*2.1. Liposome Preparation and Characterization*

Liposomes were prepared using the thin-film hydration method [31] and were composed of 20 mM soy PC (L-α-phosphatidylcholine) and 0.6 mM mPEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-*N*-[(polyethylene glycol)-2000]) (Avanti Polar Lipids, Alabaster, AL, USA), with the addition of either 5 mM cholesterol (Sigma-Aldrich, St. Louis, MO, USA) to form empty

liposomes, or 5 mM 5,7-dibromo-*N*-(*p*-hydroxymethylbenzyl)isatin (*N*-AI) (prepared in-house [23]) to form *N*-AI-loaded liposomes. *N*-AI encapsulation efficiency was determined by high-performance liquid chromatography (HPLC). Here, *N*-AI-loaded liposomes were mixed with water/acetonitrile (60:40 *v*/*v*) and centrifuged. The *N*-AI concentration was determined using an Atlantis T3 reverse-phase C18 analytical column (Waters, UK) and a Waters HPLC machine (Waters, MA, USA). Analysis was performed using an injected volume of 10 µL, with a gradient elution and monitored with a photodiode array at 435 nm. Concentration was determined by interpolating from a standard curve after analysis of standards and samples using Empower Pro V2 software (Waters, UK).

Liposomes were surface-functionalized with plasminogen activator inhibitor type 2 (PAI-2), as described by our group previously [31]. Unbound PAI-2 was removed from liposomes by sizeexclusion chromatography (SEC), using Sepharose CL-4B (Sigma-Aldrich, MO, USA). Western blotting and fluorogenic uPA activity assays were used to detect and quantify PAI-2 conjugated to liposomes and the activity of PAI-2-functionalized liposomes [32]. For Western blotting, PAI-2 in SDS–PAGE gels (reducing conditions) were transferred to PVDF membranes using Bio-Rad transfer equipment (Bio-Rad Laboratories, Hercules, CA, USA) at 100 V for 1.5 h. Membranes were rinsed in TBST (1× TBS buffer with 0.05% *v*/*v* Tween-20) and blocked using 10% skim milk in TBST for 1 h at RT. After the rinsing membranes were incubated with primary antibody (anti-SerpinB2; Abcam, Cambridge, UK) at 1:2000 dilution in 2% skim milk/TBST at 4 ◦C overnight. Membranes were washed with TBST four times (10 min each wash) and then incubated with secondary antibody (anti-rabbit-HRP; Abcam, Cambridge, UK) at 1:5000 dilution in 2% skim milk/TBST for 2 h at RT. Membranes were then washed in TBST, three times, for 5 min and then in TBS (no Tween-20), three times, for 5 min. Membranes were developed using ECL peroxidase reaction (Pierce PicoWest ECL reagent; Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Membranes were visualized using X-ray film, after developing and fixing (Bio-Rad Laboratories, CA, USA) or using a Gel Logic 2200 Digital Imager (Carestream Molecular Imaging, Woodbridge, CT, USA). Band intensities were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
