*2.3. Assessment of Cellular Uptake and Localization of Liposomes*

MCF-7 and MDA-MB-231 cells were used to assess the cellular uptake of liposomes through flow cytometry and cellular localization by confocal microscopy. For flow cytometry, MCF-7 and MDA-MB-231 cells (2 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) were seeded into 12-well plates and allowed to attach for 24 h at 37 ◦C. Liposomes containing 1% (mol/mol) FITC-PEG2000-DSPE were added to wells at dilutions ranging from 1:20 to 1:5. At specified time intervals ranging between 15 and 60 min, the supernatant was removed, the cells were washed once with PBS and then harvested using PBS containing 5 mM EDTA (pH 7.4). The cells were then centrifuged (300× *g* for 5 min) and washed three times with PBS, before being resuspended in 200 µL PBS for analysis. The fluorescence intensity was determined by flow cytometry (LSR II flow cytometer; BD Biosciences, CA) (excitation 488 nm, emission collected with 515/20 band-pass filter). FlowJo software (V10; Tree Star Inc., OR, USA) was used to evaluate the mean fluorescence intensity (MFI), to determine the cellular uptake of liposomes.

For confocal microscopy, cells (50,000 per well) were seeded into 8-well µ-Slide chambered coverslips (ibidi, Germany) and incubated for 24 h at 37 ◦C. Cells were allowed to reach 80% confluence before the addition of liposomes. Liposomes containing 1% or 10% (mole % of liposome phospholipid) FITC-PEG2000-DSPE, or 0.625% (mole % of liposome phospholipid) octadecyl rhodamine B chloride (R18; Invitrogen, Carlsbad, CA, USA) were added to cells at dilutions ranging from 1:5 to 1:10, and incubated for 30 min to 2 h at 37 ◦C. The supernatant was removed and the wells were rinsed three times with PBS, before LysoTracker Green DND-26 (excitation/emission 504/511 nm; Thermo Fisher Scientific, MA, USA) was added to each well (50 nM final concentration), immediately prior to imaging. Live imaging of cells in PBS was performed using a Leica TCS SP5 Confocal Microscope (Leica Microsystems, Wetzlar, Germany) and the images were acquired using a 63× oil immersion lens. Images were analyzed using the Leica Application Suite software (V10; Leica Microsystems, Germany).
