Dual-Pathway Sequential Transcytosis Unlocks Enhanced Tumor Penetration and Efficacy of Mitochondria-Targeted Nanodrugs

The limited penetration of nanomedicines into tumor tissues remains a major obstacle to their therapeutic efficacy. To overcome this barrier, we designed a novel nanodrug that leverages receptor/cation dual pathway-mediated transcytosis to achieve deep tumor penetration and targeted disruption of mitochondria, resulting in significantly enhanced antitumor outcomes. The multifunctional carrier, P1, was constructed through the synthesis of an amphiphilic block copolymer, terminal conjugation of the CRGDK peptide, and side-chain modification with 7-diethylaminocoumarin (DEAC) for mitochondrial targeting. Dynamic light scattering analyses confirmed the pH/ROS-responsive behavior of P1 micelles, including acid-triggered charge reversal. Drug release kinetics, cellular uptake and endocytic mechanisms, lysosomal escape efficiency, mitochondrial colocalization, induction of ROS generation, mitochondrial membrane potential (ΔΨ m ) depolarization, apoptosis induction, penetration in multicellular tumor spheroids (MTSs) and in vivo tumors, and antitumor efficacy (in vitro and in vivo) were systematically evaluated. Results indicated that DOX/P1 micelles initially target tumor tissue via CRGDK binding, followed by NRP-1-mediated transcytosis. Subsequent acidity-induced charge reversal activates a secondary cation-mediated transcytosis pathway, synergistically promoting deep tumor infiltration. Upon mitochondrial localization, the carrier undergoes ROS-triggered degradation, leading to concurrent release of doxorubicin (DOX) and cinnamaldehyde (CA) within mitochondria. This dual release acts synergistically to amplify oxidative stress, collapse ΔΨ m, and induce mitochondrial DNA damage, collectively precipitating irreversible apoptosis. This study establishes a programmable platform for developing tumor-penetrating nanotherapeutics with precise subcellular organelle-targeting capabilities.