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Preparation of ENBSe-based photoredox catalysts for O2-independent phototherapy in living systems

Nature Protocols (2026)Cite this article

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Abstract

Photodynamic therapy using an appropriate photocatalyst results in the production of cytotoxic reactive oxygen species for tumor ablation. However, the inherent O2 dependence of conventional photodynamic therapy limits its clinical translation. To overcome this challenge, here we developed a selenium-substituted Nile blue derivative (ENBSe) as a versatile O2-independent photocatalyst. Under near-infrared light irradiation, ENBSe can drive the biological oxidation of NADH to NAD+ while simultaneously triggering the cascade reduction of cytochrome c (Fe³⁺ to Fe²⁺), even in the absence of O2. To improve tumor specificity and targeting, we further developed a conditionally activatable photoredox catalysis (ConAPC) system. ENBSe is covalently attached to 4-nitrobenzyl chloride via a carbonic anhydride bond, wherein the nitro group can be specifically cleaved by nitroreductase (NTR), an enzyme overexpressed in hypoxic tissues. Such ConAPC design prevents reaction with NADH and quenches the fluorescences of ENBSe, which means that the drug molecule, ENBSe–NTR, is catalytically inactive. ENBSe–NTR is, to our knowledge, the first tumor microenvironment-responsive ConAPC molecule that enables O2-free, tumor-specific catalytic therapy. By replacing the 4-nitrobenzyl chloride group, it might be possible to target cells with different microenvironmental conditions. This protocol presents a standardized workflow encompassing the synthesis of ENBSe and its application for photocatalytic modulation of cellular electron flow in the mitochondrial electron transport chain via an O2-independent mechanism of action. The outlined protocol specifies a synthesis period of ~4 d for ENBSe, ~4 h for photoredox spectroscopic characterization and 4–5 weeks for photodiagnostic assessment in cancer cell and mice models.

Key points

  • ENBSe can initiate an O2-independent photoredox catalysis to trigger a cascaded NADH and cytochrome c (Fe3+) conversion, thereby specifically disrupting the mitochondrial electron transport chain. This offers a novel O2-independent photocatalytic antitumor strategy.

  • The procedure covers the synthesis and characterization, the target sensitivity and specificity, the photocatalytic efficiency, as well as the validation of the phototheranostics in vitro and in vivo of the photoredox catalyst ENBSe.

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Fig. 1: A photochemical reaction in PDT.
Fig. 2: An overview of ENBSe-mediated O2-independent photoredox catalysis inducing mitochondrial metabolism dysfunction against a hypoxic tumor.
Fig. 3: Synthetic routes of ENBSe and ENBSe–NTR.
Fig. 4: The design plan for the hypoxic tumor-specific ConAPC system, that is, ENBSe–NTR, selectively triggered by NTR.
Fig. 5: An overview of applying ENBSe for photoredox catalytic therapy.
Fig. 6: The photoredox catalytic properties of ENBSe.
Fig. 7: Schematic illustration of ENBSe-mediated photoredox catalysis under hypoxia and normoxia.
Fig. 8: Schematic for the ConAPC system ENBSe–NTR.
Fig. 9: ENBSe-mediated photocatalytic mitochondrial morphology changes.
Fig. 10: Photocatalytic theranostic performance of ENBSe and ENBSe–NTR in vivo.

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Data availability

The main data discussed in this protocol are available in the supporting primary research paper. All other data are available for research purposes from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Jassim, A., Rahrmann, E. P., Simons, B. D. & Gilbertson, R. J. Cancers make their own luck: theories of cancer origins. Nat. Rev. Cancer 23, 710–724 (2023).

    Article  CAS  PubMed  Google Scholar 

  2. Zemek, R. M., Anagnostou, V., Pires da Silva, I., Long, G. V. & Lesterhuis, W. J. Exploiting temporal aspects of cancer immunotherapy. Nat. Rev. Cancer 24, 480–497 (2024).

    Article  CAS  PubMed  Google Scholar 

  3. Filho, A. M. et al. The GLOBOCAN 2022 cancer estimates: data sources, methods, and a snapshot of the cancer burden worldwide. Int. J. Cancer 156, 1336–1346 (2025).

    Article  PubMed  Google Scholar 

  4. Vickerman, B. M., Zywot, E. M., Tarrant, T. K. & Lawrence, D. S. Taking phototherapeutics from concept to clinical launch. Nat. Rev. Chem. 5, 816–834 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Huang, L. & Han, G. Triplet-triplet annihilation photon upconversion-mediated photochemical reactions. Nat. Rev. Chem. 8, 238–255 (2024).

    Article  CAS  PubMed  Google Scholar 

  6. Li, M. et al. New guidelines and definitions for type I photodynamic therapy. Chem. Soc. Rev. 54, 7025–7057 (2025).

    Article  CAS  PubMed  Google Scholar 

  7. Obaid, G. et al. Engineering photodynamics for treatment, priming and imaging. Nat. Rev. Bioeng. 2, 752–769 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Shatskiy, A., Stepanova, E. V. & Karkas, M. D. Exploiting photoredox catalysis for carbohydrate modification through C–H and C–C bond activation. Nat. Rev. Chem. 6, 782–7805 (2022).

    Article  CAS  PubMed  Google Scholar 

  9. Cabanero, D. C. & Rovis, T. Low-energy photoredox catalysis. Nat. Rev. Chem. 9, 28–45 (2024).

    Article  PubMed  Google Scholar 

  10. Twilton, J. et al. The merger of transition metal and photocatalysis. Nat. Rev. Chem. 1, 0052 (2017).

    Article  CAS  Google Scholar 

  11. Huang, H. et al. Targeted photoredox catalysis in cancer cells. Nat. Chem. 11, 1041–1048 (2019).

    Article  CAS  PubMed  Google Scholar 

  12. Liu, Z. et al. Bioorthogonal photocatalytic proximity labeling in primary living samples. Nat. Commun. 15, 2712 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Seath, C. P. et al. Tracking chromatin state changes using nanoscale photo-proximity labelling. Nature 616, 574–580 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Oslund, R. C. et al. Detection of cell–cell interactions via photocatalytic cell tagging. Nat. Chem. Biol. 18, 850–858 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Rosenberger, J. E. et al. Ligand-directed photocatalysts and far-red light enable catalytic bioorthogonal uncaging inside live cells. J. Am. Chem. Soc. 145, 6067–6078 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bloom, S. et al. Decarboxylative alkylation for site-selective bioconjugation of native proteins via oxidation potentials. Nat. Chem. 10, 205–211 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Xu, Y. et al. Lutetium texaphyrin: a photocatalyst that triggers pyroptosis via biomolecular photoredox catalysis. Proc. Natl Acad. Sci. USA 121, e2314620121 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Li, M. et al. Photoredox catalysis may be a general mechanism in photodynamic therapy. Proc. Natl Acad. Sci. USA 119, e2210504119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhao, X., Liu, J., Fan, J., Chao, H. & Peng, X. Recent progress in photosensitizers for overcoming the challenges of photodynamic therapy: from molecular design to application. Chem. Soc. Rev. 50, 4185–4219 (2021).

    Article  CAS  PubMed  Google Scholar 

  20. Cheng, J. et al. Biofilm heterogeneity-adaptive photoredox catalysis enables red light-triggered nitric oxide release for combating drug-resistant infections. Nat. Commun. 14, 7510 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ryu, K. A., Kaszuba, C. M., Bissonnette, N. B., Oslund, R. C. & Fadeyi, O. O. Interrogating biological systems using visible-light-powered catalysis. Nat. Rev. Chem. 5, 322–337 (2021).

    Article  CAS  PubMed  Google Scholar 

  22. Buksh, B. F. et al. Map-red: proximity labeling by red light photocatalysis. J. Am. Chem. Soc. 144, 6154–6162 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Prier, C. K., Rankic, D. A. & MacMillan, D. W. Visible light photoredox catalysis with transition metal complexes: applications in organic synthesis. Chem. Rev. 113, 5322–5363 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Spinelli, J. B. & Haigis, M. C. The multifaceted contributions of mitochondria to cellular metabolism. Nat. Cell Biol. 20, 745–754 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nakai, R. et al. Mitochondria transfer-based therapies reduce the morbidity and mortality of Leigh syndrome. Nat. Metab. 6, 1886–1896 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Xiong, T. et al. Pyrazolone-protein interaction enables long-term retention staining and facile artificial biorecognition on cell membranes. J. Am. Chem. Soc. 146, 24158–24166 (2024).

    Article  CAS  PubMed  Google Scholar 

  27. Xiong, T. et al. Lipid droplet targeting type I photosensitizer for ferroptosis via lipid peroxidation accumulation. Adv. Mater. 36, e2309711 (2024).

    Article  PubMed  Google Scholar 

  28. Chen, Y. et al. Self-reporting photodynamic nanobody conjugate for precise and sustainable large-volume tumor treatment. Nat. Commun. 15, 6935 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li, M. et al. Conditionally activatable photoredox catalysis in living systems. J. Am. Chem. Soc. 144, 163–173 (2022).

    Article  CAS  PubMed  Google Scholar 

  30. Li, X., Lovell, J. F., Yoon, J. & Chen, X. Clinical development and potential of photothermal and photodynamic therapies for cancer. Nat. Rev. Clin. Oncol. 17, 657–674 (2020).

    Article  PubMed  Google Scholar 

  31. Brown, S. B., Brown, E. A. & Walker, I. The present and future role of photodynamic therapy in cancer treatment. Lancet. Oncol. 5, 497–508 (2004).

    Article  CAS  PubMed  Google Scholar 

  32. Nguyen, V. N., Zhao, Z., Tang, B. Z. & Yoon, J. Organic photosensitizers for antimicrobial phototherapy. Chem. Soc. Rev. 51, 3324–3340 (2022).

    Article  CAS  PubMed  Google Scholar 

  33. Li, M. et al. Near-infrared light-initiated molecular superoxide radical generator: rejuvenating photodynamic therapy against hypoxic tumors. J. Am. Chem. Soc. 140, 14851–14859 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Deng, Z. et al. Near-infrared-activated anticancer platinum(IV) complexes directly photooxidize biomolecules in an oxygen-independent manner. Nat. Chem. 15, 930–939 (2023).

    Article  CAS  PubMed  Google Scholar 

  35. Tang, Y. et al. Oxygen-independent organic photosensitizer with ultralow-power NIR photoexcitation for tumor-specific photodynamic therapy. Nat. Commun. 15, 2530 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wang, Z. et al. A nitroreductase-activatable metabolic reporter for covalent labeling of pathological hypoxic cells in tumorigenesis. Angew. Chem. Int. Ed. 63, e202411636 (2024).

    Article  CAS  Google Scholar 

  37. Liu, H. W. et al. Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging. Chem. Soc. Rev. 47, 7140–7180 (2018).

    Article  CAS  PubMed  Google Scholar 

  38. Kim, J. et al. Bioorthogonal activation of deep red photoredox catalysis inducing pyroptosis. J. Am. Chem. Soc. 147, 701–712 (2025).

    Article  CAS  PubMed  Google Scholar 

  39. Zhu, J. et al. Icing on the cake: integrating optical fiber with second near-infrared aggregation-induced emission luminogen for exceptional phototheranostics of bladder cancer. Adv. Mater. 37, e2502452 (2025).

  40. Mouli, K. et al. Oxidized carbon nanoparticles enhance cellular energetics with application to injured brain. Adv. Healthc. Mater. 14, e2401629 (2025).

    Article  PubMed  Google Scholar 

  41. Zhao, L. et al. A host–guest approach to combining enzymatic and artificial catalysis for catalyzing biomimetic monooxygenation. Nat. Commun 11, 2903 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Reza et al. Mechanoresponsive protein crystals for NADH recycling in multicycle enzyme reactions. J. Am. Chem. Soc. 28, 18817–18822 (2024).

    Google Scholar 

  43. Zhao, Y. et al. Enhancing biosafety in photodynamic therapy: progress and perspectives. Chem. Soc. Rev. 54, 7749–7768 (2025).

    Article  CAS  PubMed  Google Scholar 

  44. Shi, Z. et al. NIR-dye bridged human serum albumin reassemblies for effective photothermal therapy of tumor. Nat. Commun. 14, 6567 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zhao, M. et al. An activatable phototheranostic probe for anti-hypoxic type I photodynamic- and immuno-therapy of cancer. Adv. Mater. 36, 2305243 (2023).

    Article  Google Scholar 

  46. Chagri, S., Ng, D. Y. W. & Weil, T. Designing bioresponsive nanomaterials for intracellular self-assembly. Nat. Rev. Chem. 6, 320–338 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Nestoros, E., Sharma, A., Kim, E., Kim, J. S. & Vendrell, M. Smart molecular designs and applications of activatable organic photosensitizers. Nat. Rev. Chem. 9, 46–60 (2025).

    Article  CAS  PubMed  Google Scholar 

  48. Yan, D., Wang, D. & Tang, B. Z. In vivo, clinical and translational aspects of aggregation-induced emission. Nat. Rev. Bioeng. 3, 976–991 (2025).

    Article  Google Scholar 

  49. Cincotta, L., Szeto, D., Lampros, E., Hasan, T. & Cincotta, A. H. Benzophenothiazine and benzoporphyrin derivative combination phototherapy effectively eradicates large murine sarcomas. Photochem. Photobiol. 63, 229–237 (2008).

    Article  Google Scholar 

  50. Yi, B. et al. Imaging heterogeneous patterns of aminopeptidase N activity in hierarchical tissue structures through high-resolution whole-organ 3D apping. Angew. Chem. Int. Ed. 64, e202504668 (2025).

    Article  CAS  Google Scholar 

  51. Gong, H. et al. Hyaluronidase to enhance nanoparticle-based photodynamic tumor therapy. Nano Lett 16, 2512–2521 (2016).

    Article  CAS  PubMed  Google Scholar 

  52. Gebremedhin, K. H. et al. Benzo[a]phenoselenazine-based NIR photosensitizer for tumor-targeting photodynamic therapy via lysosomal-disruption pathway. Dyes Pigments 170, 107617 (2019).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the financial support received from the National Natural Science Foundation of China (grant nos. 22308220 and 22090011), Shenzhen University 2035 Program for Excellent Research (grant nos. 00000208 and 00000225), The Guangdong Basic and Applied Basic Research Foundation (grant no. 2023B1515120001) and the Guangdong Province Key Areas Special Project for Regular Colleges and Universities (grant no. 2024ZDZX2018). We also thank Shenzhen University’s Third-Phase Project of Constructing High-Level University (grant no. 000001032104), the Research Team Cultivation Program of Shenzhen University (grant no. 2023QNT005), Shenzhen Science and Technology Program (grant no. RCBS20231211090515015) and the China Postdoctoral Science Foundation (grant no. 2024M752099).

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Authors and Affiliations

  1. College of Materials Science and Engineering, Shenzhen University, Shenzhen, China

    Yingying Zhang  (张莹莹), Yingnan Wu  (吴蓥男), Zehao Jing  (荆泽昊), Xiaoqiang Chen  (陈小强), Mingle Li  (李明乐) & Xiaojun Peng  (彭孝军)

  2. Key Laboratory of Optoelectronic Devices and Systems of the Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen, China

    Yingying Zhang  (张莹莹) & Yingnan Wu  (吴蓥男)

  3. The State Key Library of Fine Chemicals, Dalian University of Technology, Dalian, China

    Xiaojun Peng  (彭孝军)

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Contributions

M.L. and X.P. conceived and initiated the project. M.L. contributed to the experimental work described in this protocol. Y.Z., Y.W., Z.J. and X.C. investigated, wrote and edited the protocol. M.L. and X.P. supervised the study and the manuscript preparation. All authors reviewed and edited the manuscript and approved the final draft.

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Correspondence to Mingle Li  (李明乐).

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Key references

Li, M. et al. J. Am. Chem. Soc. 144, 163–173 (2022): https://doi.org/10.1021/jacs.1c07372

Li, M. et al. Proc. Natl Acad. Sci. USA 119, e2210504119 (2022): https://doi.org/10.1073/pnas.2210504119

Kim, J. et al. J. Am. Chem. Soc. 147, 701–712 (2025): https://doi.org/10.1021/jacs.4c13131

Xu, Y. et al. Proc. Natl Acad. Sci. USA 121, e2314620121 (2024): https://doi.org/10.1073/pnas.2314620121

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Zhang, Y., Wu, Y., Jing, Z. et al. Preparation of ENBSe-based photoredox catalysts for O2-independent phototherapy in living systems. Nat Protoc (2026). https://doi.org/10.1038/s41596-025-01328-4

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