*3.3. Cytotoxicity of N-AI PAI-2 Liposomes against Breast Cancer Cells*

Treatment of MCF-7 and MDA-MB-231 cells for 72 h with *N*-AI PAI-2 liposomes but not EMP PAI-2 liposomes (at an equivalent phospholipid concentration), resulted in a dose-dependent decrease in cell viability for both cell lines, consistent with intracellular delivery of the cytotoxic *N*-AI (Supplementary Information Figure S3). The cytotoxic effect of *N*-AI PAI-2 liposomes against MDA-MB-231 cells (IC<sup>50</sup> of 5.40 ± 1.14 µM) was significantly greater (*p* < 0.01) than the MCF-7 cells (IC<sup>50</sup> of 31.84 ± 8.20 µM). EMP PAI-2 liposomes elicited some degree of cytotoxicity in both cell lines, at the highest liposome concentrations tested.

Figure 2a).

that PAI-2 liposomes were fully active.

effective at inhibiting uPA activity, as the unconjugated PAI-2 (95–100% inhibition) demonstrating

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 ±

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

**Figure 2.** Cell surface expression of uPA/uPAR and cellular uptake of PAI-2 FITC-labelled liposomes by breast cancer cells. (**a**) MCF-7 cells and MDA-MB-231 cells were incubated with antibodies against human urokinase plasminogen activator (uPA), its receptor (uPAR), or an isotype control antibody (IgG) and cell surface expression analyzed by flow cytometry. (**b**) MCF-7 cells (left) and MDA-MB-231 cells (right) were incubated with empty non-functionalized (EMP) FITC liposomes or empty PAI-2-functionalized (PAI-2) FITC liposomes for 45 min, and were analyzed by flow cytometry. MFI = mean fluorescence intensity Data are the mean ± s.d. (*n* = 3). \*: *p* < 0.05; \*\*\*: *p* < 0.001; \*\*\*\*: *p* < 0.0001; n.s. = not significant (*p* > 0.05). (**c**) EMP liposomes and PAI-2 liposomes were labelled with R18 and incubated with cells at a liposome concentration of 2.5 mM for 1 h. LysoTracker green was added to visualize lysosomes. Arrows point to white foci, which indicate colocalization of green and magenta signals. Representative images are shown. Scale bars are 25 µm.
