D 78224

Emulsion droplets were prepared by dissolving 10 mg of DPPC in 1 mL of chloroform. The solution was evaporated onto the surface of a round-bottomed flask at 50 °C for 15 min. The procedure was conducted under a vacuum using a rotary evaporator (rotovap). The dried film was hydrated by introducing 1.2 mL of PBS (phosphate-buffered saline). 0.6 mL of PFC₅ (perfluoro pentane) was added to the solution. The assembly was rotated on an iced bath for 20 min and subsequently sonicated through pulses of 30 s in an active state, followed by a 1-min pause interval between each sonication cycle. Sonication was done using a 40-kHz sonicator bath (Elma D-78224, Melrose Park, IL, USA). The size of nanoemulsion droplets was reduced by extruding through polycarbonate membrane filters with a pore size of 0.05 μm (Hamilton, Reno, NV) [109 (link),125 (link)]. Fig. 8 Provides a schematic representation of the experimental setup and preparation for nanoemulsion droplets.Fig. 8

Comprehensive schematic representation illustrating the experimental setup and steps involved in the preparation of nanoemulsion droplets.

Fig. 8

The types of equipment used in this study were a gas chromatography-mass spectrometry (GC/MS; Spectra lab Agilent‐7890, Markham, ON, Canada) instrument, an Ultrasound sonicator (Elma D-78224 Singen/htw, Germany), a rotary evaporator (Buchi rotovapor, R‐124 digital, New Castle, DE, USA), Elisa microplate reader (BioBase EL-10A, China). All organic solvents and chemicals used during the study were of analytical grade. Water, when used, was distilled using distillation apparatus.

Liposomes were prepared using the thin-film hydration method⁴⁶ . Briefly, liposomes were prepared using cholesterol, DPPC, and DSPE-PEG(2000)-NH₂ at molar ratios of 30:65:5, respectively. The lipids were dissolved in 4 ml chloroform in a round-bottom flask. The chloroform was then evaporated using a rotary evaporator under vacuum at 50 °C for 15 min until a thin film was observed on the walls. Next, the lipid film was hydrated using 2 ml of a 50-mM calcein solution, and the pH was adjusted to 7.4. To obtain unilamellar vesicles, the solution was sonicated for 2 min using a 40-kHz sonication bath (Elma D-78224, Melrose Park, IL, USA). Whereas for particle size reduction, liposomes were extruded thirty times using 200-nm polycarbonate filters (Avanti Polar Lipids, Inc., Alabaster, AL, USA)^{30 (link),31 (link),41 (link),47} . The purification of the formulation involved gel exclusion chromatography using a Sephadex G-100 column (after equilibrating it with PBS (pH 7.4)). Finally, the collected fractions were stored at 4 °C. The encapsulated calcein concentration was estimated using a calibration curve of the fluorescence intensity versus different concentrations of calcein dissolved in PBS (pH 7.4). Calcein is self-quenched at high concentrations showing no fluorescent properties. At lower concentrations, calcein does not exhibit self-quenching.