High-Throughput Discovery of Near-Infrared Oxazine Probes for Fluorescence-Guided Glioblastoma Surgery

Background/Objectives: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor in adults, characterized by highly infiltrative growth and poorly defined margins that hinder complete surgical resection. Fluorescence-guided surgery (FGS) can enhance intraoperative tumor visualization; however, currently available fluorophores often exhibit limited tumor specificity and inconsistent labeling. This study aimed to identify near-infrared (NIR) probes with improved glioblastoma selectivity using a high-throughput discovery approach. Methods: A chemically diverse library of 127 NIR oxazine probes was screened using automated fluorescence imaging across four GBM cell lines and a sarcoma control line. Top-performing probes were further evaluated in an orthotopic U251MG-GFP glioblastoma mouse model to assess blood–brain barrier penetration and tumor localization in vivo. Results: Five candidate probes exhibited strong, selective NIR fluorescence in GBM cells. In vivo imaging revealed that the lead probe, LGW01-44, achieved the highest tumor-to-brain contrast with minimal background signal. Ex vivo analysis of brain sections confirmed preferential accumulation of LGW01-44 within intracranial tumor tissue. Conclusions: These findings establish a scalable high-throughput platform for the discovery of tumor-selective NIR imaging agents and identify the oxazine probe LGW01-44 as a promising candidate for fluorescence-guided glioblastoma surgery.