Pt3100

Lungs were placed in 1 ml PBS containing 0.1% (v/v) protease and phosphatase inhibitor cocktail and stored at -80°C until use. Lungs were homogenized on ice in 5 mL CHAPS buffer containing protease and phosphatase inhibitors using a homogenizer (PT3100-POLYTRON, Kinematica, NY). Homogenates were centrifuged at 15,000 rpm for 15 min at 4°C, and the supernatant was stored at -80°C until use. Total protein concentrations were determined using bicinchoninic acid (BCA) assay. Proteins (50 μg) were separated on Novex 4–12% Tris-Glycine gels, transferred onto nitrocellulose membranes, blocked with SuperBlock™ for 2 h at room temperature (RT) and incubated with primary antibodies diluted in TRIS buffered saline pH = 7.4 (TBS) containing 0.1% Tween-20 following manufacturer’s instructions. After washing with TBS-Tween, membranes were incubated with goat anti-rabbit IgG HRP-linked secondary antibody (1:10,000) for 1 h at RT and proteins were visualized by enhanced chemiluminescence.

Based on the obtained pseudo-ternary diagrams, we prepared W/O/W double nanoemulsion using olive oil, Labrasol and ethanol as oil, surfactant, and co-surfactant, respectively. First, methotrexate was mixed in oil at a ratio of 1:2 (w/v). Next, to prepare W/O emulsion, the mixture of oil and S_mix was titrated with DIW at a rate of 1 mL/min under stirring at 500 rpm with a magnetic stirrer (GCMS-G, Global Lab, Siheung-si, Gyeonggi-do, Republic of Korea). Then, the obtained mixture was stirred at 24,000 rpm for 5 min by high speed homogenization (PT-3100, Kinematica AG, Luzern, Switzerland) in an ice bath; we performed this homogenization process a total of three times. Afterward, a fine W/O nanoemulsion was obtained using a microfluidizer (LV1, Microfluidics, Westwood, MA, USA) for three passes. W/O/W double emulsion was prepared by mixing the obtained W/O emulsion and the external water phase with homogenization at 10,000 rpm for 10 min. The preparation conditions of methotrexate-loaded nanoemulsion were optimized through the following tests: (1) surfactant/co-surfactant ratio (v/v) in water-in-oil (W₁/O) emulsion; (2) volume ratio (v/v) of internal water phase to oil phase (W₁:O), and W₁/O emulsion phase to external water phase to external water phase (W₁/O:W₂); (3) Ratio (w/v) of methotrexate:oil in nanoemulsion; (4) number of microfluidizer passes.

O/W emulsions were prepared by homogenizing the 10% (w/w) disperse phase (LM, SB, or CP) with a 90% (w/w) aqueous phase. The disperse phase consisted of either LM pure or blended with CP at weight ratios of 5:5 and with SB at different weight ratios, as follows: 2.5:7.5, 5:5, 7.5:2.5, and 9:1, respectively. Besides, the aqueous phase was comprised of ultrapure water containing 1.0% (w/w) polyoxyethylene (20) sorbitan monooleate (Tween 80) as the emulsifier, and 0.02% (w/w) sodium azide to inhibit microbial proliferation during storage. A coarse emulsion premix was prepared using a high-speed homogenizer (Polytron, PT3100, Kinematica-AG, Luzern, Switzerland) at 7000 rpm for 5 min. Further homogenization was done by passing the coarse emulsion through a high-pressure homogenizer (NanoVater, NV 200, Yoshida Kikai, Nagoya, Japan) at 100 MPa for three passes.