*3.2. PAI-2 Liposomes Are Taken up by Cells through RME-Dependent and Non-Dependent Mechanisms*

Prior to assessing cellular uptake, the MCF-7 and MDA-MB-231 breast cancer cell lines were profiled for cell surface uPA and uPAR expression through flow cytometry. MDA-MB-231 cells showed a significantly (*p* < 0.001) higher mean fluorescent intensity for uPAR and uPA (MFI; 11.82 ± 0.90 and 6.66 ± 0.97, respectively) than MCF-7 cells (MFI; 0.26 ± 0.03 and 2.49 ± 0.10, respectively; Figure 2a).

The cellular uptake of liposomes was determined by flow cytometry, using FITC-PEG-DSPE incorporated into the liposome bilayer. PAI-2-functionalized (EMP PAI-2; 152.6 ± 8.7 nm) and non-functionalized (EMP; 152.8 ± 11.7 nm) FITC liposomes were incubated with MCF-7 cells (low uPA/uPAR) and MDA-MB-231 cells (high uPA/uPAR) for 45 min. A significant increase in the EMP PAI-2 liposome uptake was observed in the MDA-MB-231 cells at 5 mM and 2.5 mM liposome concentrations (*p* < 0.0001 and *p* < 0.001, respectively) relative to the EMP liposomes, but not at 1.25 mM liposome concentration. No significant differences were observed between the uptake of EMP and EMP PAI-2 liposomes in the MCF-7 cells, at any liposome concentrations (*p* > 0.05; Figure 2b).

For the cellular localization of EMP PAI-2 liposomes through confocal microscopy, the intensely fluorescent fluorophore R18 was used to label liposomes. Dynamic light scattering revealed average diameters of 131.3 ± 2.5 nm and 131.2 ± 6.6 nm for EMP and EMP PAI-2 R18-labelled liposomes, respectively. A strong fluorescent signal from R18-labelled liposomes was detected at the cell membrane, within the cytoplasm and within lysosomes (indicated by colocalization of liposome and LysoTracker), 1 h post-incubation for both cell lines (Figure 2c), indicating cellular uptake for both EMP PAI-2 liposomes and EMP liposomes.
