Radiosynthesis and preclinical evaluation of [ 68 Ga]Ga-NOTA-folate for PET imaging of folate receptor β-positive macrophages.

Folate receptor β (FR-β), a marker expressed on macrophages, is a promising target for imaging of inflammation. Here, we report the radiosynthesis and preclinical evaluation of [ ⁶⁸ Ga]Ga-NOTA-folate ( ⁶⁸ Ga-FOL). After determining the affinity of ⁶⁸ Ga-FOL using cells expressing FR-β, we studied atherosclerotic mice with ⁶⁸ Ga-FOL and ¹⁸ F-FDG PET/CT. In addition, we studied tracer distribution and co-localization with macrophages in aorta cryosections using autoradiography, histology, and immunostaining. The specificity of ⁶⁸ Ga-FOL was assessed in a blocking study with folate glucosamine. As a final step, human radiation doses were extrapolated from rat PET data. We were able to produce ⁶⁸ Ga-FOL with high radiochemical purity and moderate molar activity. Cell binding studies revealed that ⁶⁸ Ga-FOL had 5.1 nM affinity for FR-β. Myocardial uptake of ⁶⁸ Ga-FOL was 20-fold lower than that of ¹⁸ F-FDG. Autoradiography and immunohistochemistry of the aorta revealed that ⁶⁸ Ga-FOL radioactivity co-localized with Mac-3-positive macrophage-rich atherosclerotic plaques. The plaque-to-healthy vessel wall ratio of ⁶⁸ Ga-FOL was significantly higher than that of ¹⁸ F-FDG. Blocking studies verified that ⁶⁸ Ga-FOL was specific for FR. Based on estimations from rat data, the human effective dose was 0.0105 mSv/MBq. Together, these findings show that ⁶⁸ Ga-FOL represents a promising new FR-β-targeted tracer for imaging macrophage-associated inflammation.