Development and Evaluation of [ 68 Ga]Ga-FA-PSMA: A Folic Acid-Conjugated PSMA-Targeting Radiotracer for Enhanced PET Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a critical biomarker for prostate cancer. While current radiotracers like [ 68 Ga]Ga-PSMA-11 are clinically useful, enhancing tumor uptake and contrast remains a goal. Capitalizing on PSMA’s enzymatic hydrolysis of folate polyglutamates, we report the design, synthesis, and biological evaluation of a novel heterobivalent PSMA-targeted radiotracer, [ 68 Ga]Ga-FA-PSMA. In vitro experiments revealed a significantly enhanced binding affinity of FA-PSMA for human PSMA ( K D = 3.268 nM) compared to PSMA-11 ( K D = 47.88 nM) and free FA ( K D = 424.3 nM). In vivo micro-PET/CT imaging and biodistribution studies in PSMA-positive (22RV1) xenograft models demonstrated superior performance over [ 68 Ga]Ga-PSMA-11. [ 68 Ga]Ga-FA-PSMA exhibited significantly higher peak tumor SUV max (1.06 ± 0.09 vs 0.80 ± 0.08 at 210 min p.i.) and tumor accumulation (2.74 ± 0.40 vs 1.75 ± 0.74 %ID/g at 2 h p.i.), alongside significantly enhanced tumor-to-background ratios, particularly tumor-to-blood (10.41 ± 3.41 vs 2.11 ± 0.89) and tumor-to-muscle (10.07 ± 0.73 vs 6.03 ± 1.33) at 2 h p.i. Furthermore, [ 68 Ga]Ga-FA-PSMA demonstrated reduced renal uptake (30.80 ± 8.54 vs 38.71 ± 9.72 %ID/g at 2 h p.i.), high in vitro stability, and favorable hydrophilicity (Log P = −3.76). These findings demonstrate that the novel heterobivalent tracer [ 68 Ga]Ga-FA-PSMA offers significantly enhanced PSMA-targeting affinity, tumor uptake, and imaging contrast compared to the clinical standard, validating the FA-conjugation strategy and positioning it as a highly promising next-generation diagnostic agent for precise prostate cancer imaging.