Characterization and modulation of the transferrin receptor on brain capillary endothelial cells

Purpose. The expression level of the transferrin receptor (TfR) on brain capillary endothelial cells (BCECs) and the endocytosis of I-125-transferrin (I-125-Tf) by this receptor was investigated. Furthermore, the influence of iron, the iron scavenger deferoxamine mesylate (DFO), astrocytic factors, a GTP-ase inhibitor (tyrphostin-A8, T8), lipopolysaccharide ( LPS), and the radical scavenger N-acetyl-L-cysteine (NAC) on the TfR expression was studied to gain insight in the use and optimization of the TfR for drug targeting to the brain. Methods. Experiments were performed with primary cultured bovine BCECs that were incubated with I-125-Tf at 4degreesC (to determine binding) or at 37degreesC (to determine endocytosis) in the absence or presence of the modulators. For full saturation curves in the absence or presence of iron or DFO, analysis was performed with a population approach using NONMEM, allowing us to estimate a single value for affinity (K-d, concentration of 50% receptor occupancy) and separate values for maximum receptor occupancy (B-max). Results. On BCECs, the TfR is expressed extracellularly (B-max of 0.13 fmol/mug cell protein), but also has a large intracellular pool (total B-max of 1.37 fmol/mug cell protein), and is actively endocytosing Tf via clathrin-coated vesicles. At 4degreesC, a K-d of 2.38 mug/ml was found, whereas the K-d at 37degreesC was 5.03 mug/ml. Furthermore, DFO is able to increase both the extracellular as well as the total binding capacity to 0.63 and 3.67 fmol/mug cell protein, respectively, whereas it had no influence on K-d. B-max at 37degreesC after DFO preincubation was also increased from 0.90 to 2.31 fmol/mug cell protein. Other modulators had no significant influence on the TfR expression levels, though LPS increased cellular protein concentrations after 2-h preincubation. Conclusions. The TfR is expressed on BCECs and actively endocytoses Tf, making it a suitable target for drug delivery to the blood-brain barrier and the CNS. DFO up-regulates the TfR expression level, which may influence targeting efficiency.