Antitumor Activity of Liposomal Nanoparticles Co-Encapsulating Ceramides and Doxorubicin in In Vitro Nucleolin-Expressing Neuroblastoma Models

Background: Neuroblastoma (NB) causes about 15% of cancer deaths in childhood. Recently, we suggested cell-surface nucleolin (NCL) as a novel target for preclinical therapy against NB. Methods: Here, a broad range of human NB cell lines were evaluated for NCL expression. PEGylated liposomal nanoparticles, co-encapsulating C6- or C18-ceramides and doxorubicin (DXR) and functionalized with the F3 peptide (F3-lipoC6-DXR or F3-lipoC18-DXR), were tested against NCL-expressing NB cell lines, grown in monolayers (2D) and as multicellular tumor spheroids (3D). Untargeted liposomes were used as the control. Cytotoxicity and apoptotic/necrotic deaths were evaluated. Results: All NB cell lines expressed cell-surface NCL. Compared to untargeted formulations, F3-lipoC6-DXR and F3-lipoC18-DXR showed enhanced cellular association and antitumor effects against NB cells. Compared to F3-lipoC18-DXR, F3-lipoC6-DXR was significantly more effective in reducing 2D and 3D NB cell lines’ viability (2D: IC50 range 313–995 nM and 239–629 nM, respectively; 3D: IC50 range 202–416.2 nM and 62.61–398.6 nM, respectively) and in inducing apoptotic cell death. F3-lipoC6-DXR also led to a greater cytotoxicity compared to liposomal DXR alone, highlighting the benefit of co-encapsulation. Conclusions: NCL is a promising target in NB, and F3-targeted liposomes enable the selective delivery of their cargo. F3-lipoC6-DXR showed superior antitumor activity, supporting ceramide–DXR co-encapsulation as a potential treatment strategy, which needs to be further validated.