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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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(A,B) Excitation-emission plots of peptides 2L (A) and 2D (B). The peaks labeled as “FRET” are the peaks that show acceptor emission (670 nm) with donor excitation (550 nm); (C) Luminescence decay traces at 600 nm of 3L, 3D, and reference compound Ir(ppy)3-COOH in water. All compounds were excited with a 372 nm laser at 2.5 MHz.
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sensors-15-11076-f002: (A,B) Excitation-emission plots of peptides 2L (A) and 2D (B). The peaks labeled as “FRET” are the peaks that show acceptor emission (670 nm) with donor excitation (550 nm); (C) Luminescence decay traces at 600 nm of 3L, 3D, and reference compound Ir(ppy)3-COOH in water. All compounds were excited with a 372 nm laser at 2.5 MHz.

Mentions: In the reference compounds 2L and 2D, the close proximity of the donor (Cy3) and acceptor (Cy5) yielded a very clear FRET interaction, as expected [34,38]. In the excitation-emission plots, a peak that corresponds to donor excitation (550 nm) and acceptor emission (670 nm) can be seen for both compounds (Figure 2A,B). However, there are clear differences in quenching efficiency between 2L (43% quenching) and 2D (88% quenching) (Figure 2). Based on the fluorescence intensities, we calculated the Cy3-Cy5 distances to be 53 Å for 2L and 38 Å for 2D. This difference in calculated distance between the dyes can be caused by a slightly different structural configuration of the hairpin structure with the D-amino acids compared to the one with L-amino acids. Since FRET efficiency depends to the sixth power on the donor-acceptor distance, these relatively small differences in the hairpin configuration may explain the much larger differences in FRET efficiency. Another possibility is that the difference in amino acid stereochemistry leads to a difference in dipole orientation. This also has a pronounced effect on the correct calculation of the distances [33].


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)

(A,B) Excitation-emission plots of peptides 2L (A) and 2D (B). The peaks labeled as “FRET” are the peaks that show acceptor emission (670 nm) with donor excitation (550 nm); (C) Luminescence decay traces at 600 nm of 3L, 3D, and reference compound Ir(ppy)3-COOH in water. All compounds were excited with a 372 nm laser at 2.5 MHz.
© Copyright Policy
Related In: Results  -  Collection

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

sensors-15-11076-f002: (A,B) Excitation-emission plots of peptides 2L (A) and 2D (B). The peaks labeled as “FRET” are the peaks that show acceptor emission (670 nm) with donor excitation (550 nm); (C) Luminescence decay traces at 600 nm of 3L, 3D, and reference compound Ir(ppy)3-COOH in water. All compounds were excited with a 372 nm laser at 2.5 MHz.
Mentions: In the reference compounds 2L and 2D, the close proximity of the donor (Cy3) and acceptor (Cy5) yielded a very clear FRET interaction, as expected [34,38]. In the excitation-emission plots, a peak that corresponds to donor excitation (550 nm) and acceptor emission (670 nm) can be seen for both compounds (Figure 2A,B). However, there are clear differences in quenching efficiency between 2L (43% quenching) and 2D (88% quenching) (Figure 2). Based on the fluorescence intensities, we calculated the Cy3-Cy5 distances to be 53 Å for 2L and 38 Å for 2D. This difference in calculated distance between the dyes can be caused by a slightly different structural configuration of the hairpin structure with the D-amino acids compared to the one with L-amino acids. Since FRET efficiency depends to the sixth power on the donor-acceptor distance, these relatively small differences in the hairpin configuration may explain the much larger differences in FRET efficiency. Another possibility is that the difference in amino acid stereochemistry leads to a difference in dipole orientation. This also has a pronounced effect on the correct calculation of the distances [33].

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
Related in: MedlinePlus