2.3. Preparation and characterization of ELNE

The physical complex ETP/LMC was prepared to enhance the solubility of ETP in water. First, LMC was prepared by enzymatic degradation of methylcellulose, as described previously (Chung et al., 2018). To prepare the ETP/LMC complex, ETP and LMC were dissolved in hexafluro-2-propanol at a concentration of 100 mg/mL with feed weight ratios of ETP to LMC of 1:1, 1:5, and 1:10, respectively. The admixture of ETP and LMC was then evaporated at 37 °C using a rotary evaporator, forming a thin film in a round-bottom flask. Subsequently, the thin film was dispersed in 3 mL distilled water at 50 °C with continuous sonication for 10 minutes. The ETP/LMC solution was then incubated at −4 °C for 30 minutes and filtered through a 0.22-µm polyvinylidene difluoride filter. Finally, the filtered solution was freeze-dried to obtain a powdered form of the ETP/LMC complex.

To improve the intestinal permeability of ETP via bile acid transporter-mediated uptake, the DCK–PA complex was prepared as follows. First, DCK was synthesized by conjugating deoxycholic acid to a positively charged lysine, as described previously (Pangeni et al., 2016). Then, 100 mg PA was dispersed in 20 mL water, vortex mixed, and sonicated for 20 minutes. Subsequently, 114.6 mg DCK was completely dissolved in water and then added gradually in a dropwise manner to the PA dispersion with continuous stirring at a 1:1 molar ratio. The DCK–PA dispersion was freeze-dried at −70 °C to obtain the DCK–PA complex in powder form.

Next, the ETP/LMC complex was incorporated into the multiple w/o/w nanoemulsion, with or without DCK–PA complex. A two-step spontaneous emulsification method was used to prepare w/o/w nanoemulsion based on the pseudo-ternary phase diagram (Supporting Information, Figure S1). First, the primary w/o nanoemulsion was prepared. Briefly, ETP/LMC was dispersed in water followed by oil phase titration using Capryol 90, Labrasol, and Transcutol HP as the aqueous, oil, surfactant, and co-surfactant phases, respectively. The primary nanoemulsion was comprised of a 25.9% (w/w) aqueous solution of ETP/LMC, 68.3% surfactant/co-surfactant mixture (S_mix; 1:2), and 5.80% oil phase (Supporting Information, Table S1). Second, the w/o/w ETP/LMC was prepared by aqueous titration using the primary nanoemulsion, Tween 80, and deionized water as the secondary oil, surfactant, and aqueous phases, respectively. The final composition of w/o/w nanoemulsion was as follows: 70.0% (w/w) w/o nanoemulsion (oil phase), 9.69% (w/w) surfactant, and 19.3% (w/w) deionized water (Supporting Information, Table S1) (Pangeni et al., 2016).

The average droplet size, polydispersity index (PDI), and zeta potential of ELNE were determined using a dynamic laser light scattering analyzer (Malvern Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK). Morphological evaluation of the selected ELNE was performed by transmission electron microscopy (JEM-200; JEOL, Tokyo, Japan).