Elyra 7 els

Briefly, hCMEC/D3 cells were seeded (45,000 cells/ml) one day before the experiment. The following day, cells were overnight incubated with Cy5-labeled BTL (empty) to a final concentration of 2.5 mM total lipids, corresponding to 2.20×10¹³ liposomes/ml. Subsequently, the culture medium was removed, and cells were thrice rinsed with PBS and fixed with cold 4% paraformaldehyde (PFA) for 10 min. The cells were then permeabilized (0.1% Triton X100 for 5 min), blocked (10% FBS in 0.05% Tween20 in PBS for 30 min), and stained with Anti-Transferrin Receptor Antibody (ab84036; Abcam) at 5 μg/ml in blocking serum for 1 h. Next, cells were stained with Goat Anti-Rabbit IgG H&L conjugated Alexa Fluor 488 (ab150077; Abcam) at a dilution of 1:1000 in blocking serum for 1 h and then thrice rinsed with PBS (Figure S9). Acquisition and processing were performed using super-resolution (SR) microscopy (Elyra 7 eLS, Zeiss, Germany) and ZEN software, applying 405-, 488-, 561-, and 642-nm lasers.

Seven days before experimentation, cells were seeded (75,000 cells/ml) and treated with RA and BDNF reagents for full differentiation. Next, cells were incubated with 2.8 μg/ml recombinant human AS protein aggregates (active) (ab218819; Abcam) labeled with an Alexa Flour 488 Conjugation Kit (ab236553; Abcam) for 6 h to establish PD neurons. The culture medium was then removed, and cells were rinsed with PBS (3 × times) and incubated overnight with Cy5-labeled BTL loaded with Cy3-labeled SynO4 (2.5 mM), free Cy3-labeled SynO4 (3.65 μg/ml), or Cy5-labeled BTL (empty) (2.5 mM, control). The following day, the medium was removed, and the cells were rinsed with PBS (3 × times) (Figure S14a and b) and stained with Hoechst staining (1 μg/ml) (63493; Sigma-Aldrich) for nuclei labeling in both experiments. Image acquisition and processing were performed using SR microscopy (Elyra 7 eLS, Zeiss, Germany) and ZEN software, applying 405-, 488-, 561-, and 642-nm lasers.