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Background: Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood-brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA)., Methods: Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS., Results: In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression., Conclusions: VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain., (© 2020 Federation of American Societies for Experimental Biology.)

Obtaining efficient drug delivery to the brain remains the biggest challenge for the development of therapeutics to treat diseases of the central nervous system. The main obstacle is the blood-brain barrier (BBB), which impedes the entrance of most molecules present in the systemic circulation, especially large molecule drugs and nanomedicines. To overcome this obstacle, targeting strategies binding to nutrient receptors present at the luminal membrane of the BBB are frequently employed. Amongst the numerous potential targets at the BBB, the transferrin receptor (TfR) remains the most common target used to ensure sufficient drug delivery to the brain. In this review, we provide a full account on the use of the TfR as a target for brain drug delivery by describing the function of the TfR in the BBB, the historical background of its use in drug delivery, and the most recent evidence suggesting TfR-targeted medicines to be efficient for brain drug delivery with a clear clinical potential., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Transport of the majority of therapeutic molecules to the brain is precluded by the presence of the blood-brain barrier (BBB) rendering efficient treatment of many neurological disorders impossible. This BBB, nonetheless, may be circumvented by targeting receptors and transport proteins expressed on the luminal surface of the brain capillary endothelial cells (BCECs). The transferrin receptor (TfR) has remained a popular target since its original description for this purpose, although clinical progression of TfR-targeted drug constructs or nanomedicines remains unsuccessful. One proposed issue pertaining to the use of TfR-targeting in nanomedicines is the efficient tuning of the ligand density on the nanoparticle surface. We studied the impact of TfR antibody density on the uptake and transport of nanoparticles into the brain, taking a parallel approach to investigate the impact on both antibody-functionalized gold nanoparticles (AuNPs) and cargo-loaded liposomes. We report that among three different low-range mean ligand densities (0.15, 0.3, and 0.6 ∗ 10 3 antibodies/μm 2 ), the highest density yielded the highest ability towards both targeting of the BCECs and subsequent transport across the BBB in vivo, and in vitro using primary cultures of the murine BBB. We also find that TfR-targeting on liposomes in the mouse may induce severe adverse effects after intravenous administration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rationale: The ability to treat invalidating neurological diseases is impeded by the presence of the blood-brain barrier (BBB), which inhibits the transport of most blood-borne substances into the brain parenchyma. Targeting the transferrin receptor (TfR) on the surface of brain capillaries has been a popular strategy to give a preferential accumulation of drugs or nanomedicines, but several aspects of this targeting strategy remain elusive. Here we report that TfR-targeted gold nanoparticles (AuNPs) can accumulate in brain capillaries and further transport across the BBB to enter the brain parenchyma. Methods: We characterized our targeting strategy both in vitro using primary models of the BBB and in vivo using quantitative measurements of gold accumulation by inductively-coupled plasma-mass spectrometry together with morphological assessments using light microscopy after silver enhancement and transmission electron microscopy with energy-dispersive X-ray spectroscopy. Results: We find that the uptake capacity is significantly modulated by the affinity and valency of the AuNP-conjugated antibodies. Specifically, antibodies with high and low affinities mediate a low and intermediate uptake of AuNPs into the brain, respectively, whereas a monovalent (bi-specific) antibody improves the uptake capacity remarkably. Conclusion: Our findings indicate that monovalent ligands may be beneficial for obtaining transcytosis of TfR-targeted nanomedicines across the BBB, which is relevant for future design of nanomedicines for brain drug delivery., Competing Interests: Competing interests: All authors have declared no conflicting interest in relation to the theme and contents of the manuscript. The anti-TfRA, anti-TfRD, and anti-TfRA/BACE1 antibodies were obtained from Genentech, Inc. via a Material Transfer Agreement (MTA A15783). Genentech, Inc. had no influence on study design, experimental procedures, data analysis, or preparation of the manuscript.

Drug delivery to the brain is hampered by the presence of the blood-brain barrier, which excludes most molecules from freely diffusing into the brain, and tightly regulates the active transport mechanisms that ensure sufficient delivery of nutrients to the brain parenchyma. Harnessing the possibility of delivering neuroactive drugs by way of receptors already present on the brain endothelium has been of interest for many years. The transferrin receptor is of special interest since its expression is limited to the endothelium of the brain as opposed to peripheral endothelium. Here, we investigate the possibility of delivering immunoliposomes and their encapsulated cargo to the brain via targeting of the transferrin receptor. We find that transferrin receptor-targeting increases the association between the immunoliposomes and primary endothelial cells in vitro, but that this does not correlate with increased cargo transcytosis. Furthermore, we show that the transferrin receptor-targeted immunoliposomes accumulate along the microvessels of the brains of rats, but find no evidence for transcytosis of the immunoliposome. Conversely, the increased accumulation correlated both with increased cargo uptake in the brain endothelium and subsequent cargo transport into the brain. These findings suggest that transferrin receptor-targeting is a relevant strategy of increasing drug exposure to the brain.

An unmet need exists for therapeutic compounds to traverse the brain capillary endothelial cells that denote the blood-brain barrier (BBB) to deliver effective treatment to the diseased brain. The use of nanoparticle technology for targeted delivery to the brain implies that targeted liposomes encapsulating a drug of interest will undergo receptor-mediated uptake and transport through the BBB with a subsequent unfolding of the liposomal content inside the brain, hence revealing drug release to adjacent drug-demanding neurons. As transferrin receptors (TfRs) are present on brain capillary endothelial, but not on endothelial cells elsewhere in the body, the use of TfR-targeted liposomes - colloidal particulates with a phospholipid bilayer membrane - remains the most relevant strategy to obtain efficient drug delivery to the brain. However, many studies have failed to provide sufficient quantitative data to proof passage of the BBB and significant appearance of drugs inside the brain parenchyma. Here, we critically evaluate the current evidence on the use of TfR-targeted liposomes for brain drug delivery based on a thorough investigation of all available studies within this research field. We focus on issues with respect to experimental design and data analysis that may provide an explanation to conflicting reports, and we discuss possible explanations for the current lack of sufficient transcytosis across the BBB for implementation in the design of TfR-targeted liposomes. We finally provide a list of suggestions for strategies to obtain substantial uptake and transport of drug carriers at the BBB with a concomitant transport of therapeutics into the brain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Brain capillary endothelial cells (BCECs) express transferrin receptors. The uptake of a potential drug vector (OX26, or anti-transferrin receptor antibody IgG2a) conjugated to polyethyleneglycol-coated liposomes by BCECs was studied using in situ perfusion in 18-day-old rats in which the uptake of OX26 is almost twice as high as in the adult rat. Using radio-labeling, the uptake of OX26 by BCECs after 15-minute perfusion was approximately 16 times higher than that of nonimmune IgG2a (Ni-IgG2a). OX26 and OX26-conjugated liposomes selectively distributed to BCECs, leaving choroid plexus epithelium, neurons, and glia unlabeled. Ni-IgG2a and unconjugated liposomes did not reveal any labeling of BCECs. The labeling of BCECs by OX26 was profoundly higher than that of transferrin. Perfusion with albumin for 15 minutes did not reveal any labeling of neurons or glia, thus confirming the integrity of the blood-brain barrier. The failure to label neurons and glia shows that OX26 and OX26-conjugated liposomes did not pass through BCECs. The expression of transferrin receptors by endothelial cells selective to the brain qualifies OX26 as a candidate for blood-to-endothelium transport. A specifically designed formulation of liposomes may allow for their degradation within BCECs, leading to subsequent transport of liposomal cargo further into the brain.

The distribution of transferrin, transferrin receptor, ferritin and ferric iron was studied in the developing rat brain. Transferrin immunoreactivity (IR) was observed diffusely in the brain from E16 until P10 from where it gradually decreased. The subcellular distribution of transferrin-IR in neurons was compatible with receptor-mediated uptake from P21 and onwards. Transferrin receptor-IR was observed prenatally on cells of neuroectodermal origin in the ventricular zone and in brain capillary endothelial cells (BCECs). In postnatal rats, transferrin receptor-IR in BCECs was most pronounced in rats aged P10-P21 but thereafter decreased in intensity. The neuronal transferrin-receptor IR in postnatal brains was not consistently expressed on neurons until from P21 and onwards. Transferrin receptor-IR was not observed in astrocytes, oligodendrocytes or ramified microglial cells at any age. Ferric iron and ferritin were present in BCECs already from E16, declined from P3-P5, and was absent by P10. There results are discussed with emphasis on the age-dependent transport of transferrin into the developing brain. The upregulated expression of transferrin receptors on BCECs in the second and third postnatal week is compatible with a high need for iron at this age. The neuronal transferrin receptor expression by P21 coincides with a drop in transferrin-IR and iron transport into the brain at this age, suggesting that neuronal transferrin receptor mRNA is posttranscriptionally regulated by the lowered iron availability from this developmental stage onwards., (Copyright 2002 S. Karger AG, Basel)

Anti-transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10-fold higher than isotypic, non-immune IgG2a (Ni-IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)-day-old rats than in P0 and adult (P70) rats. Iron-deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6-9% of OX26 was identified in the post-capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni-IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood-brain barrier (BBB). However, OX26 accumulation in the post-capillary compartment was too small to justify a conclusion of receptor-mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post-capillary component may result from a diphasic transport that involves high-affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni-IgG2a, followed by a second transport mechanism that releases OX26 non-specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood-CSF barrier.

1. Iron (Fe) is an essential component of virtually all types of cells and organisms. In plasma and interstitial fluids, Fe is carried by transferrin. Iron-containing transferrin has a high affinity for the transferrin receptor, which is present on all cells with a requirement for Fe. The degree of expression of transferrin receptors on most types of cells is determined by the level of Fe supply and their rate of proliferation. 2. The brain, like other organs, requires Fe for metabolic processes and suffers from disturbed function when a Fe deficiency or excess occurs. Hence, the transport of Fe across brain barrier systems must be regulated. The interaction between transferrin and transferrin receptor appears to serve this function in the blood-brain, blood-CSF, and cellular-plasmalemma barriers. Transferrin is present in blood plasma and brain extracellular fluids, and the transferrin receptor is present on brain capillary endothelial cells, choroid plexus epithelial cells, neurons, and probably also glial cells. 3. The rate of Fe transport from plasma to brain is developmentally regulated, peaking in the first few weeks of postnatal life in the rat, after which it decreases rapidly to low values. Two mechanisms for Fe transport across the blood-brain barrier have been proposed. One is that the Fe-transferrin complex is transported intact across the capillary wall by receptor-mediated transcytosis. In the second, Fe transport is the result of receptor-mediated endocytosis of Fe-transferrin by capillary endothelial cells, followed by release of Fe from transferrin within the cell, recycling of transferrin to the blood, and transport of Fe into the brain. Current evidence indicates that although some transcytosis of transferrin does occur, the amount is quantitatively insufficient to account for the rate of Fe transport, and the majority of Fe transport probably occurs by the second of the above mechanisms. 4. An additional route of Fe and transferrin transport from the blood to the brain is via the blood-CSF barrier and from the CSF into the brain. Iron-containing transferrin is transported through the blood-CSF barrier by a mechanism that appears to be regulated by developmental stage and iron status. The transfer of transferrin from blood to CSF is higher than that of albumin, which may be due to the presence of transferrin receptors on choroid plexus epithelial cells so that transferrin can be transported across the cells by a receptor-mediated process as well as by nonselective mechanisms. 5. Transferrin receptors have been detected in neurons in vivo and in cultured glial cells. Transferrin is present in the brain interstitial fluid, and it is generally assumed that Fe which transverses the blood-brain barrier is rapidly bound by brain transferrin and can then be taken up by receptor-mediated endocytosis in brain cells. The uptake of transferrin-bound Fe by neurons and glial cells is probably regulated by the number of transferrin receptors present on cells, which changes during development and in conditions with an altered iron status. 6. This review focuses on the information available on the functions of transferrin and transferrin receptor with respect to Fe transport across the blood-brain and blood-CSF barriers and the cell membranes of neurons and glial cells.

Neuronal transferrin receptor protein expression is highly upregulated widely in CNS following iron deficiency. Using the medial habenular nucleus as a model of neuronal transferrin receptor mRNA expression, the present study examined 17-day-old rats subjected to variations in dietary iron. Changing the iron availability resulted in alterations in plasma and cerebrospinal fluid (CSF) levels of transferrin and iron. The iron-binding capacity of transferrin in CSF was exceeded in normal and iron-overloaded rats. In spite of a lowering of the concentration of brain iron by approximately 22% in iron-deficient rats, neuronal transferrin receptor mRNA was not affected when measured by quantitative densitometry. Brain iron and neuronal transferrin receptor mRNA expression was unaltered in iron overloaded rats. The absence of a rise in transferrin receptor mRNA during iron deficiency suggests that neuronal transferrin receptor mRNA expression is regulated by another mechanism than the post-transcriptional regulation mechanism, which has been attributed to cells of non-neural tissue.

In order to characterize the mechanism by which Iron (Fe) is taken up by neurons, we examined the neuronal expression of transferrin receptor (TR) in rats during development and iron (Fe) deficiency by using immunohistochemistry, in vitro receptor autoradiography and in situ hybridization. In contrast to the continuous expression of TR in brain capillary endothelial cells regardless of the age of the animals studied, the expression of neuronal TR was almost absent at late embryonic and early postnatal ages but increased with increasing age to reach a plateau from postnatal (P) 21 through adulthood as verified by immunohistochemical staining. Reducing the Fe stores potentiated the expression of TR immunoreactivity in neurons of both young and adult rats in several grey matter regions. Increased TR immunoreactivity was also observed in neuronal extensions of neurons of the medial habenular nucleus, reticular neurons of the brainstem, and fibers projecting to the area postrema. TR immunoreactivity was never observed in white matter regions, except for that recorded in brain capillaries. In vitro receptor autoradiography verified the increased capacity for transferrin binding during Fe deficiency. By contrast, TR mRNA expression was not affected by Fe deficiency. These findings demonstrate that the expression of the neuronal TR protein is age dependent and susceptible to Fe deficiency.

Iron is essential for a variety of intracellular functions. Accordingly, the transfer of iron from blood to brain is vital for normal brain function. In the CNS, the receptor for iron-transferrin is generally accepted to be located in endothelial cells, whereas its occurrence in other cell types is less well established. I have investigated the distribution of the transferrin receptor in the adult mouse central nervous system by immunohistochemistry by using a monoclonal antibody raised against the transferrin receptor protein. Immunoreactive cell types comprised brain capillary endothelial cells, excluding those of circumventricular organs, and choroid plexus epithelial cells. Moreover, transferrin receptor immunoreactivity was detected intraneuronally in several brain regions without access to peripheral blood. The immunoreactive cell bodies were mainly confined to the cerebral cortex, hippocampus, habenular nucleus, red nucleus, substantia nigra, pontine nuclei, reticular formation, several cranial nerve nuclei, deep cerebellar nuclei, and cerebellar cortex. Transferrin receptor immunoreactivity was not detected in astrocytes, oligodendrocytes, or microglial cells. The occurrence of transferrin receptors at brain-barrier sites, i.e., the brain endothelium and choroid plexus epithelium, and the presence of the receptors intraneuronally are in accordance with the generally held belief that iron is released from liver transferrin and transported through capillaries and the choroid plexus into the brain interstitium. Subsequently, iron may be linked to brain transferrin synthesized within oligodendrocytes and choroid plexus epithelial cells followed by a concomitant uptake of iron-transferrin in neurons expressing transferrin receptors. The clinical importance of the intraneuronal transferrin receptor expression is discussed.

It has been suggested that iron-carrying transferrin exerts growth-factor-like influences on motor neurons. I have evaluated the distribution of proteins related to the intracerebral iron-homeostasis in the mouse mutant progressive motor neuronopathy (pmn/pmn); an autosomal recessive mutant with progressive caudo-cranial motor neuron degeneration. A higher immunoreactivity of transferrin and transferrin receptor in motor neurons of the pmn/pmn mutant compared to that in normal mice was demonstrated. Ferritin was not observed in motor neurons of the pmn/pmn mutant. Transferrin receptors were absent from axons and neuromuscular junctions, indicating that entry of blood-borne, liver-derived transferrin ('liver transferrin') into motor neurons due to uptake and subsequent retrograde axonal transport was unspecific. Due to the selective presence of transferrin receptors on neuronal somata, a more likely mode of entry of transferrin into the motor neurons was by receptor-mediated uptake of brain-derived transferrin ('brain transferrin') at the soma. This study provides data on transferrin accumulation and transferrin receptor expression in diseased motor neurons and adds further insights into influences of proteins related to iron-homeostasis in the diseased PNS.