Theranostics 2019; 9(9):2646-2661. doi:10.7150/thno.32219

Research Paper

Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo

Ryo Nishihara1,2#, Ramasamy Paulmurugan2#, Takahiro Nakajima3, Eiji Yamamoto4, Arutselvan Natarajan2, Rayhaneh Afjei2, Yuki Hiruta1, Naoko Iwasawa1, Shigeru Nishiyama1, Daniel Citterio1, Moritoshi Sato3, Sung Bae Kim2,5,✉, Koji Suzuki1,✉

1. Department of Applied Chemistry, Faculty of Science and Technology, Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
2. Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Palo Alto, California 94304, United States
3. Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1, Komaba Meguro-ku, Tokyo 153-8902, Japan
4. Graduate School of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan
5. Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan
# Equal contribution

Abstract

Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo.

Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ~300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named “Bottle Blue (BBlue)” and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe.

Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ~50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region.

Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

Keywords: Bioluminescence Imaging, bioluminescence resonance energy transfer (BRET), coelenterazine derivatives, blue-to-near infrared shift, metastasis

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
How to cite this article:
Nishihara R, Paulmurugan R, Nakajima T, Yamamoto E, Natarajan A, Afjei R, Hiruta Y, Iwasawa N, Nishiyama S, Citterio D, Sato M, Kim SB, Suzuki K. Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo. Theranostics 2019; 9(9):2646-2661. doi:10.7150/thno.32219. Available from http://www.thno.org/v09p2646.htm