A fluorescein-tagged pH-low insertion peptide (pHLIP) for glioblastoma imaging: Lipid membrane interaction and cancer cell targeting

Glioblastoma (GB) is the most aggressive and common malignant brain tumor, and despite current therapeutic approaches, prognosis remains poor. Given that surgical resection is frequently the sole potentially curative option, precise intraoperative tumor delineation is crucial for reducing recurrence rates and enhancing patient outcomes. In this study, we developed a novel pH-responsive imaging tool by coupling the pH-low insertion peptide (pHLIP) with fluorescein (FL) to enable targeted fluorescence-guided visualization of tumor margins. We investigated pHLIP–lipid membrane interactions using model systems, including liposomes and supported lipid bilayers (SLB), assessing peptide's pH-dependent insertion mechanism. Complementary in vitro experiments on patient-derived GB cell lines were performed to show the tracer's pH sensitivity, selective membrane targeting, and potential off-target effects. The FL-pHLIP construct showed robust, pH-dependent membrane insertion and selectivity in GB cellular models with minimal interaction under physiological conditions. These findings support FL-pHLIP as a promising candidate for fluorescence-guided surgery in GB and highlight its potential for clinical translation and for the broader development of pH-responsive diagnostic tools.