Folates are critical for central nervous system function. Folate transport is mediated by 3 major pathways, reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα/Folr1), known to be regulated by ligand-activated nuclear receptors. Cerebral folate delivery primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems can result in very low folate levels in the cerebrospinal fluid causing childhood neurodegenerative disorders. These disorders have devastating effects in young children, and current therapeutic approaches are not sufficiently effective. Our group has previously reported in vitro that functional expression of RFC at the blood–brain barrier (BBB) and its upregulation by the vitamin D nuclear receptor (VDR) could provide an alternative route for brain folate uptake. In this study, we further demonstrated in vivo, using Folr1 knockout (KO) mice, that loss of FRα led to a substantial decrease of folate delivery to the brain and that pretreatment of Folr1 KO mice with the VDR activating ligand, calcitriol (1,25-dihydroxyvitamin D3), resulted in over a 6-fold increase in [13C5]-5-formyltetrahydrofolate ([13C5]-5-formylTHF) concentration in brain tissues, with levels comparable to wild-type animals. Brain-to-plasma concentration ratio of [13C5]-5-formylTHF was also significantly higher in calcitriol-treated Folr1 KO mice (15-fold), indicating a remarkable enhancement in brain folate delivery. These findings demonstrate that augmenting RFC functional expression at the BBB could effectively compensate for the loss of Folr1-mediated folate uptake at the choroid plexus, providing a therapeutic approach for neurometabolic disorders caused by defective brain folate transport.

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In this study, we investigated convection-enhanced delivery (CED) of 23 ± 3 nm gold nanoparticles (AuNPs) labeled with the β-particle-emitting radionuclide 177 Lu ( 177 Lu-AuNPs) for treatment of orthotopic U251-Luc human glioblastoma multiforme (GBM) tumors in NRG mice. The cytotoxicity in vitro of 177 Lu-AuNPs (0.0−2.0 MBq, 4 × 10 11 AuNPs) on U251-Luc cells was also studied by a clonogenic survival assay, and DNA double-strand breaks (DSBs) caused by β-particle emissions of 177 Lu were measured by confocal immunofluorescence microscopy for γH2AX. NRG mice with U251-Luc tumors in the right cerebral hemisphere of the brain were treated by CED of 1.1 ± 0.2 MBq of 177 Lu-AuNPs (4 × 10 11 AuNPs). Control mice received unlabeled AuNPs or normal saline. Tumor retention of 177 Lu-AuNPs was assessed by single-photon emission computed tomography/ computed tomography (SPECT/CT) imaging and biodistribution studies. Radiation doses were estimated for the tumor, brain, and other organs. The effectiveness for treating GBM tumors was determined by bioluminescence imaging (BLI) and T2-weighted magnetic resonance imaging (MRI) and by Kaplan−Meier median survival. Normal tissue toxicity was assessed by monitoring body weight and hematology and blood biochemistry analyses at 14 d post-treatment. 177 Lu-AuNPs (2.0 MBq, 4 × 10 11 AuNPs) decreased the clonogenic survival of U251-Luc cells to 0.005 ± 0.002 and increased DNA DSBs by 14.3-fold compared to cells treated with unlabeled AuNPs or normal saline. A high proportion of 177 Lu-AuNPs was retained in the U251-Luc tumor in NRG mice up to 21 d with minimal re-distribution to the brain or other organs. The radiation dose in the tumor was high ( 599Gy). The dose in the normal right cerebral hemisphere of the brain excluding the tumor was 93-fold lower (6.4 Gy), and 2000−3000-fold lower doses were calculated for the contralateral left cerebral hemisphere (0.3 Gy) or cerebellum (0.2 Gy). The doses in peripheral organs were <0.1 Gy. BLI revealed almost complete tumor growth arrest in mice treated with 177 Lu-AuNPs, while tumors grew rapidly in control mice. MRI at 28 d post-treatment and histological staining showed no visible tumor in mice treated with 177 Lu-AuNPs but large GBM tumors in control mice. All control mice reached a humane endpoint requiring sacrifice within 39 d (normal saline) or 45 d post-treatment (unlabeled AuNPs), while 5/8 mice treated with 177 Lu-AuNPs survived up to 150 d. No normal tissue toxicity was observed in mice treated with 177 Lu-AuNPs. We conclude that CED of 177 Lu-AuNPs was highly effective for treating U251-Luc human GBM tumors in the brain in NRG mice at amounts that were non-toxic to normal tissues. These 177 Lu-AuNPs administered by CED hold promise for treating patients with GBM to prevent recurrence and improve long-term outcome.