Abstract
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.
Highlights
The use of luminescence in molecular imaging has many advantages
We have previously shown that covalent attachment of Ir(ppy)3 and Cy5 using a disulfide bond generates a luminophore pair in which a combination of spin-orbit coupling by the iridium atom and Förster Resonance Energy Tranfer (FRET) quenches the luminescent signal of both luminophores [29]
The cells were grown on a glass-bottom well, after which they were incubated at 37 °C in a solution of an activatable imaging agent in Dulbecco’s minimum essential medium (DMEM) for 24 h (0.2 μM for 2L and 2D; 0.5 μM for 3L and 3D)
Summary
The use of luminescence in molecular imaging has many advantages. Luminescence is a high-resolution, real-time imaging technology that requires low concentrations of label. We have previously shown that covalent attachment of Ir(ppy) and Cy5 using a disulfide bond generates a luminophore pair in which a combination of spin-orbit coupling by the iridium atom and FRET quenches the luminescent signal of both luminophores [29]. This quenching was accompanied by a shortening of the Ir(ppy) related lifetime. In this approach, the luminescence lifetime should allow for the separation of luminescence signal from autofluorescence, while the signal activation should reduce the background signal generated by non- distributed imaging agent [29,30,31]. Ir(ppy)3-Cy5 pair combined with MMP-2/9 selective signal activation has the potential to provide a new generation of molecular imaging agents
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