Cell permeable organometallic lanthanide bioprobes for time-resolved near-infrared II imaging BS69 in EBNA2-associated cancer therapy

The main problem of commercial available molecular imaging agents such as fluorescein is high background noises during microscopy study. These noises are caused by biological samples’ autofluorescence scattering light from solid substrates, and lastly the luminescence from the optical components. For this purpose, more attention has been focused on the time-resolved fluorescent technique and using lanthanide complexes as imaging labels. Time-resolved spectroscopy can determine the excited lifetimes which provide insight of the excited dynamics and the decay pathways of the excited chromophore/materials. With this technique, it is possible, for example, to extract information based on differences in their fluorescence lifetime. The autofluoresence is happened in nano to few micro-second scales. The emission lifetime of lanthanide is in micro-milli second scale which is perfect for time-resolved imaging, especially lined up with the higher tissue penetration near-infrared II (NIR-II, 1000-1700 nm) emissive lanthanide ion, such as ytterbium/erbium/neodymium. However, most reported time- resolved lanthanide probes was only available in visible to NIR-I region (700-1000 nm) and non-specific in vitro.

In this project, we are going to develop new lanthanide bioprobes (ytterbium/erbium) for time-resolved NIR-II imaging and targeting key functional protein for EBV associated cancer growth-BS69. The guideline for selection of NIR-II probe via its brightness and emission quantum yield will be demonstrated. The interference BS69 by EBNA2 protein is well known to initialize overexpressed of EBV associated cancer cells. However, the detailed in situ interacting mechanism between BS69 and EBNA2 is unclear due to lack of direct imaging agents. The lanthanide bioprobes proposed herein will be a dual function bioprobe - not only to monitor the presence of BS69 and is also able to inhibit the EBNA2-associated cancer growth via the physical interference of the BS69-EBNA2 complex. The interaction of BS69 and EBNA2 will be visualized by our proposed lanthanide probes and monitored with an in-house time-resolved microscope equipped with NIR-II camera. Four preliminary experiments have been performed to support our hypothesis, especially the binding of the proposed ytterbium complexes with the BS69 protein is confirmed by responsive NIR-II emission, Western blot and in vitro selective toxicity in the EBNA2-positive cell lines. We strongly believe this proposed study will be a pioneering work for the systematic synthesis of lanthanide-derived imaging agents and their development into time-resolved luminescent probes for NIR-II imaging. Thus, the results of this study will broaden the horizon for the vital stains and traditional heterocyclic/organic fluorophores that are currently available commercially.