The development of PSMA-targeting low-molecular-weight hybrid molecules aims at advancing preoperative imaging and accurate intraoperative fluorescence guidance for improved diagnosis and therapy of prostate cancer. In hybrid probe design, the major challenge is the introduction of a bulky dye to peptidomimetic core structures without affecting tumor-targeting properties and pharmacokinetic profiles. This study developed a novel class of PSMA-targeting hybrid molecules based on the clinically established theranostic agent PSMA-617. The fluorescent dye-bearing candidates of the strategically designed molecule library were evaluated in in vitro assays based on their PSMA-binding affinity and internalization properties to identify the most favorable hybrid molecule composition for the installation of a bulky dye. The library’s best candidate was realized with IRDye800CW providing the lead compound. Glu-urea-Lys-2-Nal-Chx-Lys(IRDye800CW)-DOTA (PSMA-927) was investigated in an in vivo proof-of-concept study, with compelling performance in organ distribution studies, PET/MRI and optical imaging, and with a strong PSMA-specific tumor uptake comparable to that of PSMA-617. This study provides valuable insights about the design of PSMA-targeting low-molecular-weight hybrid molecules, which enable further advances in the field of peptidomimetic hybrid molecule development.

In prostate cancer (PCa) surgery, precise tumor margin identification remains challenging despite advances in surgical techniques. This study evaluates the combination of tumor-specific near-infrared imaging with the PSMA-targeting molecule PSMA-914 and optical endomicroscopy (NIR-pCLE) for single-cell-level tumor identification in a preclinical proof of concept. Methods: NIR-pCLE imaging of varying PSMA-914 concentrations was performed on PSMA-positive LNCaP and PSMA-negative PC-3 cells using Cellvizio^® 100 with pCLE Confocal Miniprobes™. To identify optimal PSMA-914 dosing for in vivo imaging, different doses (0–10 nmol) were evaluated using NIR-pCLE, Odyssey CLx imaging, and confocal microscopy in an LNCaP tumor-bearing xenograft model. A proof of concept mimicking a clinical workflow was performed using 5 nmol [⁶⁸Ga]Ga-PSMA-914 in LNCaP and PC-3 tumor xenografts, including PET/MRI, in/ex vivo NIR-pCLE imaging, and microscopic/macroscopic imaging. Results: NIR-pCLE detected PSMA-specific fluorescence at concentrations above 30 nM in vitro. The optimal dose was identified as 5 nmol PSMA-914 for NIR-pCLE imaging with cellular resolution in LNCaP xenografts. PET/MRI confirmed high tumor uptake and a favorable distribution profile of PSMA-914. NIR-pCLE imaging enabled real-time, single-cell-level detection of PSMA-positive tissue, visualizing tumor heterogeneity, confirmed by ex vivo microscopy and imaging. Conclusions: This preclinical proof of concept demonstrates the potential of intraoperative PSMA-specific NIR-pCLE imaging to visualize tissue structures in real time at cellular resolution. Clinical implementation could provide surgeons with valuable additional information, potentially advancing PCa patient care through improved surgical precision.

Objectives: Fluorescence-guided surgery (FGS) targeting prostate-specific membrane antigen (PSMA) holds promise for improving surgical precision in prostate cancer. Since conjugation of fluorescent dyes to targeting vectors can substantially alter pharmacokinetic properties, we systematically evaluated a library of fluorescent dyes conjugated to a PSMA-617-derived scaffold incorporating sarcosine-based spacers to identify candidates with favorable biodistribution and optical profiles for clinical translation. Methods: Nineteen fluorescent dyes spanning NIR, large Stokes shift, and STED-compatible categories were conjugated to a dual-labeled PSMA-617-derived precursor (Glu-urea-Lys-2Nal-TXA-Sar₁₀-Lys(DOTA)-Sar₅-βAla; hereafter DP). Compounds were radiolabeled with ⁶⁸Ga or ¹⁷⁷Lu and characterized for serum stability, lipophilicity, binding affinity, and internalization in LNCaP^PSMA+ cells. In vivo pharmacokinetics were assessed in LNCaP xenograft-bearing BALB/c nu/nu mice by µPET/MRI (1 and 2 h p.i., 500 pmol ⁶⁸Ga), organ distribution (0.5, 1, and 2 h p.i., 60 pmol ¹⁷⁷Lu), and clinical-grade endoscopic fluorescence imaging. Results: All conjugates retained hydrophilic character (logD: −3.72 to −1.79), low nanomolar binding affinity (K_i: 18–87 nM), and high serum stability (94–100% intact at 24 h). Despite comparable in vitro properties, dye conjugation markedly influenced in vivo pharmacokinetics: tumor uptake at 2 h p.i. ranged from 1 to 23%ID/g and kidney accumulation from 3 to 82%ID/g. Visible-range dyes exhibited faster renal washout within the imaging window and higher tumor-to-background contrast than NIR fluorophores. Fluorescence signal intensity did not correlate with radiotracer-derived uptake, underscoring the importance of dye-specific photophysical properties. Conclusions: DP-12 (SulfoCy5), DP-15 (Alexa Fluor 647), and DP-18 (Tide Fluor 5WS) were identified as lead candidates combining favorable pharmacokinetics with strong fluorescence contrast, warranting further evaluation toward fluorescence-guided prostate cancer surgery.