Percent of particles present relative to the total particle population.

The size and morphology of *N*-AI liposomes were further confirmed by measurement of liposome diameter from cryogenic transmission electron microscopy (cryo-TEM) images (Figure 1a). The average diameter of *N*-AI liposomes (138.7 ± 18.4 nm; Figure 1b) was similar to that determined using dynamic light scattering (139.9 ± 3.9 nm; Table 1). Cryo-TEM additionally revealed *N*-AI liposomes to be spherical, monodisperse, and unilamellar. The concentration of *N*-AI encapsulated in the liposomes could not be determined by spectrophotometry, as the liposome phospholipid interfered with the peak absorbance of *N*-AI at 310 nm and 435 nm (Supplementary Information Figure S1). Therefore, the concentration of *N*-AI loaded into liposomes was determined by HPLC, which revealed an *N*-AI concentration of 2.2 mM, equating to a 43.1% entrapment efficiency based on the starting amount of *N*-AI used in the liposome preparation (Supplementary Information Figure S2). This translated into 7.3% *w*/*w N*-AI loaded per unit weight of the soy PC, indicating the percentage of mass of the liposome that is due to the encapsulated drug.

curve.

phospholipid (PEG-DSPE; molecular weight ~2940 kDa) to form PAI-2-PEG-DSPE was confirmed by a lag in gel migration of PAI-2 in the peak 1 fraction (sample #1), relative to the peak 2 fraction (sample #2), which corresponded to the 45 kDa molecular weight of free PAI-2. The amount of PAI-2

**Figure 1.** Characterization of *N*-AI-loaded liposomes. (**a**) Representative cryo-TEM image of *N*-AIloaded liposomes. (**b**) Determination of the average liposome diameter from cryo-TEM image analysis. (**c**) Size-exclusion chromatograph of the PAI-2 liposome fractions after conjugation, including PAI-2 liposomes (peak 1) and unbound PAI-2 (peak 2). (**d**) Western blot detection of PAI-2 in size-exclusion fractions (1, 2), un-purified liposomes (3), and purified PAI-2 (50, 25 and 12.5 ng). OD = optical density, M = marker, PEG = polyethylene glycol. (**e**) Kinetic inhibition curves for unconjugated PAI-2 versus PAI-2 conjugated to empty liposomes (EMP PAI-2), against uPA in solution. Empty liposomes were included as a fluorescence control. Values are means ± s.d. (*n* = 3). **Figure 1.** Characterization of *N*-AI-loaded liposomes. (**a**) Representative cryo-TEM image of *N*-AIloaded liposomes. (**b**) Determination of the average liposome diameter from cryo-TEM image analysis. (**c**) Size-exclusion chromatograph of the PAI-2 liposome fractions after conjugation, including PAI-2 liposomes (peak 1) and unbound PAI-2 (peak 2). (**d**) Western blot detection of PAI-2 in size-exclusion fractions (1, 2), un-purified liposomes (3), and purified PAI-2 (50, 25 and 12.5 ng). OD = optical density, M = marker, PEG = polyethylene glycol. (**e**) Kinetic inhibition curves for unconjugated PAI-2 versus PAI-2 conjugated to empty liposomes (EMP PAI-2), against uPA in solution. Empty liposomes were included as a fluorescence control. Values are means ± s.d. (*n* = 3).

An important step in characterizing ligand-functionalized liposomes was to confirm whether the targeting ligand(s) retain activity against the target receptor once bound to the liposome surface. The uPA inhibitory activity of PAI-2-liposomes was assessed using enzymatic assays. A significant reduction in the rate of FLU was observed for the EMP PAI-2 liposomes (43.5 ± 24.9 FLU/min) compared to the EMP liposomes (4026.9 ± 206.2 FLU/min) (Figure 1e). EMP PAI-2 liposomes were as PAI-2 was incubated with preformed liposomes containing mal-PEG2000-DSPE, to allow conjugation to the liposome surface. Unconjugated PAI-2 was removed using size-exclusion chromatography (SEC; Figure 1c). Analysis of fractions by spectrophotometry revealed that the unconjugated PAI-2 (peak 2) had separated from the covalently attached PAI-2 on the liposome surface (peak 1). As PAI-2 could not be detected or quantified using commercial biochemical protein assays, due to phospholipid interference [34] (data not shown), Western blotting was used to confirm successful conjugation of PAI-2 to liposomes (Figure 1d). Covalent conjugation of PAI-2 to liposome phospholipid (PEG-DSPE; molecular weight ~2940 kDa) to form PAI-2-PEG-DSPE was confirmed by a lag in gel migration of

PAI-2 in the peak 1 fraction (sample #1), relative to the peak 2 fraction (sample #2), which corresponded to the 45 kDa molecular weight of free PAI-2. The amount of PAI-2 associated with the liposome fraction in sample #1 was 42 ng, after interpolation from a standard curve.

An important step in characterizing ligand-functionalized liposomes was to confirm whether the targeting ligand(s) retain activity against the target receptor once bound to the liposome surface. The uPA inhibitory activity of PAI-2-liposomes was assessed using enzymatic assays. A significant reduction in the rate of FLU was observed for the EMP PAI-2 liposomes (43.5 ± 24.9 FLU/min) compared to the EMP liposomes (4026.9 ± 206.2 FLU/min) (Figure 1e). EMP PAI-2 liposomes were as effective at inhibiting uPA activity, as the unconjugated PAI-2 (95–100% inhibition) demonstrating that PAI-2 liposomes were fully active.
