Active tumor-targeting approaches using specific ligands have drawn considerable attention over the years.However,a single ligand often fails to simultaneously target the cancer cell surface and subcellular organelles,which limits the maximum therapeutic efficacy of delivered drugs.We describe a polymeric delivery system modified with the G3-C12 peptide for sequential dual targeting.In this study,galectin-3-targeted G3-C12 peptide was conjugated onto the N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer for the delivery of D(KLAKLAK)2(KLA) peptide.G3-C12-HPMA-KLA exhibited increased receptor-mediated intealization into gaiectin-3-overexpressing PC-3 cells.Furthermore,G3-C12 peptide also directed HPMA-KLA conjugates to mitochondria.This occurred because the apoptosis signal triggered the accumulation of galectin-3 in mitochondria,and the G3-C12 peptide that specifically bound to galectin-3 was trafficked along with its receptor intracellularly.As a result,G3-C12-HPMA-KLA disrupted the mitochondrial membrane,increased the generation of reactive oxygen species (ROS) and induced cytochrome c release,which ultimately resulted in enhanced cytotoxicity.An in vivo study revealed that the G3-C12 peptide significantly enhanced the tumor accumulation of the KLA conjugate.In addition,G3-C12-HPMA-KLA exhibited the best therapeutic efficacy and greatly improved the animal survival rate.Our work demonstrates that G3-C12 is a promising ligand with dual-targeting functionality.