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NIR-II aggregation-induced emission nanoparticles camouflaged with preactivated macrophage membranes for phototheranostics of pulmonary tuberculosis

Nature Protocols volume 20, pages 2560–2585 (2025)Cite this article

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Abstract

Phototheranostics, which allows simultaneous diagnosis and therapy, offers notable advantages in terms of noninvasiveness, controllability and negligible drug resistance, presenting a promising approach for disease treatment. By integrating second near-infrared (NIR-II, 1,000–1,700 nm) phototheranostic agents characterized by aggregation-induced emission (AIE) and cell membranes with specific targeting capacity, we have developed a versatile type of biomimetic nanoparticle (NP) for precise phototheranostics of pulmonary tuberculosis (TB). Coating the phototheranostic agents with preactivated macrophage membranes results in the formation of biomimetic NPs, which exhibit specific binding to TB through a lesion–pathogen dual-targeting strategy, allowing the accurate detection of Mycobacterium tuberculosis via NIR-II fluorescence imaging and precise photothermal therapy using the irradiation of a 1,064 nm laser. In comparison with traditional treatments, small individual granulomas (0.2 mm in diameter) in TB-infected mice are visualized, and improved antibacterial effects are achieved upon NP administration. Here we present a standardized workflow for the synthesis of the NIR-II AIE agents, their use for the fabrication of the biomimetic NPs and their in vitro and in vivo applications as phototheranostics against M. tuberculosis. The preparation and characterization of the NIR-II AIE agents requires ~8 d, the synthesis and characterization of the phototheranostic NPs requires ~8 d, the validation of in vitro targeting capacity and photothermal eradication requires ~26 d, and the in vivo NIR-II fluorescence imaging and imaging-guided photothermal therapy requires ~74 d. All procedures are straightforward and suitable for clinicians or researchers with prior training in organic synthesis and biomedical engineering.

Key points

  • The procedure covers the synthesis of the photothermal agents, the preactivation of macrophages, membrane extraction and coextrusion to create the final biomimetic nanoparticles, as well as validation of the in vitro and in vivo image-guided photothermal therapy of pulmonary TB.

  • Alternative clinical approaches include chemotherapy, immunotherapy, photodynamic therapy and vaccines.

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Fig. 1: A precise theranostic strategy for TB based on Mycobacterium preactivated macrophage membrane-coated photothermal NPs.
Fig. 2: Fabrication of macrophage-like BBTD@PM NPs.
Fig. 3: Overview of using BBTD@PM NPs for pulmonary TB phototheranostics.
Fig. 4: Characterization of BBTD@PM NPs.
Fig. 5: In vitro validation of targeting and photothermal sterilization activity of BBTD@PM NPs.
Fig. 6: In vivo NIR-IIb FLI of tuberculous granulomas and PTT against tuberculous granulomas.

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Data availability

The main data discussed in this protocol are available in the supporting primary research paper45. 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 partially supported by the National Natural Science Foundation of China (52122317, 22175120, 22307080), the Developmental Fund for Science and Technology of Shenzhen Government (RCYX20200714114525101), The Shenzhen Science and Technology Program (RCBS20221008093224016, JCYJ20220531101201003, 20220809130438001), the Guangdong Basic and Applied Basic Research Fund (2023A1515010558), the Pearl River Talent Recruitment Program (2019QN01Y103), the Research Team Cultivation Program of Shenzhen University (2023QNT003), the Medical-Engineering Interdisciplinary Research Foundation of Shenzhen University (2023YG021) and the Medicine Plus Program of Shenzhen University (2024YG004). The authors also acknowledge the Instrumental Analysis Center of Shenzhen University.

Author information

Authors and Affiliations

  1. Center for AIE Research, Guangdong Provincial Key Laboratory of New Energy Materials Service Safety, College of Materials Science and Engineering, Shenzhen University, Shenzhen, China

    Dingyuan Yan, Xue Li, Ben Zhong Tang & Dong Wang

  2. Institute for Engineering Medicine, Kunming Medical University, Kunming, China

    Huanhuan Wang, Bin Li, Wei Wang & Yuhui Liao

  3. School of Science and Engineering, Shenzhen Institute of Aggregate Science and Technology, The Chinese University of Hong Kong, Shenzhen (CUHK–Shenzhen), Guangdong, China

    Ben Zhong Tang

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  1. Dingyuan Yan
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  2. Xue Li
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  4. Bin Li
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  5. Wei Wang
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  6. Yuhui Liao
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Contributions

D.Y., D.W. and B.Z.T. conceived and initiated the project. D.Y., H.W. and B.L. contributed to the experimental work displayed in this protocol. D.Y., X.L., H.W., Y.L., W.W. and D.W. wrote the protocol. D.W. and B.Z.T. supervised the study and the manuscript preparation. All authors reviewed and edited the manuscript and approved the final draft. Lab links: https://www.kmmc.cn/Pages_2592_53259.aspx; https://tangbenz.people.ust.hk/; https://cmse.szu.edu.cn/szdw1/jsml/gfzclygc/j_s/wd.htm.

Corresponding authors

Correspondence to Yuhui Liao, Ben Zhong Tang or Dong Wang.

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Nature Protocols thanks Kanyi Pu and Fan Zhang for their contribution to the peer review of this work.

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Key references

Li, B. et al. Nat. Nanotechnol. 19, 834–845 (2024): https://doi.org/10.1038/s41565-024-01618-0

Wang, W. et al. Matter 7, 1187–1206 (2024): https://doi.org/10.1016/j.matt.2024.01.004

Li, B. et al. Adv. Sci. 8, 2003556 (2021): https://doi.org/10.1002/advs.202003556

Wang, M. et al. Adv. Funct. Mater. 32, 2205371 (2022): https://doi.org/10.1002/adfm.202205371

Yan, D. et al. Angew. Chem. Int. Ed. 60, 26769–26776 (2021): https://doi.org/10.1002/anie.202111767

Supplementary information

Supplementary Information

Supplementary Figs. 1–11 and procedures for characterizing the intermediates and TPE-BT-BBTD, NIR-II imaging and biotoxicity evaluation.

Source data

Source Data Figs. 4 and 5

Statistical Source Data.

Source Data Fig. 4

Parent Figure.

Source Data Supplementary Fig. 11

Statistical Source Data.

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Yan, D., Li, X., Wang, H. et al. NIR-II aggregation-induced emission nanoparticles camouflaged with preactivated macrophage membranes for phototheranostics of pulmonary tuberculosis. Nat Protoc 20, 2560–2585 (2025). https://doi.org/10.1038/s41596-025-01146-8

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