Target Identification of the Hepatocellular Carcinoma-Specific Aptamer JHIT2e and Construction of Engineered Nanotrains for Synergistic Chemoradiotherapy

Hepatocellular carcinoma (HCC) demands advanced multimodal therapies to overcome heterogeneity-driven treatment resistance. Although aptamers exhibit superior targeting advantages over antibodies, their clinical translation remains severely hindered by ambiguous target identification and the limited drug-loading capacity of direct aptamer-drug conjugates. To address this, we first identified vascular endothelial growth factor receptor 2 (VEGFR2) as the target of the HCC-specific aptamer JHIT2e using an integrated approach combining 3D photo-cross-linking chip technology, surface plasmon resonance (SPR), and liquid chromatography-mass spectrometry (LC-MS). Building on this, we engineered a multifunctional DNA nanotrain (¹³¹I-NTs-Dox, namely, ¹³¹I-labeled DNA nanotrains loaded with doxorubicin) comprising three key components: the JHIT2e aptamer as a navigation module for tumor-specific targeting, hybridization chain reaction (HCR)-assembled dsDNA duplexes as "carriages" intercalated with doxorubicin (Dox), and carboxyfluorescein (FAM)-terminated termini radiolabeled with iodine-131 (¹³¹I) via an optimized chloramine T method. Notably, this FAM-mediated labeling strategy significantly enhanced radiolabeling efficiency to 93%, surpassing conventional tyrosine-based methods (70-85%). Each nanotrain entity achieved an unprecedented payload capacity of 50 Dox molecules and 80 ¹³¹I atoms. In vitro studies demonstrated that ¹³¹I-NTs-Dox exhibits specific affinity for HepG2 cells, rapid lysosomal endocytosis, and enhanced tumor accumulation. Furthermore, the platform synergistically inhibited HCC cell viability through dual chemoradiotherapeutic mechanisms: Dox-induced DNA repair inhibition and ¹³¹I-mediated DNA damage. Collectively, ¹³¹I-NTs-Dox represents a scalable nanoplatform with high-precision HCC targeting, dual-modal therapeutic efficacy, and promising clinical potential for chemoradiotherapy.