Abstract

Several visible and near-IR fluorescent nanoparticles have recently been developed and commercialized under the brand name KODAK X-SIGHT Imaging Agents. These nanoparticles (549, 650, 691, and 761) have the advantage of being made of organic “non-toxic” materials and contain multiple fluorochromes that are embedded into the core of the nanoparticle, providing a brighter reporter and a more stable environment, when compared to standard fluorochrome. The nanoparticle size is sufficiently small to enable near-IR fluorescence resonant energy transfer (NIR-FRET) between different particles having appropriate excitation-emission spectra. We have used these nanoparticles to create a robust 96

well-plate-binding assay utilizing FRET. Interaction between either donor nanoparticles 549 or 650 (goat anti- rabbit IgG conjugated) and rabbit IgG conjugated acceptor nanoparticle 761, produced fluorescence energy transfer as imaged using the KODAK In-Vivo Imaging System FX Pro. The dynamic range of proximity measures presented here indicates the potential of this assay system to be adapted within any high-throughput screening for single- chain antibody libraries, therapeutic, and pharmaceutical compounds. The protected nature of the fluorochromes inside the nanoparticles helps eliminate physical artifacts and enables assays in conditions that are unfavorable for typical fluorescence dyes. Potential applications and limitations of these nanoparticle-based FRET assays for in vitro and in vivo use will be presented.

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400 500 600 700 800 900

Wavelength (nm)

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Nanoparticle 549Nanoparticle 650Nanoparticle 691Nanoparticle 761

Spectral Characteristics of the Nanoparticles

FRET Modeling: Spectral Comparison /Overlaps of

650, 691 and 761 nanoparticles

Excitation (nm)550 600 650 700 750

Net

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nsity

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1e+5

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4e+5761 IgG761IgG Interaction 650 IgG650 IgGS(761 IgG and 650 IgG spectra)(761 IgG Interaction 650 IgG) minus

S(761 IgG and 650 IgG spectra)

5 nM IgG conjugated nanoparticles (549, 650, or 761) was used for interaction studies in a final volume of 0.2 ml. The 96-well plate (Black/clear bottomed) was imaged for 30sec (4X4 Binning). Inset: The brightest (1st lane) well is a result of FRET between 650 IgG and 761 IgG. The first well in the 3rd lane is 650 IgG alone. The four wells to the right of both the above two lanes are controls, where >100 fold excess of free IgG was used to inhibit FRET resulting interaction.

Branding/Trademark Group Comments:Official product name: KODAK In-Vivo Imaging System FX Pro

FRET emission of 761 nanoparticle caused by excitation of proximal 650 nanoparticle is projected to be well resolved by deconvolving the excitation spectrum. Also, the figure of merit for the 650/761 combination is the highest compared to any other nanoparticle combinations.

Wavelength (nm)400 500 600 700 800 900

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1.0650abs 650fluor 691abs 691fluor 761abs 761fluor (650f)(691a) (650f)(761a) (691f)(761a)

Net

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nsity

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-2e+5

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8e+5X761 IgG + X650 IgGCumulative intensity ofX761IgG and X650 IgGObserved FRET(Bar (1) minus Bar (2)

X761 IgG + X650 IgG in the presence of free Rabbit IgGX761 IgG + X650 IgG in the presence of free Goat IgG

CWL, Bandpass in nm750, 30 790, 30 830, 30

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D650/A691D650/A761D690/A761

Estimated Figure of Meritfor FRET Measure usingDonor/acceptors forDiffering Fluorescent windows

Calculated from spectra ofdonor (D) and acceptor (A):{(Aabs/Aabd)}*{(Afluor^2/Dfluor)}

FRET

635nm

830nm650anti-rabbit + 761 rabbit IgG

650 anti-rabbit + 761 anti-rabbit IgGNo FRET

635nm

X

FRET Schematic

ConclusionsThe embedded nature of the fluorochrome in these nano-particles are ideally suited in detecting FRET assuring reliable spectral properties in changing environments.

FRET measurements of low concentration interactantsshown here reiterate the possibilities of high-throughputscreening of single-chain antibody libraries, therapeuticproteins, pharmaceutical compounds, and not the leastin-vivo detection of protein-protein interactions.

The nanoparticles- donor 650/ acceptor761 (FRET pair)with the highest figure of merit, is ideal for small animalFRET near-IR (NIRF) imaging.

Projections: Expected signal-to-background

NIR-FRET Imaging with KODAK Nanospheres for In Vitro and In Vivo Applications

Rao V. L. Papineni*, Douglas L. Vizard, Tao Ji, Gilbert D.Feke, William E. McLaughlin, Douglas O. S. Wood, and Elizabeth L.White

Carestream Health, Inc. Contact: rao.papineni1@carestreamhealth.com0400

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