Sfx150

Hippocampus and cortical regions were lysed for protein extraction by utilizing the BioPlex Cell Lysis Kit (BIO-RAD, CA, USA) with modifications. Briefly, 500 μl of complete cell lysis buffer was added to frozen tissue and mechanically homogenized (Kinematica Polytron 1300D, Luzern, CHE). The homogenates were then stored at −80 °C overnight. Frozen homogenates were then allowed to thaw on ice and immediately sonicated (Branson SFX 150, CT, USA), and centrifuged for 10 min at 10,000 × g, followed by supernatant collection. Protein concentrations were measured using the Pierce Bicinchoninic Acid Assay Kit (Thermo Fisher Scientific, MA, USA) with minor modifications. Protein lysates were diluted to 1000 ng/μl with a complete cell lysis buffer and stored at −80 °C until further processing.

SLNs loaded with PEA (PEA-SLNs) were prepared by the melt emulsification technique [33 (link),34 (link),35 (link),36 (link)]. PEA (15 mg) was dissolved in a stearic acid (40 mg)/cholesteryl stearate (30 mg) blend containing Span 85 (30 mg) melted at a temperature of 85 °C. Then, the aqueous phase (5 mL Milli-Q water) containing 0.3% Pluronic F68 was heated at the same temperature and added to the lipid phase. The emulsification was performed by ultrasounds (SFX150 Branson, Milan, Italy) for 1 min, followed by homogenization by Ultra-Turrax (T-25 basic, Ika Labortechnik, Staufen im Breisgau, Germany) at 24,000 rpm for 1.5 min and sonication for 1 min. The obtained oil-in-water emulsion was cooled in an ice bath under magnetic stirring for 15 min and then purified by dialysis membrane (MWCO 12–14,000 Da) for 1 h in 300 mL Milli-Q water.
The same procedure was used to prepare unloaded SLNs (U-SLNs). Labeled SLNs for the cell internalization study were obtained by adding 0.01% w/w Nile Red in the melted stearic acid and following the same method described above for both unloaded (U-SLNs-NR) and PEA-loaded (PEA-SLNs-NR) SLNs.