Erasmus Medical Center, Optical molecular Imaging, Department of Radiology and Nuclear Medicine, Rotterdam, Netherlands; Erasmus Medical Center, Department of Molecular Genetics, Rotterdam, Netherlands; Percuros B.V., Leiden, Netherlands.
Promega Biosciences L.L.C., San Luis Obispo, United States.
J Photochem Photobiol B. 2021 Mar;216:112128. doi: 10.1016/j.jphotobiol.2021.112128. Epub 2021 Jan 26.
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.
纳米萤光素酶最近因其体积小和优越的生物发光性能而受到关注。对于体内成像应用，纳米萤光素酶受到其底物 furimazine 的限制，furimazine 的水溶性和生物利用度低。在此，我们比较了最近报道的用于小鼠体内成像的纳米萤光素酶底物的性能。两种水溶性得到改善的底物，氢 furimazine 和氟 furimazine，以及三种稳定的 O-乙酰化 furimazine 类似物 hikarazines 一起进行了评估。在体外测试时，所有 5 种类似物与标准的纳米萤光素酶底物 furimazine 相比，显示出更高的信号强度和反应持续时间。在体外研究中表现最好的两种类似物被选中进行进一步的体内测试。纳米萤光素酶/氟 furimazine 对在静脉注射后表现出最高的生物发光强度。与纳米萤光素酶/furimazine 和纳米萤光素酶/hikarazine-003 对相比，其亮度分别提高了约 9 倍和 11 倍，当腹膜内注射底物时，平均光发射强度提高了 3 倍，在皮下模型中。令人兴奋的是，尽管纳米萤光素酶/氟 furimazine 主要发射蓝光，但我们证明可以通过纳米萤光素酶/氟 furimazine 对来可视化被困在小鼠肺部血管中的细胞，并将结果与 AkaLuc/AkaLumine 系统进行比较。因此，在所测试的类似物中，氟 furimazine 能够实现更高的底物加载和小动物光学成像灵敏度的提高，从而升级了用于深层组织成像的基于纳米萤光素酶的生物发光系统的使用。
