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 (Φf < 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.
| [1] |
A. Vogel, T. Meyer, G. Sapisochin, R. Salem, and A. Saborowski, “Hepatocellular Carcinoma,” The Lancet 400 (2022): 1345–1362.
|
| [2] |
L. Wu, Z. Li, K. Wang, et al., “Advances in Organic Small Molecule-Based Fluorescent Probes for Precision Detection of Liver Diseases: A Perspective on Emerging Trends and Challenges,” Journal of the American Chemical Society 147 (2025): 9001–9018.
|
| [3] |
L. van Manen, H. J. M. Handgraaf, M. Diana, et al., “A Practical Guide for the Use of Indocyanine Green and Methylene Blue in Fluorescence-Guided Abdominal Surgery,” Journal of Surgical Oncology 118 (2018): 283–300.
|
| [4] |
J. S. D. Mieog, F. B. Achterberg, A. Zlitni, et al., “Fundamentals and Developments in Fluorescence-guided Cancer Surgery,” Nature Reviews Clinical Oncology 19 (2022): 9–22.
|
| [5] |
D. Chen, T. Xiao, L. Wang, et al., “A Simple “Spraying” Fluorescence-Guided Surgery by AIE Probes for Liver Tumor Resection Through Configuration-Induced Cross-Identification,” Aggregate 5 (2024): e550.
|
| [6] |
C. E. S. Hoogstins, Q. R. J. G. Tummers, K. N. Gaarenstroom, et al., “A Novel Tumor-Specific Agent for Intraoperative Near-Infrared Fluorescence Imaging: A Translational Study in Healthy Volunteers and Patients With Ovarian Cancer,” Clinical Cancer Research 22 (2016): 2929–2938.
|
| [7] |
Y. Chen, T. Xiong, J. Du, J. Fan, and X. Peng, “Tumor-Specific Cascade Recognition of Activatable Probes for Fluorescence Navigation Surgery,” CCS Chemistry 7 (2025): 740–751.
|
| [8] |
Z. Li, P.-Z. Liang, L. Xu, et al., “In Situ Orderly Self-assembly Strategy Affording NIR-II-J-aggregates for in Vivo Imaging and Surgical Navigation,” Nature Communications 14 (2023): 1843.
|
| [9] |
H. Li, Q. Yao, W. Sun, et al., “Aminopeptidase N Activatable Fluorescent Probe for Tracking Metastatic Cancer and Image-Guided Surgery via in Situ Spraying,” Journal of the American Chemical Society 142 (2020): 6381–6389.
|
| [10] |
C. Chai, T. C. Owyong, Y. Zhang, et al., “Perspective on Intraoperative Real-Time Biliary Tract Navigation: From Radiological to Fluorescent Imaging,” Chemical and Biomedical Imaging 4 (2025): 684–694.
|
| [11] |
N. M. Cotto, N. Chauhan, B. Adriano, et al., “Milk Exosome-Glow Nanosystem for Cancer Cellular and Tissue Bioimaging,” Chemical and Biomedical Imaging 2 (2024): 711–720.
|
| [12] |
M. Liang, L. E. Zhang, B. Yu, et al., “H 2 S-Activated near-Infrared Emission Chemiluminescent Probes for Precise Diagnosis of Inflammations and Tumors,” Aggregate 6 (2025): e742.
|
| [13] |
K. Wu, Z. Chao, S. Aji, et al., “Peroxynitrite Responsive Second Near-Infrared Cyanine Dye J -Aggregate for Drug-Induced Hepatotoxicity Monitoring,” Chemical and Biomedical Imaging 3 (2025): 379–386.
|
| [14] |
Y. Jing, J. Lu, J. Hong, et al., “An NIR-II Anionic Heptamethine Cyanine Probe Enables Real-time in Vivo Imaging of Tumor-associated Glutathione,” Dyes and Pigments 247 (2026): 113482.
|
| [15] |
N. Su, Y. Lin, B. Zhang, et al., “Charge-Regulation Activable Theranostic Probe for Tumor-Specific Fluorescence Imaging and Chemo-Phototherapy,” Advanced Healthcare Materials (2026): e05434, https://doi.org/10.1002/adhm.202505434.
|
| [16] |
L. Yin, P. Xu, Y. Huang, et al., “Glutathione-Responsive near-Infrared-II Fluorescence Probe for Early and Accurate Detection of in Situ and Metastatic Tumors,” Small 21 (2025): 2503257.
|
| [17] |
P. Huang, W. Zhang, F. Huo, and C. Yin, “Based on GSH Aggregating AIE and Disaggregating ACQ to Achieve Non-competitive Discriminative Detection of Cys and GSH in Living Cells,” Chinese Chemical Letters 37 (2026): 111163.
|
| [18] |
C. Zhao, X. Lai, Y. Zhang, et al., “Programmed Activatable Photosensitizer for Acidity/Glutathione Dual-Locked Photodynamic Theranostics of Tumors,” Aggregate 7 (2026): e70300.
|
| [19] |
Y. Pan, S. Lei, Q. Liu, et al., “Multiple GSH-depleting Dimeric Cyanine for Photothermal-ferroptosis Induction With NIR-II Fluorescence Visualization and Renal Clearance,” Cell Reports Physical Science 7 (2026): 103027.
|
| [20] |
J. Ma, R. Sun, K. Xia, Q. Xia, Y. Liu, and X. Zhang, “Design and Application of Fluorescent Probes to Detect Cellular Physical Microenvironments,” Chemical Reviews 124 (2024): 1738–1861.
|
| [21] |
Q. Zhang, Z. Zhang, J. Liu, et al., “Advances in Aggregation-induced Emission Fluorescent Probes for Reactive Sulfur Species Detection,” Coordination Chemistry Reviews 547 (2026): 217122.
|
| [22] |
Z. An, X. Zhang, and J. Jing, “Recent Advances in Fluorescent Probes Development for Interrogating Protein Misfolding and Aggregation,” Trends in Analytical Chemistry 183 (2025): 118066.
|
| [23] |
J. Liu, W. Zhang, C. Zhou, et al., “Precision Navigation of Hepatic Ischemia–Reperfusion Injury Guided by Lysosomal Viscosity-Activatable NIR-II Fluorescence,” Journal of the American Chemical Society 144 (2022): 13586–13599.
|
| [24] |
Y. Hou, J. Li, G. Jiang, et al., “Synergistic Inter- and Intramolecular Aggregation of Dimeric Cyanine Dyes Affords Highly Efficient In Vivo Self-Delivery and Photothermal Therapy,” Advanced Materials 34 (2024): 2316452.
|
| [25] |
X. Zhang, Y. Qin, H. Zhang, et al., “An Aggregation-Independent and Rotor-specific TPE-cyanine Probe for in Vivo near-infrared Fluorescent Imaging,” Chinese Chemical Letters 36 (2025): 110715.
|
| [26] |
S. Yu, D. Tu, F. Zhao, et al., “Synergistic Ligand Engineering Suppresses Dye Aggregation Quenching and Photobleaching in Lanthanide Upconversion Nanoparticles,” Aggregate 7 (2026): e70351.
|
| [27] |
Z. Y. Zhang, W. W. Xu, W. S. Xu, J. Niu, X. H. Sun, and Y. Liu, “A Synergistic Enhancement Strategy for Realizing Ultralong and Efficient Room-Temperature Phosphorescence,” Angewandte Chemie International Edition 59 (2020): 18748–18754.
|
| [28] |
D. H. Li, C. L. Schreiber, and B. D. Smith, “Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance near-Infrared Fluorescence Imaging,” Angewandte Chemie International Edition 59 (2020): 12154–12161.
|
| [29] |
X. Zhao, Y. Ma, J. Di, et al., “Synergetic Pyroptosis With Apoptosis Improving Phototherapy of Mitochondria-Targeted Cyanines With Superior Photostability,” ACS Applied Materials & Interfaces 16 (2024): 12310–12320.
|
| [30] |
X. Zhao, H. Zhao, S. Wang, et al., “A Tumor-Targeting near-Infrared Heptamethine Cyanine Photosensitizer With Twisted Molecular Structure for Enhanced Imaging-Guided Cancer Phototherapy,” Journal of the American Chemical Society 143 (2021): 20828–20836.
|
| [31] |
Y. Zhang, M. Wu, W. Li, et al., “Protein-Triggered Reassembly of Quinocyanine Nanosheets for Intraoperative NIR-II Cholangiography,” Angewandte Chemie International Edition 65 (2025): e22772.
|
| [32] |
H. Bian, D. Ma, Y. Chen, et al., “Molecular Fluorophore Dimerization: a new Paradigm for Precision Phototheranostics,” Chemical Society Reviews 55 (2026): 3139–3187.
|
| [33] |
B. He, Z. Wang, Y. Sun, et al., “Dendritic NIR-II Cyanines Enable Deep Imaging and Precise Surgery,” Small 21 (2025): e09826.
|
| [34] |
D. Zhai, W. Xu, L. Zhang, and Y.-T. Chang, “The Role of “Disaggregation” in Optical Probe Development,” Chemical Society Reviews 43 (2014): 2402–2411.
|
| [35] |
P. Huang, W. Zhang, F. Huo, and C. Yin, “Based on GSH Aggregating AIE and Disaggregating ACQ to Achieve Non-Competitive Discriminative Detection of Cys and GSH in Living Cells,” Chinese Chemical Letters 37 (2025): 111163.
|
| [36] |
Y. Cai, D. Ni, W. Cheng, et al., “Enzyme-Triggered Disassembly of Perylene Monoimide-Based Nanoclusters for Activatable and Deep Photodynamic Therapy,” Angewandte Chemie International Edition 59 (2020): 14014–14018.
|
| [37] |
S.-Y. Lim, K.-H. Hong, D. I. Kim, H. Kwon, and H.-J. Kim, “Tunable Heptamethine–Azo Dye Conjugate as an NIR Fluorescent Probe for the Selective Detection of Mitochondrial Glutathione Over Cysteine and Homocysteine,” Journal of the American Chemical Society 136 (2014): 7018–7025.
|
| [38] |
Z. Yuan, L. Gui, J. Zheng, et al., “GSH-Activated Light-Up Near-Infrared Fluorescent Probe With High Affinity to αvβ3 Integrin for Precise Early Tumor Identification,” ACS Applied Materials & Interfaces 10 (2018): 30994–31007.
|
| [39] |
W.-L. Xia, X.-Y. Ran, K.-P. Xie, et al., “Optimized Indocyanine Green Nanopreparations for Biomedical Applications,” Coordination Chemistry Reviews 528 (2025): 216422.
|
| [40] |
X. Zhang, J. Gao, Y. Tang, et al., “Bioorthogonally Activatable Cyanine Dye With Torsion-induced Disaggregation for in Vivo Tumor Imaging,” Nature Communications 13 (2022): 3513.
|
| [41] |
R. A. Skolik, C. Noud, S. Oliver, et al., “Biochemical Control of the Mitochondrial Protein MitoNEET by Biological Thiols and Lipid-derived Electrophiles,” Advances in Redox Research 7 (2023): 100059.
|
| [42] |
Y. Yue, F. Huo, X. Li, et al., “pH-Dependent Fluorescent Probe That Can Be Tuned for Cysteine or Homocysteine,” Organic Letters 19 (2016): 82–85.
|
| [43] |
Y. Yue, F. Huo, X. Pei, Y. Wang, and C. Yin, “Fluorescent Imaging of Resveratrol Induced Subcellular Cysteine Up-Regulation,” Analytical Chemistry 92 (2020): 6598–6603.
|
| [44] |
D. Lee, G. Kim, J. Yin, and J. Yoon, “An Aryl-thioether Substituted Nitrobenzothiadiazole Probe for the Selective Detection of Cysteine and Homocysteine,” Chemical Communications 51 (2015): 6518–6520.
|
| [45] |
W. Ding, S. Yao, Y. Chen, et al., “A Near-Infrared Fluorescent and Photoacoustic Probe for Visualizing Biothiols Dynamics in Tumor and Liver,” Molecules 28 (2023): 2229.
|
| [46] |
J. Cai, C. Lian, Z. Lu, et al., “FGF19-Based Mini Probe Targeting FGFR4 for Diagnosis and Surgical Navigation of Hepatocellular Carcinoma,” Journal of Medicinal Chemistry 67 (2024): 3764–3777.
|
| [47] |
Y. Tang, Y. Li, C. He, et al., “NIR-II-excited off-on-off Fluorescent Nanoprobes for Sensitive Molecular Imaging in Vivo,” Nature Communications 16 (2025): 278.
|
| [48] |
X. Wu, Y. Deng, R. Wang, et al., “Rational Design of an Activatable Near-Infrared Fluorogenic Platform for in Vivo Orthotopic Tumor Imaging and Resection,” Angewandte Chemie International Edition 64 (2024): e202416877.
|
| [49] |
K. Li, X. Duan, Z. Jiang, et al., “J-aggregates of Meso-[2.2]paracyclophanyl-BODIPY Dye for NIR-II Imaging,” Nature Communications 12 (2021): 2376.
|
Rights & permissions
2026 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.
PDF
(6216KB)
