Evaluation of NanoLuc substrates for bioluminescence imaging of transferred cells in mice

Natasa Gaspar, JR Walker, Giorgia Zambito, Kranthi Panth, Clemens Löwik, TA Kirkland, Laura Mezzanotte

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NanoLuc luciferase recently gained popularity due to its small size and superior bioluminescence performance. For in vivo imaging applications, NanoLuc has been limited by its substrate furimazine, which has low solubility and bioavailability. Herein, we compared the performances of recently reported NanoLuc luciferase substrates for in vivo imaging in mice. Two substrates with improved aqueous solubility, hydrofurimazine and fluorofurimazine, were evaluated along with three stabilized O-acetylated furimazine analogues, the hikarazines. All 5 analogues, when tested in vitro, displayed greater signal intensity and reaction duration, in comparison to the standard NanoLuc substrate, furimazine. The two best-performing analogues from the in vitro study were selected for further in vivo testing. The NanoLuc/fluorofurimazine pair demonstrated the highest bioluminescence intensity, post intravenous administration. It was found to be around 9-fold brighter compared to the NanoLuc/furimazine and 11-fold more intense than the NanoLuc/hikarazine-003 pair, with an average of 3-fold higher light emission when the substrate was injected intraperitoneally, in a subcutaneous model. Excitingly, despite the fact that NanoLuc/fluorofurimazine emits mostly blue light, we prove that cells trapped in mice lungs vasculature could be visualised via the NanoLuc/fluorofurimazine pair and compare the results to the AkaLuc/AkaLumine system. Therefore, among the tested analogues, fluorofurimazine enables higher substrate loading and improved optical imaging sensitivity in small animals, upgrading the use of NanoLuc derived bioluminescent systems for deep tissue imaging.

Original languageEnglish
Article number112128
JournalJournal of Photochemistry and Photobiology B: Biology
Publication statusPublished - Mar 2021

Bibliographical note

Funding Information:
We acknowledge the funding for this work provided by the European Commission under the H2020-MSCA-RISE award grant number 777682 (CANCER) and under the H2020-MSCA-ITN award, grant number 675743 (ISPIC).Yves L. Janin is acknowledged for kindly providing a sample of the luciferase prosubstrates; hikarazine-001, hikarazine-003 and hikarazine-097. This work was supported by the European H2020 MSCA award under proposal number 675743 (project acronym: ISPIC) and 777682 (project acronym: CANCER) and the Applied Molecular Imaging Erasmus MC (AMIE) facility.

Publisher Copyright:
© 2021 The Authors

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