Elucidating the kinetics of sodium fluorescein for fluorescence-guided surgery of glioma

Abstract

OBJECTIVE The use of the optical contrast agent sodium fluorescein (NaFl) to guide resection of gliomas has been under investigation for decades. Although this imaging strategy assumes the agent remains confined to the vasculature except in regions of blood-brain barrier (BBB) disruption, clinical studies have reported significant NaFl signal in normal brain tissue, limiting tumor-to-normal contrast. A possible explanation arises from earlier studies, which reported that NaFl exists in both pure and protein-bound forms in the blood, the former being small enough to cross the BBB. This study aims to elucidate the kinetic binding behavior of NaFl in circulating blood and its effect on NaFl accumulation in brain tissue and tumor contrast. Additionally, the authors examined the blood and tissue kinetics, as well as tumor uptake, of a pegylated form of fluorescein selected as a potential optical analog of gadolinium-based MRI contrast agents. METHODS Cohorts of mice were administered one of the following doses/forms of NaFl: 1) high human equivalent dose (HED) of NaFl, 2) low HED of NaFl, or 3) pegylated form of fluorescein. In each cohort, groups of animals were euthanized 15, 30, 60, and 120 minutes after administration for ex vivo analysis of fluorescein fluorescence. Using gel electrophoresis and fluorescence imaging of blood and brain specimens, the authors quantified the temporal kinetics of bound NaFl, unbound NaFl, and pegylated fluorescein in the blood and normal brain tissue. Finally, they compared tumor-to-normal contrast for NaFl and pegylated-fluorescein in U251 glioma xenografts. RESULTS Administration of NaFl resulted in the presence of unbound and protein-bound NaFl in the circulation, with unbound NaFl constituting up to 70% of the signal. While protein-bound NaFl was undetectable in brain tissue, unbound NaFl was observed throughout the brain. The observed behavior was time and dose dependent. The pegylated form of fluorescein showed minimal uptake in brain tissue and improved tumor-to-normal contrast by 38%. CONCLUSIONS Unbound NaFl in the blood crosses the BBB, limiting the achievable tumor-to-normal contrast and undermining the inherent advantage of tumor imaging in the brain. Dosing and incubation time should be considered carefully for NaFl-based fluorescence-guided surgery (FGS) of glioma. A pegylated form of fluorescein showed more favorable normal tissue kinetics that translated to higher tumor-to-normal contrast. These results warrant further development of pegylated-fluorescein for FGS of glioma.

On the use of fluorescein-based contrast agents as analogs to MRI-gadolinium agents for imaging brain tumors

“…In a recent study, we reported on a comprehensive examination of the kinetics of NaFl in blood, normal brain, and tumor. 15 Specifically, we found that NaFl, which is a relatively small molecule (400 Da) binds only weakly to blood proteins, with a large component remaining in the free state. 15 This unbound NaFl crosses the blood brain barrier while the protein-bound dye remains in the blood.…”

“…15 Specifically, we found that NaFl, which is a relatively small molecule (400 Da) binds only weakly to blood proteins, with a large component remaining in the free state. 15 This unbound NaFl crosses the blood brain barrier while the protein-bound dye remains in the blood. This behavior confounds tumor imaging as the unbound dye in the brain reduces the potential tumor-to-normal contrast.…”

“…Dynamic imaging (fast T1W images) began immediately after the administration of gadobutrol in the tail vain and was run for about 70 minutes after administration at a rate of 89 seconds per frame. Animals in the fluorescence imaging group were administered NaFl or Pegylated fluorescein in the tail vein and then sacrificed at the pre-determined time point after administration, as describe previously 15 . Brains ware harvested, gross sectioned, and imaged with a Typhoon flatbed scanner.…”

Blood-Brain Barrier, Blood-Brain Tumor Barrier, and Fluorescence-Guided Neurosurgical Oncology: Delivering Optical Labels to Brain Tumors

“…Unspecific leakage of unbound FLS through a heterogeneous and dynamic BBB/BBTB explains reports of unspecific FLS staining in peritumoral areas (238). Compared to unbound FLS, a PEGylated form of FLS with a molecular weight similar to that of gadolinium-based contrast agents (939 Da) showed improved specificity of accumulation in U251 orthotopic mouse gliomas 1 h after administration (95). An albumin-bound formulation of 5-aminofluorescein (66,950 Da) was investigated in a Phase I/II study in Germany (96,97), where bright fluorescence was observed in 10 of 10 patients with high-grade gliomas 0.5 to 4 days after IV administration.…”

“…Increasing the dose of FLS (5 vs. 20 mg/kg human equivalent) results in an increase in unbound FLS in the blood and normal brain tissue (95). Therefore, the dose and timing of FLS administration should be considered to optimize fluorescent contrast during surgery.…”