Anticancer drugs loaded into tumor- and vasculature-targeted nanocarriers (NC) can reduce side-effects and improve therapeutic efficacy in pre-clinical studies. However, poorly perfused and dysfunctional tumor vessels and lymphatics limit the transport of the payload into the parenchyma of solid tumors. The use of NC decorated with tumor-penetrating peptides (TPPs) might enhance tumor penetration and antitumor effects.
A previously characterized neuroblastoma (NB)-targeting peptide ligand was here modified (now referred as TPP-NB) by adding a consensus motif as a mediator of cell, vascular and tissue penetration via neuropilin-1 (NRP-1) receptor recognition. NPR-1 expression was validated by FACS analysis in NB cell lines and by IHC staining in tumor cells and tumor stroma from NB-bearing mice. Recombinant NRP-1 was used to validate TPP-NB specificity. In vitro and in vivo cell association and internalization of TPP-NB, either free or coupled to Liposomes (L) were tested by FACS and confocal microscopy. Vascular permeability assay after treatment with TPP-NB-targeted, doxorubicin-loaded Liposomes (TPP-NB-L[DXR]) was performed evaluating the in vivo accumulation of Evans Blue dye within the tumor mass. Therapeutic experiments with TPP-NB-L[DXR] were performed in mice orthotopically injected with human NB cells.
NRP-1 expression is validated in a panel of NB cells and in tumors from NB-bearing mice. Differently from the original peptide and some control ones, TPP-NB is able to recognize recombinant NRP-1. The addition of the NRP-1-recognizing sequence to the original peptide significantly increases its NB cellular association in vitro. Interestingly, the results seem to indicate that the enhanced capability by TPP-NB in binding NB cells is related to the combination of the NRP-1-recognizing and the original sequence. Importantly, TPP-NB coupled at the external surfaces of L[DXR] significantly increases their cellular association on NB cells in vitro. Competitive binding assay reveals that binding of TPP-NB is specific and can be inhibited by an excess of the unlabeled free peptide. The localization and the cellular distribution of L evaluated by confocal microscopy in vitro and in mouse models of NB, confirm the binding specificity, showing an increased selective internalization of TPP-NB-L-FITC compared to that obtained with either untargeted L or L decorated with the scrambled peptide. Moreover, TPP-NB-L[DXR] further increases the vascular permeability into the NB tumor mass, but not in non-tumor tissues. The therapeutic efficacy of TPP-NB-L[DXR] has been investigating in terms of overall survival. On running results indicate that the novel NC exerts an increased anti-NB effect compared to DXR-loaded L decorated with the original peptide.
Our findings demonstrate that the achieved penetrating features by a NB-targeting peptide might increase liposomal drug binding, homing and antitumor efficacy.
Citation Format: Fabio Pastorino, Chiara Brignole, Laura Emionite, Silvia Bruno, Flavio Curnis, Daniela Di Paolo, Patrizia Perri, Alessandro Gori, Renato Longhi, Michele Cilli, Angelo Corti, Mirco Ponzoni. Tumor-penetrating peptide-coated nanoparticles as a novel strategy for the targeted therapy of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2017-5130