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 [⁶⁸Ga]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, [⁶⁸Ga]Ga-FA-PSMA. In vitro experiments revealed a significantly enhanced binding affinity of FA-PSMA for human PSMA (<i>K</i>_D = 3.268 nM) compared to PSMA-11 (<i>K</i>_D = 47.88 nM) and free FA (<i>K</i>_D = 424.3 nM). In vivo micro-PET/CT imaging and biodistribution studies in PSMA-positive (22RV1) xenograft models demonstrated superior performance over [⁶⁸Ga]Ga-PSMA-11. [⁶⁸Ga]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, [⁶⁸Ga]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<i>P</i> = -3.76). These findings demonstrate that the novel heterobivalent tracer [⁶⁸Ga]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.