Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary tumor of the brain with a median survival of 15 months. Maximal safe resection is the initial standard-of-care, but resection is limited by the diffuse invasiveness of the tumor. Fluorescence imaging has been shown to aid intra-operative tumor identification, but the current options fail to do so consistently. Recent literature has shown fatty acid (FA) metabolism and uptake to be altered in glioblastoma. For this reason, we introduce a novel, near-infrared fluorescent dye, fatty-acid indocyanine green (FA-ICG), that aims to target the enhanced fatty acid uptake of glioblastoma. We evaluate this dye in in vitro and in vivo models. MATERIAL AND METHODS FA-ICG consists of palmitic acid, a long-chain, saturated fatty acid, that has been covalently linked to indocyanine green (ICG), as described previously. We validated FA-ICG in vitro in one animal cell line and four (two primary and two recurrent) patient-derived neurosphere glioblastoma cell lines. We performed in vitro temporal uptake dynamics and oleic acid based inhibition assays for FA-ICG and ICG on all cell lines. Time uptake was investigated by starving and then incubating cells with either dye for an increasing length of time. Similarly, inhibition was tested by incubating cells with increasing concentrations of oleic acid (in addition to either FA-ICG or ICG). Subsequently, the dye was tested in an NRMI mouse model without tumor for pharmacokinetic properties. Mice were injected with the dye and blood was collected at multiple time points post-injection. Quantification of fluorescence signals in serum was performed using a fluorescent standard curve. RESULTS FA-ICG was taken up by all glioblastoma cells lines. Across all cell cultures, uptake of FA-ICG showed a stepwise increase of up to 5-30 times higher uptake (compared to uptake at baseline) when cells were starved longer, while ICG uptake showed the same trend in only three out of five cell lines (and only up to 5 times higher uptake than at baseline). Oleic acid was found to inhibit FA-ICG uptake by 40% at low concentrations and up to 80-90% at higher concentrations across all cell lines, while ICG was unaffected. In vivo pharmacokinetics showed FA-ICG to have a mean half-life of 65 minutes in blood. CONCLUSION Our findings indicate that the uptake of FA-ICG is affected by starvation and oleic acid competition across all cell lines while ICG uptake is only partially affected by starvation. In vivo pharmacokinetic shows the half-life of FA-ICG to be significantly longer than that of ICG in literature. Taken together, these findings might point to an enhanced targeting and retention mechanism of FA-ICG in glioblastoma. Therefore, these results support the further translation of FA-ICG for fluorescence guided surgery in GBM.