A divalent
knottin containing two separate
integrin binding
epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a
4-nitrophenyl 2-(18)F-fluoropropinate ((18)F-NFP) group and the resulting divalent positron emission tomography (PET) probe, (18)F-FP-3-4A, was evaluated as a novel imaging probe to detect
integrin αvβ3 positive
tumors in living animals.
Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with (18/19)F-NFP to produce the fluorinated
peptide (18/19)F-fluoropropinate-3-4A ((18/19)F-FP-3-4A). The stability of (18)F-FP-3-4A was tested in both
phosphate buffered saline (PBS)
buffer and mouse serum. Cell uptake assays of the radiolabeled
peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of (18)F-FP-3-4A were performed in U87MG
tumor-bearing mice. The receptor targeting specificity of the radiolabeled
peptide was also verified by coinjecting the probe with a blocking
peptide cyclo(RGDyK). Our study showed that (18)F-FP-3-4A exhibited excellent stability in PBS
buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that (18)F-FP-3-4A exhibited rapid and good
tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/g at 0.5 and 1 h, respectively). (18)F-FP-3-4A was rapidly cleared from the normal tissues, resulting in excellent
tumor-to-normal tissue contrasts. For example, liver uptake was only 0.39 ± 0.07% ID/g and the
tumor to liver ratio was 5.69 at 1 h p.i. Furthermore, coinjection of cyclo(RGDyK) with (18)F-FP-3-4A significantly inhibited
tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in the
tumor. In summary, the divalent probe (18)F-FP-3-4A is characterized by rapid and high
tumor uptake and excellent
tumor-to-normal tissue ratios. (18)F-FP-3-4A is a highly promising
knottin based PET probe for translating into clinical imaging of
tumor angiogenesis.