Integrins are cell surface heterodimeric
glycoproteins involved in cell-to-cell
and cell-to-matrix interactions[1].
The recognition motif, RGD (Arg-Gly-Asp),
is a tripeptide sequence used to bind
integrin receptors including αvβ3.
Interest in using a labeled RGD peptide
ligand for the study and/or monitoring
of diseases related to the αvβ3 receptor
over-expression is increasing. Several
groups have labeled RGD successfully
with fluorescent dyes for in vitro
and in vivo imaging[2,3].
Characterization of IRDye 800CW RGD
included in vitro and in
vivo binding to a variety of
tumor cell lines (i.e., U87, A431, PC3M-LN4,
and 22Rv1). Results from U87 and 22Rv1
cells are shown in Image A. Specificity
of the conjugate for the integrin
receptor was confirmed by competition
with either unlabeled RGD peptide
or unlabeled RAD, non-specific peptide,
in U87 cells (Image B). In addition,
Images C and D confirm IRDye 800CW
RGD maintains specificity to the target
tissue in in vivo applications.
Images of nude mice bearing two subcutaneously
implanted tumors on the hips illustrate
IRDye 800CW RGD localized to tumor
tissue (Image C). A dose of unlabeled
RGD (given intravenously via the tail
vein) prior to IRDye 800CW RGD gave
reduced signal in both tumors confirming
specificity (Image D).
Figure: A) The On-Cell Western cell-based
assay was used to evaluate binding
affinity of IRDye 800CW RGD to U87
(glioblastoma) and 22Rv1 (prostate
carcinoma) tumor cell lines. B) Binding
of the IRDye 800CW RGD peptide was
blocked by unlabeled RGD but not by
a non-specific RAD peptide in U87
tumor cells. Nude mice bearing subcutaneous
tumors, U87 (left hip) and A431(right
hip), were imaged 24 hours post intravenous
injection of IRDye 800CW RGD (1 nmole;
C) or IRDye 800CW RGD (1 nmole) following
a pre-injection 1 hour prior to an
injection of unlabeled RGD peptide
(100 nmoles; D) Images were captured
on the Pearl Imager; 800nm signal
is presented in pseudo-color overlaid
on a white light image of the mouse.
The images are normalized to the same
look-up-table.
Advantages of NIR Optical
Agents
- High signal-to-noise ratio.
- Low NIR autofluorescence from
tissues, cells, and biological materials.
- IRDye 800CW absorption/emission
near 800 nm matches NIR absorption
minima for bodily fluids and tissues,
resulting in excellent tissue penetration.
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