Manipulating H-aggregation of Cyanine Dimers by Side-chain Engineering for High-contrast Tumor Surgical Navigation

Abstract

Accurate tumor boundary delineation is critical for successful surgical resection, yet achieving high-contrast imaging of hepatocellular carcinoma (HCC) remains challenging due to nonspecific liver accumulation and high intrinsic fluorescence of optical agents. Herein, we reported a molecular design strategy exploiting enhanced cyanine H-aggregation by dimerization, which enabled glutathione (GSH) responsive high-contrast HCC imaging and precise surgical navigation. Four cyanine dimers with different side-chains were constructed via molecular engineering. Among them, the propyl-substituted dimer DCy-Pr exhibited exceptionally low intrinsic fluorescence quantum yield (Φ_f < 0.01%) due to strong H-aggregation-caused quenching, achieving the highest GSH-triggered fluorescence turn-on (151.0-fold). However, the other three cyanine dimers with hydrophilic side chains (polyethylene glycol, anionic SO₃⁻, or cationic ammonium) showed unsatisfactory properties, such as absence of H-aggregation, no reactivity with GSH, and high intrinsic fluorescence. DCy-Pr displayed a remarkable fluorescence quenching, which outperformed its monomeric counterpart Cy-Azo (16.0-fold higher background fluorescence). Moreover, in vivo studies revealed that DCy-Pr efficiently accumulated in tumor regions, achieving remarkable fluorescence contrast with 9.7-fold tumor-to-normal tissue and 5.1-fold tumor-to-liver fluorescence ratios. Finally, DCy-Pr enabled precise delineation of orthotopic hepatic tumor margins and facilitated successful identification and resection of peritoneal metastatic nodules. In summary, this work not only presented a molecular design paradigm through modulating cyanine H-aggregation but also advanced precision surgery of HCC by providing credible real-time tumor margins.