments were conducted with a model nonspecific fluorescently labeled OND. Nevertheless, the structural features involved in the key procedures, such as the preparation of ion pairs with cationic lipids and, thus, hydrophilicity reduction, are applicable for potential therapeutic OND sequences. Therefore, the easily detectable fluorescently labeled model OND was used to investigate this innovative formulation process. Monovalent cationic lipids with two lipophilic chains, DDAB and DOTAP, are known to be less toxic than their single-tailed counterparts [53]. Both aliphatic chains of DDAB are saturated, unlike the chains of DOTAP, which have a double bond in the C9 Cationic lipid-DNA complexes have been shown to be a less immunogenic but, also, a less efficient alternative to viral vectors for the delivery of nucleic acids [4]. These macromolecules are often formulated as lipoplexes with preformed liposomes to enhance the cell transfection of large DNA molecules (several kb) [51], as well as shorter siRNA (tenths of bp) [52], for parenteral delivery to the systemic circulation. In this study, we prepared hydrophobic complexes consisting of cationic lipids and OND in order to load them into SEDDS, a well-established lipid-based oral delivery system. The presented experiments were conducted with a model nonspecific fluorescently labeled OND. Nevertheless, the structural features involved in the key procedures, such as the preparation of ion pairs with cationic lipids and, thus, hydrophilicity reduction, are applicable for potential therapeutic OND sequences. Therefore, the easily detectable fluorescently labeled model OND was used to investigate this innovative formulation process.

Monovalent cationic lipids with two lipophilic chains, DDAB and DOTAP, are known to be less toxic than their single-tailed counterparts [53]. Both aliphatic chains of DDAB are saturated, unlike the chains of DOTAP, which have a double bond in the C9 position. This influences their physicochemical properties; DDAB exists in the gel state and DOTAP in the liquid crystalline state at room temperature [54]. In the present study, it was found that the more flexible DOTAP chains seem to be more easily assembled around the OND core, leading to CE > 95% already at the charge ratio 2:1 (N<sup>+</sup> :PO<sup>2</sup> <sup>−</sup>). At the ratio 3:1 (N<sup>+</sup> :PO<sup>2</sup> −), there was no significant difference between the CE of the cationic lipids (Table 3). At all employed ratios, no lipid was observed at the interface during the Bligh–Dyer extraction, suggesting

that most of the lipid contributed to the complexation. The ability of DDAB to create a more ordered structure was shown also by DSC, as several melting peaks of crystalline assembles were observed (Figure 4). The more ordered structure and, consequently, tighter molecular packing could explain the smaller size of the DDAB-OND complexes, as detected by AFM (Figure 3).

The formation of complexes between a cationic lipid added as a monomer or in micellar form and plasmid DNA (pDNA) has already been described and well-characterized [23,55]. In both published preparation methods, a sufficient recovery of pDNA was reported at the charge ratio 1:1. The recovery process was more efficient (>90%) if a lipid was added in excess of 1.5:1 (N<sup>+</sup> :PO<sup>2</sup> −) [55]. For shorter siRNA, the need of higher lipid/OND ratio was reported [52,56]. It is widely accepted that the mechanism of complex formation is common for any type of nucleic acid and cationic lipid [57]. In other words, both components of the complex self-assemble into a core–shell structure based on the ion pairing—more specifically, primarily on electrostatic interactions between the cationic headgroup of a lipid and anionic phosphate of the nucleic acid backbone.

The ATR-FTIR analysis (Figure 5 and Table 5) showed complex-specific bands that occurred mostly in the region characteristic of changes in the phosphate-deoxyribose backbone and confirmed the interactions with the OND backbone in the complex formation process. These interactions could not be observed in physical mixtures prepared with the same cationic lipid:OND ratio. Previous studies showed that electrostatic interactions help to form inverted micelles, encapsulating the nucleic acid in the core surrounded by the shell of the cationic lipid [23]. This is in agreement with the AFM topography images showing collapsed lipid shells around the cores, as observed in both the DDAB and DOTAP complexes (Figure 3).
