Targeted Noninvasive Near-infrared Fluorescence Imaging of GLP-1 Receptors: Appropriate Arsenal for the Deliveryman.

Rao V.L. Papineni*, Tao Ji, William Mclaughlin, Gilbert Feke, and Sean Orton.
Carestream Health, Inc.
*E.Mail: rao.papineni1@carestreamhealth.com B20

Time Lapse Imaging

To enhance targeted molecular diagnostic efficacy Exendin-4 in Non-Invasive NIRF Imaging

KODAK in-Vivo Multispectral FX system.
and therapeutics, the carrier’s nonspecific interactions Image X-ray, NIRF EM:650, and EM 750

need to be minimal. Apart from the stability issues, easy

conjugation chemistries and sensitive reporter detection
system are essential. Here we exploited the high affinity
between the neutravidin and biotin for in vivo near- NIRF imaging of GLP-1 binding. Biotin-Exendin-4 (Anaspec) was added to the
Kodak-LSS 640 dye conjugated neutravidin and incubated for 30 min on ice.
infrared fluorescence imaging (NIRF imaging) of Thesamples were injected (tail-vein) and imaged as follows: X-Ray: 1 min
exposure 2X2 binning, f-stop 2.8;Fluorscence: 5 min exp, no binning f-stop 2.8
pancreas. Dyes that have a large stokes shift (LSS-Dye)
(>80 nm) in the near infrared range was conjugated to NIRF signals enrich at the abdominal
region as observed in the image obtained
neutravidin and utilized for targeted molecular imaging. at 120 min (right panel). Ex vivo
imaging and multivalent targeting agents
Biotinylated exendin-4, a potent glucagons-like peptide to pancreas are in the process.
1 (GLP1) receptor antagonist was linked to the LSS
conjugated neutravidin to target the GLP-1 rich
Avidin/Streptavidin/
pancreas. LSS dyes attached delivery system enabled Neutravidin

maximal fluorescence signal while overcoming Four (X) biotinylated

Exendin-4

autofluorescence issues and minimizing nonspecific Kodak LSS Dye

30 min 45 min 120 min
binding. NIRF imaging of the target positioned through
various axial rotation angles obtained further
exemplifies the potential for LSS Dye-neutravidin NIRF Images of GLP-1 Binding
scaffold as an efficient in vivo imaging agent. Apart liver
liver pancreas

spleen

from insulinomas, GLP-1 receptors are enriched in Multiple Angle Imaging kidney

other tumors. Further, such a diagnostic and therapy

KODAK in-Vivo Multispectral FX system. Cline et al Presented at SMI-2006, Rhode Island

monitoring system can be easily applied with emerging Image X-ray, NIRF EM:650, and EM 790 99mTC–X-Sight 761 Nanosphere 99mTC–X-Sight 761 Nanosphere plus

Exendin-4

cancer biomarkers. Our prior experiments conducted together with Dr.

Gary Cline at yale university shows enrichment of the
exendin-4 conjugated nanoparticle in multiple organs
apart form the pancreas. The above image was
obtained with microSPECT-CT scanner.
Neutravidin- NIRF Tagged Delivery Vehicle Representative images to demonstrate the advantages of NIRF multiple angle imaging.
GLP-1 binding - Biotin-Exendin-4 (Anaspec) added to the Kodak-LSS 640 dye
conjugated neutravidin and injected (tail-vein). X-Ray and NIRF imaging as follows: X-
Ray: 1 min exposure 2X2 binning, f-stop 2.8;Fluorscence: 5 min exp, no binning f-stop
Kodak X-Sight 640 Large Stokes Shift Dye 2.8
1.2
Normalized Intensity

Absorption 635 nm 733 nm

1.0
Emission
0.8 The Large Stokes Shift Dye displays 98 nm
stokes shift and is amine reactive
0.6 Biotin Bombesin and Conclusions
0.4 Neutravidin Delivery System
0.2
To confirm the feasibility of the easy link methodology • Biotin-interaction based non invasive near-infrared imaging is feasible. Targeting
0.0
for NIRF Imaging- Biotin-bonbesin attached to avidin agents linked to macromolecules or nanoparticles seem to be performing with equal
450 500 550 600 650 700 750 800 850 900 950 conjugate Kodak dye encapsulated nanoparticle (~16 nm) vigor.
Wavelength (nm) is shown here. The samples were injected (tail-vein). The
0.6 bombesin receptors are widely distributed in CNS and • Organic dyes with extremely large stokes shift allows and improves imaging both
Absorbance (a.u)

Neutravidin a deglycosylated version of gastrointestinal tract.

0.5 in terms of overcoming autofluorescence and depth issues.
avidin, a tetramer, with a strong affinity NIRF imaging as follows: 30 sec exp, f-stop 4.0 FOV
0.4
for biotin (kD10-15) is used here as a 200mm
delivery tool for biotinylated ligands.
•The presence of neutravidin allows not only the delivery of targets that tend to be
0.3
Neutravidin is initially conjugated to the degraded during circulation but potentially acts as a high-volume/high-capacity
0.2 coupler. We are in the process of utilization of such a vehicle in image guidable
Kodak X-Sight 640 Large Stokes Shift
Dye (NHS attachment group). The 0.1 gene silencing nanoparticles.
One X-Sight nanosphere
conjugates were characterized by UV
0.0 Avidin/Streptavidin/
absorbance ( right panel). 200 400 600 800 Neutravidin
Four (X) biotinylated
Wavelength (nm) Bombesin

"Molecular Imaging - Wisdom To See For Maladies To Flee"

Dr. Rao V. L. Papineni