Three
amino acids residues,
Arg-Gly-Asp (RGD), in
vitronectin and
fibronectin show affinity for alpha(V)beta(3)
integrins expressed in vascular endothelial cells. That
tumor growth can upregulate the expression of these
integrins on
tumor cells for invasion and
metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD
peptides as antagonists of alpha(V)beta(3)
integrins for broad spectrum
tumor specific imaging. The
polypeptide RGD-4C, which contains four
cysteine residues for cyclization, has shown preferential localization on
integrins at sites of
tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with
6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated
peptides by a C18 Sep-Pak cartridge with 20%
methanol, both
peptides were radiolabeled using
tricine. For cell binding studies, both 99mTc
peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling
solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)
beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the
integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human
renal adenocarcinoma (ACHN) and human
colon cancer cell line (LS174T) nude mouse
tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of
alpha(V)beta(3) integrin receptors per
tumor cell and low binding affinity, radiolabeled RGD
peptides may have limitations as
tumor imaging agents.