Development of novel Near-Infrared fluorescent contrast agents for intraoperative imaging

Abstract

Surgery is the primary treatment for most patients with solid tumors and can be curative if complete resection is achieved. While visual inspection and palpation are usually effective for identifying the bulk tumor intraoperatively, they often prove inadequate for the delineation of small tumor remnants or nerves, increasing the risk of recurrence or iatrogenic nerve injury. Fluorescence-guided surgery (FGS) aims to visualize these critical structures in real time. It involves the administration of fluorescent contrast agents, typically composed of a ligand targeting the tissue of interest, conjugated to a Near-Infrared (NIR) fluorescent fluorophore. However, preclinical findings suggest that frequently utilized heptamethine cyanines like IRDye800 are affected by instability towards nucleophiles and, furthermore, are associated with non-specific retention in vivo, which compromises the agent’s efficacy in generating contrast and delays imaging. In this PhD work, targeted contrast agents were developed that overcome the afore-mentioned limitations due to the incorporation of the NIR fluorophore “sNIR”, discovered by explorative studies on a Zincke salt-based approach toward substituted hep-tamethine cyanines. Besides reduced off-target retention in vivo, it also displays faster clearance and greater photostability compared to IRDye800. sNIR was excessively characterized for its optical and chemical properties. Conjugation to established ligands for targeting biomarkers in certain tissue types afforded contrast agents to overcome cancer- and anatomy-related surgical challenges. As the main project, a meningioma-specific contrast agent was developed, which addresses the overexpressed somatostatin receptor subtype 2 (SSTR2) using the octapeptide TATE. TATE sNIR was extensively validated against controls in vitro in SSTR2-expressing cells and in vivo in physiologically SSTR2-expressing mouse tissues and three tumor mouse models, yielding outstanding contrast ratios. Further translational work included a proof-of-concept study in a pig as well as compatibility assessments with NIR fluorescence imaging devices already established in clinical practice. Explorative studies on additional sNIR-based probes addressing a variety of molecular markers expressed by other tu-mor types further suggest that sNIR holds great potential for FGS, regardless of the size of the targeting moiety