*3.10. Caco-2 Cell Monolayer Transport Study*

Several parameters of the prepared formulations were investigated in vitro in the Caco-2 monolayer. As an indicator of permeation enhancement, the TEER values were determined by an Endohm chamber at 25 ◦C before and after the experiment. After 120 min, the TEER decreased significantly if incubated with the Citrem or Standard SEDDS. No changes occurred after incubation with OND solution or the MES-HBSS buffer serving as the control. There were no significant changes among the formulations based on the Citrem SEDDS nor Standard SEDDS, indicating that the TEER changes can be attributed to the two SEDDS. Nevertheless, the extent to which the two SEDDS decreased the TEER differed significantly. The final TEER at 120 min of the Citrem SEDDS and Standard SEDDStreated wells represented 66% ± 10% and 28% ± 4% of the initial TEER values, respectively (*p* < 0.01) (Figure 8A). In addition, the TEER values seem to be influenced differently by the Citrem and the Standard SEDDS.

**Formulation** 

**Figure 8.** (**A**) Percent of initial transepithelial electrical resistance (TEER) measured by Endohm chamber after 120 min of incubation in both Citrem and Standard SEDDS. \*\* denotes a significant difference of TEER decline (*p* < 0.01) between the Citrem and Standard SEDDS. All SEDDS-incubated cells showed significantly lower TEER relative to the respective CTRL (buffer) and OND solution. (**B**) Percent of viability by lactate dehydrogenase (LDH) assay after 120 min of incubation with Citrem and Standard SEDDS. \* and \*\* denote significant differences in the viability between different SEDDS (\* *p* <0.05 and \*\* *p* < 0.01). (**C**,**D**) Relative TEER values measured by chopstick electrodes at 37 °C for Citrem SEDDS (**C**) and Standard SEDDS (**D**). (**D**) ### represents a significant difference of the relative TEER values of SEDDS-treated cells relative to the CTRL and OND solution (*p* < 0.001). Results are presented as mean ± SD (*n* = 5 to 6). **Figure 8.** (**A**) Percent of initial transepithelial electrical resistance (TEER) measured by Endohm chamber after 120 min of incubation in both Citrem and Standard SEDDS. \*\* denotes a significant difference of TEER decline (*p* < 0.01) between the Citrem and Standard SEDDS. All SEDDS-incubated cells showed significantly lower TEER relative to the respective CTRL (buffer) and OND solution. (**B**) Percent of viability by lactate dehydrogenase (LDH) assay after 120 min of incubation with Citrem and Standard SEDDS. \* and \*\* denote significant differences in the viability between different SEDDS (\* *p* < 0.05 and \*\* *p* < 0.01). (**C**,**D**) Relative TEER values measured by chopstick electrodes at 37 ◦C for Citrem SEDDS (**C**) and Standard SEDDS (**D**). (**D**) ### represents a significant difference of the relative TEER values of SEDDS-treated cells relative to the CTRL and OND solution (*p* < 0.001). Results are presented as mean ± SD (*n* = 5 to 6).

**Table 8.** Transported OND accumulated in the basolateral compartment after 120 min presented as the total mass (pmol) and % of the initial apical mass. **Citrem SEDDS Standard SEDDS Transported OND Accumulated Basolaterally at 120 min Transported OND Accumulated Basolaterally at 120 min pmol % pmol %**  This aspect was further evaluated by chopstick electrodes throughout the experiment at 37 ◦C starting from 30 min of the experiment and related to this time point. The cells incubated with the Citrem SEDDS had already reached the final TEER values by this time point, with the values remaining unchanged until the end of the experiment. This contrasts with the Standard SEDDS, in which TEER after 60 min reached a plateau at 80% of the TEER at 30 min (Figure 8C,D).

OND solution 0.15 ± 0.16 0.03 0.21 ± 0.06 0.04 DDAB-OND in SEDDS 0.45 ± 0.10 \* 0.09 1.37 ± 0.21 \*\* 0.28 DOTAP-OND in SEDDS 0.70 ± 0.23 \*\*\* 0.14 1.72 ± 0.06 \*\*\* 0.34 DDAB in SEDDS 0.91 ± 0.31 \*\* 0.18 2.40 ± 0.53 \*\* 0.48 DOTAP in SEDDS 0.88 ± 0.41 \*\* 0.17 2.43 ± 0.25 \*\*\* 0.49 Nonloaded SEDDS 1.19 ± 0.55 \*\* 0.24 2.32 ± 0.26 \*\*\* 0.46 Results are presented as mean ± SD (*n* = 5 to 6). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 = statistically significant differences Figure 9A,B shows the cumulative OND transport into the basolateral compartment. At 120 min, both SEDDS displayed a significant increase of transported OND compared to the OND solution (Citrem SEDDS # *p* < 0.05 and Standard SEDDS ## *p* < 0.01 and ### *p* < 0.001). In the case of Citrem SEDDS (Figure 9A), no difference was observed between the two complexes. For the Standard SEDDS (Figure 9B), DDAB and DOTAP in the SEDDS and, also, the nonloaded SEDDS enabled the transport of higher amounts of OND than DDAB-OND and DOTAP-OND in SEDDS over 120 min (\* *p* < 0.05). The total transported mass of OND after 120 min is summarized in Table 8.

in transported OND formulated into SEDDS in comparison to the OND solution.

**Figure 9.** The transported OND accumulated in the basolateral compartment for Citrem SEDDS (**A**) and Standard SEDDS (**B**). The apparent permeability coefficient (Papp) values for specified time intervals in the Citrem SEDDS (**C**) and Standard SEDDS (**D**). (**A**,**B**) #, ## and ### represent significant differences of the flux curves of SEDDS-treated cells relative to the OND solution (# *p* <0.05, ## *p* < 0.01 and ### *p* < 0.001). (**B**) \* represents a significant difference between complex-loaded Standard SEDDS and a group of other Standard SEDDS formulations (\* *p* < 0.05). (**C**,**D**) Bars marked by # showed a significant difference in the Papp relative to the respective bars of the OND solution (*p* < 0.05). (**D**) The significant differences between the time intervals in the Standard SEDDS is denoted as \* *p* < 0.05. The results are presented as mean ± SD (*n* = 5 to 6). **Figure 9.** The transported OND accumulated in the basolateral compartment for Citrem SEDDS (**A**) and Standard SEDDS (**B**). The apparent permeability coefficient (Papp) values for specified time intervals in the Citrem SEDDS (**C**) and Standard SEDDS (**D**). (**A**,**B**) # , ## and ### represent significant differences of the flux curves of SEDDS-treated cells relative to the OND solution (# *p* <0.05, ## *p* < 0.01 and ### *p* < 0.001). (**B**) \* represents a significant difference between complex-loaded Standard SEDDS and a group of other Standard SEDDS formulations (\* *p* < 0.05). (**C**,**D**) Bars marked by # showed a significant difference in the Papp relative to the respective bars of the OND solution (*p* < 0.05). (**D**) The significant differences between the time intervals in the Standard SEDDS is denoted as \* *p* < 0.05. The results are presented as mean ± SD (*n* = 5 to 6).

The viability of the Caco-2 cells was evaluated by an LDH assay after 120 min of treatment with all SEDDS (Figure 8B). The OND solution was confirmed to be nontoxic. For both the Citrem and Standard SEDDS, there were no significant differences between the applied SEDDS formulations, which suggests that the main contribution to the cytotoxicity is from the surfactants in SEDDS and not the cationic lipids or the complexes. All Citrem SEDDS had viability values close to 100%, while the Standard SEDDS had a decreased viability to about 70% (\* *p* < 0.05 and \*\* *p* < 0.01). These data might indicate in-

*3.11. In Vitro Toxicity Study* 

*3.12. Uptake Study* 

creased cell stress due to compromised integrity of the cell membrane.


**Table 8.** Transported OND accumulated in the basolateral compartment after 120 min presented as the total mass (pmol) and % of the initial apical mass.

Results are presented as mean ± SD (*n* = 5 to 6). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001 = statistically significant differences in transported OND formulated into SEDDS in comparison to the OND solution.

> A linear steady state of the flux curves was taken as the basis for the calculation of Papp. In order to investigate in detail changes in permeability across the Caco-2 monolayer during 120 min, the time frame of the experiment was divided into three intervals, i.e., ∆15–45 min, ∆30–60 min and ∆60–120 min, with the Papp values calculated for each interval. As can be seen in Figure 9C, the Papp was stable for all Citrem SEDDS formulations at all time intervals. OND solutions containing dispersions of the Citrem SEDDS with or without DDAB or DOTAP already showed a significant increase at the interval of ∆30–60 min relative to the OND solution. At the final interval of ∆60–120 min, all Citrem SEDDS significantly improved the permeability of OND relative to the respective time intervals of the OND solution (*p* < 0.05). In contrast, for the Standard SEDDS, the Papp increased over time for all formulations (*p* < 0.05) (Figure 9D). Only the OND solution with dispersions of DDAB and DOTAP in the Standard SEDDS already increased the Papp at the initial time interval ∆15–45 min. At the following interval of ∆30–60 min, the Papp of DOTAP-OND in the Standard SEDDS and the nonloaded Standard SEDDS was also enhanced significantly. Finally, at the interval of ∆60–120 min, all formulations of the Standard SEDDS showed an increase in Papp relative to the OND solution.
