Abstract
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Objectives The prostate-specific membrane antigen (PSMA) is a cell surface protein over-expressed in prostate cancer and neovasculature of most solid tumors. It is an attractive target for imaging and therapy of prostate and potentially other cancers. Previously, we demonstrated successful PET and SPECT imaging of PSMA-expressing xenografts using radiolabeled urea-based PSMA inhibitors and near infrared fluorescent (NIRF) imaging using an IRDye800CW/2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid conjugate. Here we extend that work by investigating the effect of different fluorophores and dye linkers on tumor uptake by preparing and testing 14 new NIRF imaging agents in mice with PSMA+ PC-3 PIP and PSMA- PC-3 flu tumors.
Methods The new imaging agents were prepared by conjugating the activated ester of IRDye800RS, IRDye800CW, ICG, Cy7 or Cy5.5 with urea precursor 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid 1, 2-(3-{5-[2-(2-aminomethoxy-ethoxy)-acetylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 2 and 2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 3. Mice bearing a PSMA+ PC-3 PIP tumor and a PSMA- PC-3 flu tumor in opposite flanks were injected intravenously prior to imaging and biodistribution.
Results The chemical yields for conjugation of the dye-containing activated esters with urea precursors ranged from 53% to 90%. All agents demonstrated varying degrees of PSMA+ PC-3 PIP tumor uptake with little PSMA- PC-3 flu tumor uptake, indicating target selectivity in vivo. Using the identical linker the degree of tumor uptake was: Cy7 ≈ IRDye800CW > IRDye800RS > Cy5.5 > ICG. For the Cy7-urea conjugates, the degree of tumor uptake was Cy7-3 ≈ Cy7-2 > Cy7-1 with Cy7-3 having lower normal organ uptake.
Conclusions This study shows that the choices of fluorophore and linker have significant effects on tumor uptake and pharmacokinetics in vivo. Within this series, Cy7-3 is the most promising NIRF imaging agent