**1. Introduction**

Breast cancer is the most common invasive cancer in women worldwide and remains a leading cause of cancer-related morbidity and mortality [1]. While overall survival has improved steadily over the last several decades, breast cancer still accounts for almost half a million deaths each year [2]. Numerous studies and clinical evidence indicate that the urokinase plasminogen activator system (uPAS) play a key role in breast cancer metastasis [3,4]. In this system uPA, secreted as a zymogen, is activated upon binding to its specific cell surface receptor uPAR. Once activated, uPA catalyzes the activation of co-localized plasminogen to plasmin, which in turn directly degrades the components of the extracellular matrix (ECM), promoting further degradation and tissue remodeling, by activating pro-metalloproteinases and activating latent growth factors from the ECM [5]. Localization of the uPAS at the invasive front of tumors thus facilitates cell migration and invasion. In breast cancer, progression-free survival is inversely correlated with uPA and uPAR expression [6,7]. Patients with high uPA mRNA levels are more likely to suffer from metastatic disease [8], and overexpression of uPAR by tumor cells or stromal cells is associated with a poor prognosis for metastatic breast cancer [9].

Amplification and overexpression of uPA and uPAR are thus recognized biomarkers of metastasis and are indicative of an overall poor patient prognosis for breast and several other cancer types [6,7,10,11].

In the triple-negative breast cancer (TNBC) subtype, uPAR was also shown to increase the malignant potential [12] and was identified as a possible novel target for treatment [13,14]. As this subtype lacks the three main breast cancer molecular biomarkers (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2)), there are no targeted therapy options for TNBC, and the use of conventional chemotherapies remains the standard of care. In addition, TNBC is a markedly heterogeneous subtype that can make treatment problematic. The prognosis of TNBC is generally poor, with high rates of disease recurrence and relapse. The progression-free survival and overall survival rates of TNBC patients are significantly shorter than those of non-TNBC patients [15].

Given the role of uPAS in the promotion of metastasis, targeting uPA/uPAR is a promising therapeutic strategy for TNBC [16,17]. uPA is efficiently and specifically inhibited by the serpin plasminogen activator inhibitor-2 (PAI-2/SerpinB2), forming a covalent complex with uPA/uPAR, which is rapidly internalized via endocytosis receptors [18,19]. Previous work by our group showed that PAI-2 could be used as a targeting ligand for the intracellular delivery of covalently attached cytotoxins to uPAR-positive tumor cells [5,20,21]. Specifically, PAI-2 was conjugated to an *N*-alkylisatin (*N*-AI)-based cytotoxin, a potent microtubule destabilizing agent [22], which could evade P-glycoprotein (P-gp)-mediated efflux in multi-drug resistant cancer cell lines [23,24]. The *N*-AI-PAI-2 conjugate was efficacious in vivo, reducing MDA-MB-231 tumor growth at 1/20th of the dose of free *N*-AI [25]. Given the potency and previous validation of *N*-AI as a cytotoxin for use in anticancer applications, *N*-AI is a promising candidate for further development in drug delivery research. As a hydrophobic molecule, *N*-AI has a low aqueous solubility that limits the amount of drug that can be administered intravenously [26]. However, *N*-AI is amenable to encapsulation within liposomes, in order to improve solubility and physicochemical stability.

Liposomes emerged as a useful delivery system for the transport of drugs and other molecules to solid tumors. Their unique structure allows for the encapsulation of hydrophobic or hydrophilic drugs in the lipid bilayer or aqueous core, respectively [27]. Encapsulated drugs can then be delivered to target cells for intracellular drug release and anti-tumor effect. The inclusion of hydrophilic polymers, most commonly PEG, at the outer surface of the liposome can increase the in vivo circulation time, by reducing recognition and clearance by the MPS [28]. For this reason, PEGylated liposomes were long considered a clinically useful nanoparticle for drug delivery applications. In addition to their versatile drug encapsulation capabilities, liposomes permit the active targeting of specific cell types via the conjugation of ligands to the liposome surface, for drug delivery to cells expressing the target surface receptor(s) of interest [29,30]. Following liposome extravasation into the tumor interstitial space, subsequent ligand-directed surface binding and internalization promotes liposome and drug entry into specific cell types. As targeted liposome formulations combine both passive and active drug delivery mechanisms, ligand-directed liposomes should show superior drug delivery, compared to non-ligand liposomes, depending on the tumor type [31].

Herein, we describe the preparation and characterization of novel *N*-AI-loaded liposomes surface-functionalized with PAI-2 as a ligand for targeting uPA/uPAR. We further evaluated the cellular targeting and in vivo biological properties of these liposomes, using relevant models of uPAR-positive TNBC breast cancer.
