A Radiofluorinated Divalent Cystine Knot Peptide forTumor PET Imaging

摘要

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 2-¹⁸F-fluoropropinate (¹⁸F-NFP) group and the resulting divalent positron emission tomography (PET) probe, ¹⁸F-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 ^18/19F-NFP to produce the fluorinated peptide ^18/19F-fluoropropinate-3-4A (^18/19F-FP–3-4A). The stability of ¹⁸F-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 ¹⁸F-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 ¹⁸F-FP–3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistributiondata revealed that ¹⁸F-FP–3-4A exhibited rapid andgood tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/gat 0.5 and 1 h, respectively). ¹⁸F-FP–3-4A was rapidlycleared from the normal tissues, resulting in excellent tumor-to-normaltissue 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 ¹⁸F-FP–3-4A significantlyinhibited tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/gat 2.5 h) in U87MG xenograft models, demonstrating specific accumulationof the probe in the tumor. In summary, the divalent probe ¹⁸F-FP–3-4A is characterized by rapid and high tumor uptakeand excellent tumor-to-normal tissue ratios. ¹⁸F-FP–3-4Ais a highly promising knottin based PET probe for translating intoclinical imaging of tumor angiogenesis.