Intramolecular π-π stacking-regulated ROS amplification in water-soluble GalNAc-functionalized tetraphenylethylene photosensitizers for hepatocellular carcinoma.

Herein, a modular geminal-tetraphenylethylene (gem-TPE) photosensitizer platform is reported, integrating asialoglycoprotein receptor (ASGPR)-targeting GalNAc units with aryl-rich organelle-directing motifs to regulate intramolecular packing and photophysical behavior. A series of derivatives (gem-TPEVP-TsG, gem-TPEVP-FBG, and gem-TPEVP-TPPG) together with a non-stacking control (gem-TPEVP-G2) were systematically investigated. ROS probe assays and EPR spectroscopy show that gem-TPEVP-G2 mainly proceeds through a Type I pathway, whereas the organelle-modified derivatives exhibit dual Type I/Type II photoreactivity, with efficiencies ranked as follows: gem-TPEVP-TsG > gem-TPEVP-FBG > gem-TPEVP-TPPG > gem-TPEVP-G2. Notably, gem-TPEVP-TsG produced ∼3.6-fold higher Type I ROS than the control. Theoretical calculations reveal that aryl-rich substituents promote intramolecular π-π stacking with the 4-N-methylvinylpyridinium acceptor (approximately 3.4 Å), inducing conformational rigidity and enhancing intersystem crossing in the non-aggregated state. In contrast, gem-TPEVP-G2 exhibits ineffective stacking and displays weaker charge-transfer properties. Confocal microscopy confirms efficient ASGPR-mediated uptake and a predominant lysosomal distribution, likely arising from π-π-driven molecular folding. Nevertheless, all derivatives exhibit light-dependent cytotoxic responses that qualitatively track trends in ROS generation, while maintaining negligible dark effects. Overall, this work demonstrates that intramolecular π-π interactions can emulate aggregation-induced intersystem crossing at the molecular level, providing a general design strategy to enhance ROS generation while preserving aqueous solubility.