*3.7. Dynamic In Vitro Lipolysis of SEDDS*

Human intestinal digestion of SEDDS under fasted-state conditions was simulated by dynamic in vitro lipolysis. Lipolysis of the SEDDS was performed with the nonloaded Citrem and Standard SEDDS, as well as both SEDDS with orlistat, in order to evaluate the degree of digestion (Figure 6). The amount of released free fatty acids was the same for both SEDDS. The addition of orlistat significantly decreased lipolysis to ~10% of the formulations with no orlistat (\*\*\* *p* < 0.001). No significant difference was observed between the Citrem and Standard SEDDS upon the addition of orlistat. Moreover, lipolysis of DDAB in SEDDS and DOTAP in SEDDS was performed. None of cationic lipids had any impact on the digestion process (data not shown).

loaded SEDDS. Results are presented as mean ± SD (*n* = 3).

Human intestinal digestion of SEDDS under fasted-state conditions was simulated by dynamic in vitro lipolysis. Lipolysis of the SEDDS was performed with the nonloaded Citrem and Standard SEDDS, as well as both SEDDS with orlistat, in order to evaluate the degree of digestion (Figure 6). The amount of released free fatty acids was the same for both SEDDS. The addition of orlistat significantly decreased lipolysis to ~10% of the formulations with no orlistat (\*\*\* *p* < 0.001). No significant difference was observed between

DOTAP-OND 240 ± 9 \* 183 ± 27 0.23 0.25 14.0 ± 0.9 ### 0.3 ± 0.3

\* *p* < 0.05 statistical difference relative to nonloaded Standard SEDDS in DI water, \*\*\* *p* < 0.001 statistical difference relative to nonloaded Standard SEDDS in MES-HBSS buffer and ### *p* < 0.001 statistical difference relative to the respective non-

*3.7. Dynamic In Vitro Lipolysis of SEDDS* 

impact on the digestion process (data not shown).

**Figure 6.** In vitro profile of digestion of SEDDS in fasted-state simulated intestinal fluid presented as the amount of free fatty acids titrated by sodium hydroxide over 60 min. Data are presented as mean ± SD (*n* = 3). \*\*\* *p* < 0.001—significant effect of orlistat on SEDDS lipolysis. **Figure 6.** In vitro profile of digestion of SEDDS in fasted-state simulated intestinal fluid presented as the amount of free fatty acids titrated by sodium hydroxide over 60 min. Data are presented as mean ± SD (*n* = 3). \*\*\* *p* < 0.001—significant effect of orlistat on SEDDS lipolysis.

All formulations dispersed into nanosized structures. Predominantly, spherical uni-

#### *3.8. Cryo-TEM 3.8. Cryo-TEM*

SEDDS were dispersed in FaSSIF at 37 °C in the ratio 1:100 (*v*/*v*). The final concentration of OND in dispersion of loaded SEDDS was 1 nmol/mL. In total, 69 images were evaluated, with one representative image per formulation presented in Figure 7. Figure 7A–C depicts the dispersed Citrem SEDDS, nonloaded, DDAB-OND and DOTAP-ONDloaded, respectively. Figure 7D–F shows the Standard SEDDS, nonloaded, DDAB-OND and DOTAP-OND-loaded, respectively. SEDDS were dispersed in FaSSIF at 37 ◦C in the ratio 1:100 (*v*/*v*). The final concentration of OND in dispersion of loaded SEDDS was 1 nmol/mL. In total, 69 images were evaluated, with one representative image per formulation presented in Figure 7. Figure 7A–C depicts the dispersed Citrem SEDDS, nonloaded, DDAB-OND and DOTAP-OND-loaded, respectively. Figure 7D–F shows the Standard SEDDS, nonloaded, DDAB-OND and DOTAP-ONDloaded, respectively. *Pharmaceutics* **2021**, *13*, x 17 of 27

**Figure 7.** Cryo-TEM images of SEDDS, dilution 1:100 (*v*/*v*) in fasted-state simulated intestinal fluid (FaSSIF). (**A**) Nonloaded Citrem SEDDS, (**B**) DDAB-OND-loaded Citrem SEDDS (100 nmol/g), (**C**) DOTAP-OND-loaded Citrem SEDDS (100 nmol/g), (**D**) nonloaded Standard SEDDS, (**E**) DDAB-OND-loaded Standard SEDDS (100 nmol/g) and (**F**) DOTAP-OND-loaded Citrem SEDDS (100 nmol/g). Full arrows indicate uni- and multilamellar vesicles, dashed arrows indicate more complex vesicular structures and open arrows indicate oil droplets. **Figure 7.** Cryo-TEM images of SEDDS, dilution 1:100 (*v*/*v*) in fasted-state simulated intestinal fluid (FaSSIF). (**A**) Nonloaded Citrem SEDDS, (**B**) DDAB-OND-loaded Citrem SEDDS (100 nmol/g), (**C**) DOTAP-OND-loaded Citrem SEDDS (100 nmol/g), (**D**) nonloaded Standard SEDDS, (**E**) DDAB-OND-loaded Standard SEDDS (100 nmol/g) and (**F**) DOTAP-OND-loaded Citrem SEDDS (100 nmol/g). Full arrows indicate uni- and multilamellar vesicles, dashed arrows indicate more complex vesicular structures and open arrows indicate oil droplets.

> *3.9. Protection Against Nuclease Degradation*  The S1 nuclease completely degraded OND in an aqueous solution of naked OND (data not shown). Dispersions of both nonloaded SEDDS in the aqueous solution of naked OND showed the same pattern. This confirmed that neither the composition of Citrem All formulations dispersed into nanosized structures. Predominantly, spherical uniand multilamellar vesicles (darker and more pronounced borders) were observed, while oil droplets occurred less frequently (darker spheres with brighter borders). Wu et al. described similar differences between vesicles and oil droplets in a soybean oil-in-water

> > **DDAB-OND in SEDDS DOTAP-OND in SEDDS**

**Table 7.** Percentage of OND protected from degradation after 30 min of incubation in the presence

Citrem 16.0 ± 1.5 15.7 ± 1.0 Standard 59.9 ± 3.4 \*\*\* 57.1 ± 3.0 \*\*\* \*\*\* *p* < 0.001 is the statistically significant difference between Citrem and Standard SEDDS. Data

**SEDDS Intact OND (%)** 

OND in any of the SEDDS.

are presented as mean ± SD (*n* = 3).

of the S1 nuclease.

If OND becomes cleaved by a nuclease, short OND fragments are detected in the supernatant that do not precipitate into pellets. The amount of OND that remain intact upon contact with the S1 nuclease was detected in the pellets and is shown in Table 7. There was a significant difference in the amount of OND protected by the Citrem and Standard SEDDS, as the Standard SEDDS showed about 3.5-fold higher protection (*p* < 0.001). On the other hand, no difference was observed between DDAB-OND and DOTAP-

emulsion stabilized by egg yolk lecithin [50]. The nanostructures varied in size from 100 to 300 nm. In addition, cryo-TEM detected more complex spherical and elongated vesicular structures (such as in Figure 5A,B,D), usually of larger sizes (>300 nm). No apparent differences between the six different SEDDS could be observed.
