Neuropilin-1-Targeted Cell-Penetrating Tandem Peptide–Drug Conjugate Exhibits Potent In Vivo Antiglioma and Antiangiogenic Activity

Most therapeutics for brain malignancies are limited by poor blood-brain barrier (BBB) transport and inadequate tumor penetration. Combining a BBB-penetrating peptide (SynB3) with a neuropilin-1 (NRP-1) receptor-targeting peptide (tuftsin) offers a promising strategy to enhance brain delivery, glioma targeting, and tumor penetration. This study demonstrated that SynB3, tuftsin, and their tandem construct bind in silico to the NRP-1 b1 pocket, corresponding to the C-end rule motif interaction site. Their cellular internalization likely involves macropinocytosis. The peptides penetrated 3D glioma spheroids, and the attached cargo did not significantly modulate their penetration. Dau-conjugates consistently inhibited proliferation in four glioblastoma cell lines and showed adequate in vitro stability in mouse plasma. In a subcutaneous murine model, Dau-SynB3 and the tandem conjugate reduced tumor volume and weight, prolonged tumor doubling time, and demonstrated improved tolerability compared with free Dau. Both conjugates exhibited antiproliferative effects, while the tandem construct additionally showed antiangiogenic activity ex vivo.