| Summary: | <p>Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Single-chain (sc)-VEGF protein containing an N-terminal Cys-tag has been designed for site-specific modification with a variety of imaging and therapeutic moieties. Site-specific labeling of scVEGF with thiol-reactive prosthetic group, <i>N</i>-[2-(4-<sup>18</sup>F-fluorobenzamido) ethyl] maleimide ([<sup>18</sup>F]FBEM) for positron emission tomography (PET) imaging of VEFGR may provide a new tracer which has great potential for clinical translation.</p><p><b>Methods</b>: [<sup>18</sup>F]FBEM-scVEGF was synthesized by site-specific conjugation of <sup>18</sup>F-FBEM to a thiol group in Cys-tag of scVEGF at room temperature. The functional activity after labeling was tested by immunofluorescence staining, cellular uptake and efflux<i>. </i>The tumor targeting and <i>in vivo</i> properties were evaluated by biodistribution and microPET studies in tumor-bearing mice.</p><p><b>Results</b>: The radiolabeling yield and specific activity of [<sup>18</sup>F]FBEM-scVEGF were 20.6 ± 15.1% (based on starting [<sup>18</sup>F]FBEM, uncorrected, n = 5) and 58.8 ± 12.4 GBq/µmol, respectively. Noninvasive microPET and direct tissue sampling experiments demonstrated that [<sup>18</sup>F]FBEM-scVEGF had VEGFR specific tumor uptake in MDA-MB-435, U87MG and 4T1 xenograft models. The optimal tumor uptake was achieved at 2 h p.i., which can be partially, but significantly blocked by co-injection of non-labeled scVEGF protein. Overall, [<sup>18</sup>F]FBEM-scVEGF showed VEGFR specific tumor uptake.</p><p><b>Conclusion</b>: The scVEGF was site-specifically labeled with <sup>18</sup>F via [<sup>18</sup>F]FBEM prosthetic group and the tracer [<sup>18</sup>F]FBEM-scVEGF exhibited high receptor binding affinity and tumor targeting efficacy. Further study of [<sup>18</sup>F] FBEM-scVEGF to evaluate angiogenesis in cancer and other disease types is warranted.</p>
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