Noninvasive Imaging of Early Acute Inflammation with Myeloperoxidase-Specific PET Ligand ⁶⁸Ga-NOTA-3G-bis-5HT

Myeloperoxidase (MPO), a well-established biomarker of neutrophil activation, plays a crucial role in early acute inflammation. In the study, we developed an MPO-specific PET ligand, ⁶⁸Ga-NOTA-3G-bis-5HT, for noninvasive imaging of early acute inflammation. ⁶⁸Ga-NOTA-3G-bis-5HT was achieved with a radiochemical yield of >70%, radiochemical purity of >95%, and molar activity of 6.10-24.4 GBq/μmol. Molecular docking and molecular dynamics (MD) simulations demonstrated the binding mode and stability between ⁶⁸Ga-NOTA-3G-bis-5HT and MPO with a binding free energy of -75.18 kcal/mol. The in vitro binding assays confirmed the oxidation binding mechanism and high affinity with an IC₅₀ of 1.67 nM. The Matrigel implantation experiment determined the sensitivity of ⁶⁸Ga-NOTA-3G-bis-5HT in the detection of human MPO in vivo. Micro-PET imaging in the mouse model revealed that the accumulation of ⁶⁸Ga-NOTA-3G-bis-5HT in the inflammation site was very rapid and decreased over time within 120 min with an uptake of 1.53 ± 0.11%ID/g and inflammation-to-muscle ratio of 2.67 ± 0.31 at 30 min. Blocking experiments demonstrated the specific binding of ⁶⁸Ga-NOTA-3G-bis-5HT to MPO. Histopathological results further validated MPO expression and neutrophil infiltration in early acute inflammation. The biodistribution manifested a rapid distribution and fast clearance from the body, supporting the favorable pharmacokinetics of ⁶⁸Ga-NOTA-3G-bis-5HT (t_1/2α = 3.55 min and t_1/2β = 24.90 min). Collectively, these findings highlighted the clinical potential of ⁶⁸Ga-NOTA-3G-bis-5HT as a tool for monitoring MPO activity, offering a valuable approach for early acute inflammation diagnosis and risk stratification.