The Enhanced Efficacy of Intracellular Delivery of Doxorubicin/C6-Ceramide Combination Mediated by the F3 Peptide/Nucleolin System Is Supported by the Downregulation of the PI3K/Akt Pathway

SIMPLE SUMMARY: Targeted nanomedicine-based approaches that aim at the simultaneous delivery of synergistic drug combinations to multiple cellular populations are of high relevance for tackling heterogeneity on solid tumors. Considering that cancer stem cells (CSC) may originate from non-stem cancer cells, single-drug regimens targeting only one of these cell populations could enable tumors to evade treatments. As such, the identification of a common marker, such as nucleolin, might result in a therapeutic advantage. The results herein generated suggested a transversal role of nucleolin in the internalization of F3 peptide-targeted pegylated pH-sensitive liposomes into bulk ovarian cancer cells, including putative CSC-enriched ovarian cells. The intracellular delivery of a drug combination such as the one tested herein was relevant in the context of cell lines with higher intrinsic resistances to doxorubicin. The enhanced efficacy of the F3 peptide-targeted liposomal combination of doxorubicin/C6-ceramide was supported by the downregulation of the Akt pathway, within a specific range of basal level of expression. ABSTRACT: Targeting multiple cellular populations is of high therapeutic relevance for the tackling of solid tumors heterogeneity. Herein, the ability of pegylated and pH-sensitive liposomes, functionalized with the nucleolin-binding F3 peptide and containing doxorubicin (DXR)/C6-ceramide synergistic combination, to target, in vitro, ovarian cancer, including ovarian cancer stem cells (CSC), was assessed. The underlying molecular mechanism of action of the nucleolin-mediated intracellular delivery of C6-ceramide to cancer cells was also explored. The assessment of overexpression of surface nucleolin expression by flow cytometry was critical to dissipate differences identified by Western blot in membrane/cytoplasm of SKOV-3, OVCAR-3 and TOV-112D ovarian cancer cell lines. The former was in line with the significant extent of uptake into (bulk) ovarian cancer cells, relative to non-targeted and non-specific counterparts. This pattern of uptake was recapitulated with putative CSC-enriched ovarian SKOV-3 and OVCAR-3 sub-population (EpCAM(high)/CD44(high)). Co-encapsulation of DXR:C6-ceramide into F3 peptide-targeted liposomes improved cytotoxic activity relative to liposomes containing DXR alone, in an extent that depended on the intrinsic resistance to DXR and on the incubation time. The enhanced cytotoxicity of the targeted combination was mechanistically supported by the downregulation of PI3K/Akt pathway by C6-ceramide, only among the nucleolin-overexpressing cancer cells presenting a basal p-Akt/total Akt ratio lower than 1.