Monitoring the binding of protein ligands and therapeutic antibodies to their respective receptors (target engagement) is crucial to compound prioritization in anti-cancer targeted drug screening. However, current in vivo optical imaging techniques cannot distinguish between co-localization and actual receptor-ligand binding at the tumor region. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The homodimeric nature of TfR allows for measuring fluorescence lifetime FRET (FLI-FRET) to quantitate the TfR-Tf binding and internalization into cancer cells, based on the reduction of donor fluorophore lifetime. Near infrared (NIR) FLI-FRET has been used to directly visualize and quantitate TfR-Tf binding and internalization by providing the fraction of donor-labeled entity that is interacting with its respective receptor. NIR FLI-FRET has been validated at multiscale, using both in vitro microscopy as well as in vivo macroscopy whole-body deep imaging assays using different NIR FRET pairs. Accuracy of NIR FLI-FRET quantitation has been compared between fluorescence intensity and lifetime measurements using both microscopy and macroscopy fluorescence imaging. NIR FLI-FRET employs well-characterized quantitative lifetime-based metrics, standard in FRET microscopy, but with the additional benefit of a seamless multiscale technological platform. In summary, we have successfully demonstrated quantitative imaging of receptor-mediated binding and uptake of Tf using NIR FLI-FRET microscopy and macroscopy imaging in vitro and in vivo, respectively. This novel approach can be extended to other receptors, currently targeted in oncology. Hence, NIR FLI-FRET can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.
Margarida Barroso, Alena Rudkouskaya, Jamie Ward, Joseph E. Mazurkiewicz, Nattawut Sinsuebphon, Sez-Jade Chen, and Xavier Intes, "Near infrared fluorescence lifetime FRET imaging of target engagement at multiscale (Conference Presentation)," Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104980R (Presented at SPIE BiOS: January 29, 2018; Published: 14 March 2018); https://doi.org/10.1117/12.2297567.5751624473001.
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Study of self-shadowing effect as a simple means to realize nanostructured thin films and layers with special attentions to birefringent obliquely deposited thin films and photo-luminescent porous silicon