Bodipy phalloidin

The HeLa cells were seeded on poly-L-lysine-coated glass coverslips (12 × 12 mm) placed inside 6-well plates at a density of 150,000 cells/well in 3 mL of DMEM medium (supplemented with 10% of fetal serum bovine, 1% penicillin-streptomycin, 1% NEAA, and 1% sodium pyruvate) and grown to 90% confluency under standard culture conditions. Then, the medium was discarded and the cells were washed with PBS with a pH of 7.4. Subsequently, the cells were exposed to FITC-PLGA/Au-HS NPs and FITC-PLGA/Au-HS-RGD NPs contained in a 3 mL DMEM medium. After 4 h of incubation, the medium with the NPs was aspirated and the cells were fixed with paraformaldehyde 4% (w/v) for 10 min, washed with PBS (three times), permeabilized with 0.2% (w/v) Triton X-100 for 10 min, washed with PBS (three times), and stained with BODIPY^® Phalloidin (Invitrogen) for 30 min. Finally, the cells were washed with PBS (three times), mounted on a glass slide, stained with ProLong^® Gold antifade DAPI (Invitrogen), and stored for 24 h at −20 °C. The samples were visualized with 20× objective using a confocal spectral microscope Leica TCS-SP2 (Leica Microsystems GmbH, Heidelberg Mannheim, Germany).

The modification of the morphology of mouse RAW 264.7 macrophages after liposaccharide (LPS) stimulation and the inhibitory effect provided by ATOR-loaded PLGA/miRNA NPs was analyzed by seeding this type of cell on poly-l-lysine-coated glass coverslips (12 × 12 mm²) placed inside 6-well plates (3 mL, 1 × 10⁵ cells per well) and grown for 24 h at standard culture conditions. Then, 300 µL of ATOR-loaded PLGA/miRNA NPs (50 mg/mL) was added to cells and incubated for 24 h. After incubation, cells were treated with 100 ng/mL LPS and incubated for an additional 72 h. Afterwards, treated and control cells were washed with cold 10 mM PBS pH 7.4 three times, fixed with paraformaldehyde 4% (w/v) for 10 min, washed with PBS again, permeabilized with 0.2% (w/v) Triton X-100, washed with PBS twice, and stained with BODIPY Phalloidin (Invitrogen, Thermo Fisher, Waltham, MA, USA) for 30 min. Cells were washed again with cold PBS, mounted on glass slides, stained with ProLong Gold antifade DAPI (Invitrogen, Thermo Fisher, Waltham, MA, USA), cured for 24 h at −20 °C, and observed by fluorescence microscopy as described above.