Liposomal Delivery of Pyronaridine as a Repurposed Inhibitor of ERCC1/XPF for the Sensitization of Colorectal Cancer Cells to Platinum Chemotherapeutics

Inhibition of ERCC1/XPF, a heterodimeric enzyme complex with endonuclease activity that participates in the repair of DNA inter- and intrastrand cross-links, is expected to make cells sensitive to DNA damage by platinum-based chemotherapeutics. Here we report on repurposing a clinically used antimalaria drug, pyronaridine (PYD), as a novel inhibitor of ERCC1/XPF for the sensitization of colorectal cancer cells (CRC) to platinum drugs. We have developed a liposomal formulation of PYD to make the chemosensitizing activity of this ERCC1/XPF inhibitor more specific to the tumor site. Liposomal PYD was optimized by changing the lipid composition and the pH gradient system within the liposomes. Prepared formulations were characterized for their average diameter, PYD encapsulation, and <i>in vitro</i> drug release. The intracellular XPF-PYD interaction and subsequent XPF protein thermal stabilization in intact HCT116 cells were confirmed by the cellular thermal shift assay (CETSA). The cytotoxic activity of free and selected PYD liposomal formulation alone or in combination with carboplatin and oxaliplatin was assessed against HCT116 and SW620 cells using MTT and colony-forming assays. The possibility of a synergistic effect between PYD or liposomal PYD and Pt-based chemotherapeutics in HCT116 was tested through analyzing the MTT assays using the Combenefit software. The results showed an average diameter of 82.32 ± 0.33 nm with a polydispersity index of 0.181 ± 0.008 along with a 99% PYD encapsulation efficiency for the optimized liposomal formulations prepared using a combination of 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and cholesterol with an inside pH of 3.5. In media consisting of PBS and bovine serum albumin, ∼50% of the loaded PYD was released within 72 h. The validation of target interaction performed via CETSA <i>in vitro</i> demonstrated the significant thermal stabilization of XPF after treatment with either free or liposomal PYD at the temperature range of 47-50 °C. Combenefit analysis showed synergy in HCT116 cells between PYD and carboplatin, which was further increased when carboplatin was combined with liposomal PYD. Oxaliplatin and PYD combination displayed less synergy in HCT116 cells, while combining oxaliplatin and liposomal PYD only showed an additive effect in this cell line. In SW620 cells, an elevated level of PYD was needed (0.6 μM) for the sensitization of cells to the effect of carboplatin or oxaliplatin. The colony-forming assay in HCT116 cells evidenced a decrease of colony formation when PYD (0.15 μM) was combined with carboplatin or oxaliplatin. In SW620, a higher PYD concentration (0.6 μM) was required to significantly lower colony formation when paired with carboplatin, suggesting lower sensitivity compared to HCT116 cells. The same trend was seen with the oxaliplatin combination. The results indicate a potential for PYD and its liposomal formulation in the chemosensitization of CRC to platinum chemotherapeutics in a cell-dependent manner.