© Carestream Health, Inc.

FLUORESCENCE QUENCHING OF NEAR-IR DYE BY NEUTRAVIDIN PEGYLATED GOLD NANOPARTICLES.

Rao V. L. Papineni*, Hans Schmitthenner, Tao Ji, Jingyi Pan.

Carestream Molecular Imaging, 4 Research Dr., Woodbridge, CT, 06525, USA. * rao.papineni1@carestreamhealth.com

AbstractActivatable probes are rapidly becoming a smart and essential tool in optical imaging of in vivo molecular targets. Addition of multimodal reporting capability to the technology along with simpler chemistry to attach substrate moieties will augment the process of developing activatable probes for novel molecular targets and pathways. In pursuit of achieving this goal, we developed a system where an organic dye conjugate can be linked by a simple step of incubation with gold nanoparticles. Biotin was conjugated to a water soluble, near-infrared (NIR) tricarbocyanine, cyclic enamine- functionalized dye. Dose-dependent fluorescence quenching was observed when biotin-NIR dye was linked to monodispersed neutravidin PEGylated gold nanoparticle (20nm). This process is reversed by the enzymatic cleavage of neutravidin by trypsin. As expected, quenching was not observed when incubating the biotin-NIR dye complex with trypsin treated neutravidin PEGylated gold nanoparticle. The high biotin-binding affinity of this fluorescence activatable system can be exploited by incorporating novel peptide substrates to the biotin-dye complex. The resultant quenching and unquenching processes of the neutravidin PEGylated gold nanoparticle and biotin-dye conjugated substrates can be monitored in vivo by NIR fluorescence, computed tomography CT and planar bench top X-ray modalities. Further, to enhance the selectivity of the activatable probe a biotinylated targeting macromolecule can be linked along with the substrate-dye complex.

Activatable Probes

ConclusionsThe large Stokes Shift NIR dye conjugated to biotin is

quenched by PEGylated neutravidin Gold

Nanoparticles.

Degradation of neutravidin by trypsin unquenches the

NIR Dye interacting with Gold Nanoparticle through

biotin linker. A property that can be well utilized in

developing Targeted activatable probes. The

resulting probes not only reports fluorescence but

also X-Ray contrast.

.

Carestream is a trademark of Carestream Health, Inc. The Kodak trademark is used under license from Kodak. Carestream

Molecular Imaging is a division of Carestream Health, Inc. Although the Carestream In-Vivo Multispectral Imaging System FX can

be used for in vivo and in vitro mol ecular imaging of materi als, researchers should be aware that the methods of preparing and

viewing the materials for molecular imaging may be subject to various patent rights.

Neutravidin PEGyl ated Gold Nanoparticl es is the trademark of Pol ysci ences Inc.

"Molecular Imaging - Wisdom To See For Maladies To Flee"Dr. Rao V. L. Papineni

NIRF Dye Conjugated Gold Nanoparticles

The Bimodal Neutravidin PEGylated Gold- Near-IR dye Conjugated Nanoparticles.Neutravidin PEGylated Gold Nanoparticles (20 nm) were obtained from Polysciences Inc. Themonodisperse nature of the nanoparticles - as confirmed from TEM image provided by Polysciences Inc (Right Panel).Step-1 PEG- Biotin was conjugated to water soluble Carestream near-infrared tricarbocyanine,cyclic enamine-functionalized dye (X-Sight 640 LSS Dye). Step-2, Biotin-NIR dye was incubatedwith the Neutravidin PEGylated Gold Nanoparticles to ensure the Biotin-Neutravidin interaction.

Quenching And Unquenching of NIRF

A representative triplicate sample of the NIRF quenched Biotin Dye- Neutravidin Gold complex as Imaged (Right three well samples in the inset and the corresponding surface Plot). NIRF unquenching upon trypsinization (Left three well samples).The fluorescence values obtained are plotted in the left two panels.

Quenching of Biotin-NIR Dye fluorescence upon interaction with the Neutravidin-Gold Nanoparticles (Upper Panel), and Unquenchingprocess of the same upon trypsin treatment (Lower Panel). Increasing concentration of Neutravidin PEGylated Gold nanoparticleswas incubated with 1 l Biotin- X-Sight LSS 640 dye (5 mM) in a final volume of 110 l PBS. The feasibility of utilizing this technique as a Smart Probe is demonstrated by unquenching of the NIRF as demonstrated by using non-specific enzymatic action of Trypsin (2.5 g/sample well). Time Lapse images of these samples in a 96 well plate were carried out using Carestream In-Vivo Multispectral FX PRO. Ex 655 EM 750; 1min exp; F-Stop: 2.8, FOV: 120 mm Time lapse Imaging Protocol- 2min interval; Total of 30 min or 100 min.

NIRF quenched Probe NIRF & CTReporting

Enzymatic Action

BiotinylatedNIRF Dye

Enzyme

Neutravidin-GoldNanoparticle

Quenched Probe NIRF & CTReporting

Enzymatic Action

Targeting Moiety

The above schematics describe the action of “ActivatableMolecular Multimodal Imaging Probes” also known asSmart Probes.The “Upper Schema” denotes the enzymatic activation and the subsequent near-IR fluorescence (NIRF) reporting capability of the Gold- NIRF dye nanoparticles throughcleavage of intermediary enzyme substrate recognition sequence.In the “Lower Schema”, additional functionality proposed with provision of added “targeting capability” through a targeting moiety. This can allow specificity and enhancement in the NIRF and Computed Tomography (CT) contrast.

939 A

+PEG-Biotin NIRF Dye Biotin-Dye

Neutravidin PEGylated Gold - Biotin- NIRF Dye NP

Step 1

Step 2

0 5 10 15 20 25 30

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Flu

ore

scen

ce (

a.u

)

Time (min)

1 l Gold NP

25 l Gold NP

0 20 40 60 80

1

2

3

4

Flu

ore

scen

ce (

a.u

)

Time (min)

Enzyme Treatment

Control Wells

L R