Radiotracer Development for Cardiac Sympathetic Neuroimaging and Vascular Apoptosis Imaging: Before the Pictures

Abstract

Introduction & Objectives: Molecular imaging is a non-invasive diagnostic tool that is particularly interesting to scientists and clinicians due to its minimal harm to patients. Unlike MRI and ultrasound imaging modalities, PET imaging can provide researchers with information such as cellular responses within tissues under disease conditions. Determining cellular mechanisms with the use of imaging probes or evaluating effectiveness of radiotracers in vitro and in tightly controlled conditions are primary steps to successfully validating molecular imaging methods. To this end, the content of this thesis is comprised of radiotracer development experiments from two projects to i) validate tracer kinetics in an isolated system, ii) develop successful bioconjugation strategies to label a protein of interest. This thesis presents two projects aimed at advancing molecular imaging: (1) investigating the intraneuronal kinetics of meta-[18F]fluorobenzylguanidine ([18F]MFBG), a PET tracer targeting the norepinephrine transporter (NET) system, and (2) developing radiolabeled annexin V (ANXV) bioconjugates for apoptosis imaging Methods: Project 1 The established SNS radiotracer [18F]MFBG was used to verify NET intraneuronal kinetics in SK-N-SH (low vesicle content) and PC12 (high vesicle content) cells through treatment with high-concentration potassium chloride (KCl) and the VMAT2 inhibitor reserpine (RSP). Project 2 Annexin V bioconjugates were synthesized using maleimide and trifluoroborate functionalities. Radiolabeling with fluorine-18 via isotopic exchange was optimized for precursor concentration and reaction time. Results: Project 1 Vesicle-rich PC12 cells showed significant [18F]MFBG washout with KCl or RSP, indicating active vesicle turnover, while vesicle-poor SK-N-SH cells showed minimal release. Increased treatment time led to higher tracer release in PC12 cells, supporting vesicular activity in retention. Project 2 Radiolabeling of ANXV bioconjugate with fluorine-18 achieved 32-34% radiochemical conversion. HPLC confirmed the product and a minor side product, and autoradiography verified binding to ANXV. Conclusion: The [18F]MFBG project enhances our understanding of intraneural tracer mechanisms, providing insights that will support more accurate interpretation of in vivo imaging data by revealing how this established tracer behaves inside neurons. Additionally, the results from the annexin V radiotracer development project demonstrate the successful synthesis of bioconjugates with varying chain linkers, improving targeting efficiency and supporting further studies to develop and evaluate this tracer in vivo.