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MMP-2/9-Specific Activatable Lifetime Imaging Agent.

Rood MT, Raspe M, ten Hove JB, Jalink K, Velders AH, van Leeuwen FW - Sensors (Basel) (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Leiden 2300RC, The Netherlands. m.t.m.rood@lumc.nl.

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.

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Confocal images of SKOV-3 cells after 24 h incubation with 2L (A–C) or 2D (D–F). (A,D) Cy3 channel in green; (B,E) Cy5 channel in red; (D,F) Overlay of differential interference contrast, nuclear stain (Hoechst 33342, blue), Cy3 (green), and Cy5 (red). Excitation and emission wavelengths are described in the Experimental Section.
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sensors-15-11076-f004: Confocal images of SKOV-3 cells after 24 h incubation with 2L (A–C) or 2D (D–F). (A,D) Cy3 channel in green; (B,E) Cy5 channel in red; (D,F) Overlay of differential interference contrast, nuclear stain (Hoechst 33342, blue), Cy3 (green), and Cy5 (red). Excitation and emission wavelengths are described in the Experimental Section.

Mentions: After successful cleavage of the activatable imaging agents by cells in suspension, SKOV-3 cells were grown on a glass slide and incubated in vitro with 2L and 2D. Confocal microscopy was used to examine the cleavage activity after 24 h of incubation. Just as it was observed for the cleavage in suspension (Figure 3), peptide 2L yielded an activated donor (Cy3) emission, which was not detected for 2D (Figure 4B,E). The emission of Cy5 was used as a control, since the acceptor fluorescence upon direct acceptor excitation is not affected by the presence or absence of the Cy3 donor. Cy5 intensities of 2L and 2D were similar (Figure 4A,D).


MMP-2/9-Specific Activatable Lifetime Imaging Agent.

Rood MT, Raspe M, ten Hove JB, Jalink K, Velders AH, van Leeuwen FW - Sensors (Basel) (2015)

Confocal images of SKOV-3 cells after 24 h incubation with 2L (A–C) or 2D (D–F). (A,D) Cy3 channel in green; (B,E) Cy5 channel in red; (D,F) Overlay of differential interference contrast, nuclear stain (Hoechst 33342, blue), Cy3 (green), and Cy5 (red). Excitation and emission wavelengths are described in the Experimental Section.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4481940&req=5

sensors-15-11076-f004: Confocal images of SKOV-3 cells after 24 h incubation with 2L (A–C) or 2D (D–F). (A,D) Cy3 channel in green; (B,E) Cy5 channel in red; (D,F) Overlay of differential interference contrast, nuclear stain (Hoechst 33342, blue), Cy3 (green), and Cy5 (red). Excitation and emission wavelengths are described in the Experimental Section.
Mentions: After successful cleavage of the activatable imaging agents by cells in suspension, SKOV-3 cells were grown on a glass slide and incubated in vitro with 2L and 2D. Confocal microscopy was used to examine the cleavage activity after 24 h of incubation. Just as it was observed for the cleavage in suspension (Figure 3), peptide 2L yielded an activated donor (Cy3) emission, which was not detected for 2D (Figure 4B,E). The emission of Cy5 was used as a control, since the acceptor fluorescence upon direct acceptor excitation is not affected by the presence or absence of the Cy3 donor. Cy5 intensities of 2L and 2D were similar (Figure 4A,D).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Leiden 2300RC, The Netherlands. m.t.m.rood@lumc.nl.

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.

Show MeSH