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Preparation of AIEgen-based near-infrared afterglow luminescence nanoprobes for tumor imaging and image-guided tumor resection

Nature Protocols volume 19, pages 2408–2434 (2024)Cite this article

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Abstract

Fluorescence imaging represents a vital tool in modern biology, oncology and biomedical applications. Afterglow luminescence (AGL), which circumvents the light scattering and tissue autofluorescence interference associated with real-time excitation source, shows remarkably increased imaging sensitivity and depth. Here we present a protocol for the design and synthesis of AGL nanoprobes with an aggregation-induced emission (AIE) effect to simultaneously red shift and amplify the afterglow signal for tumor imaging and image-guided tumor resection. The nanoprobe (AGL AIE dot) is composed of an enol ether format of Schaap’s agent and a near-infrared AIE fluorogen (AIEgen) (tetraphenylethylene-phenyl-dicyanomethylene-4H-chromene, TPE-Ph-DCM) to suppress the nonradiative dissipation pathway. Pre-irradiating AGL AIE dots with white light could generate singlet oxygen to convert Schaap’s agent to its 1,2-dioxetane format, thus initializing the AGL process. With the aid of AIEgen, the AGL shows simultaneously red shifted emission maximum (from ~540 nm to ~625 nm) and enhanced intensity (by 3.2-fold), facilitating better signal-to-background ratio, imaging sensitivity and depth. Intriguingly, the activated AGL can last for over 10 days. Compared with conventional approaches, our method provides a new solution to concurrently red shift and amplify afterglow signals for better in vivo imaging outcomes. The preparation of AGL AIE dots takes ~2 days, the in vitro characterization takes ~10 days (less than 1 day if not involving afterglow kinetic profile study) and the tumor imaging and image-guided tumor resection takes ~7 days. These procedures can be easily reproduced and amended after standard laboratory training in chemical synthesis and animal handling.

Key points

  • The protocol describes the synthesis and characterization of near-infrared afterglow nanoprobes with an aggregation-induced emission effect to simultaneously red shift and amplify the afterglow signal.

  • Due to the long-lasting and improved signal intensity, these nanoprobes represent a powerful tool for real-time in vivo imaging and have been successfully employed for intraoperative image-guided tumor resection.

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Fig. 1: Synthesis of AGL AIE dots for deep tissue afterglow imaging.
Fig. 2: A schematic of TPE-Ph-DCM and AGL AIE dot synthesis.
Fig. 3: Overview of applying AGL AIE dots for tumor imaging and image-guided tumor resection.
Fig. 4: AIE effect and 1O2 generation characterization of TPE-Ph-DCM.
Fig. 5: Characterization of AGL AIE dots.
Fig. 6: In vitro validation of afterglow imaging ability of AGL AIE dots.
Fig. 7: Afterglow imaging of tumor and afterglow image-guided tumor resection with AGL AIE dots.

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Data availability

The main data discussed in this protocol are available in the supporting primary research paper51. The raw datasets are provided in the Source Data file. The online version also contains a Supplementary Information PDF file. All other data are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC) (grant nos. 51961160730, 52225310, 22205067, 32201178, 32371449), the Fundamental Research Funds for the Central Universities and the Tianjin Science Fund for Distinguished Young Scholars (19JCJQJC61200), Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates (2023B1212060003), Xi’an Jiaotong University Young Talent Support Grant.

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Authors and Affiliations

  1. Frontiers Science Center for Cell Responses, State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin, China

    Chao Chen, Xiaoyan Zhang, Zhiyuan Gao & Dan Ding

  2. State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, School of Materials Science and Engineering, South China University of Technology, Guangzhou, P. R. China

    Guangxue Feng

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  1. Chao Chen
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Contributions

C.C., G.F. and D.D. conceived the idea of the project, C.C., Z.G. and X.Z. contributed to the experimal work invivoled in this protocol. C.C, Z.G., X.Z., G.F. and D.D. wrote the protocol, D.D. supervised the study and the prepration of the masnucript. All authors contributed to the editing and reviewing of the draft and approved the final manscript.

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Correspondence to Guangxue Feng or Dan Ding.

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Nature Protocols thanks Qian Ju, Kanyi Pu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Ni, X. et al. Nano Lett. 19, 318–330 (2019): https://doi.org/10.1021/acs.nanolett.8b03936

Chen, C. et al. J. Am. Chem. Soc. 144, 3429–3441 (2022): https://doi.org/10.1021/jacs.1c11455

Gao, Z. et al. Angew. Chem. Int. Ed. Engl. 61, e202209793 (2022): https://doi.org/10.1002/anie.202209793

Li, J. et al. Adv. Funct. Mater. 33, 2212380 (2023): https://doi.org/10.1002/adfm.202212380

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Chen, C., Zhang, X., Gao, Z. et al. Preparation of AIEgen-based near-infrared afterglow luminescence nanoprobes for tumor imaging and image-guided tumor resection. Nat Protoc 19, 2408–2434 (2024). https://doi.org/10.1038/s41596-024-00990-4

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