*N***-Alkylisatin-Loaded Liposomes Target the Urokinase Plasminogen Activator System in Breast Cancer**

#### **Lisa Belfiore 1,2, Darren N. Saunders <sup>3</sup> , Marie Ranson 1,2,4 and Kara L. Vine 1,2,4,\***


Received: 26 May 2020; Accepted: 3 July 2020; Published: 7 July 2020

**Abstract:** The urokinase plasminogen activator and its receptor (uPA/uPAR) are biomarkers for metastasis, especially in triple-negative breast cancer. We prepared anti-mitotic *N*-alkylisatin (*N*-AI)-loaded liposomes functionalized with the uPA/uPAR targeting ligand, plasminogen activator inhibitor type 2 (PAI-2/SerpinB2), and assessed liposome uptake in vitro and in vivo. Receptor-dependent uptake of PAI-2-functionalized liposomes was significantly higher in the uPA/uPAR overexpressing MDA-MB-231 breast cancer cell line relative to the low uPAR/uPAR expressing MCF-7 breast cancer cell line. Furthermore, *N*-AI cytotoxicity was enhanced in a receptor-dependent manner. In vivo, PAI-2 *N*-AI liposomes had a plasma half-life of 5.82 h and showed an increased accumulation at the primary tumor site in an orthotopic MDA-MB-231 BALB/c-Fox1nu/Ausb xenograft mouse model, relative to the non-functionalized liposomes, up to 6 h post-injection. These findings support the further development of *N*-AI-loaded PAI-2-functionalized liposomes for uPA/uPAR-positive breast cancer, especially against triple-negative breast cancer, for which the prognosis is poor and treatment is limited.

**Keywords:** *N*-alkylisatin; liposome; urokinase plasminogen activator; PAI-2; SerpinB2; breast cancer
