title: Validation of the transferrin receptor for drug targeting to brain capillary endothelial cells in vitro.
abstract: Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood-brain barrier (BBB) and its concentration in biological media can be easily quantified. Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 microg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37 degree C. This was approximately 3-fold higher compared to binding at 4 degree C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37 degree C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed. In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.
authors.affiliation: Division of Pharmacology, Leiden/Amsterdam Centre for Drug Research (LACDR) Leiden University PO Box RA 2300 Leiden The Netherlands.
authors.forename: Corine C
authors.initials: CC
authors.lastname: Visser
authors.forename: Sanja
authors.initials: S
authors.lastname: Stevanović
authors.forename: L
authors.initials: L
authors.lastname: Heleen Voorwinden
authors.forename: Pieter J
authors.initials: PJ
authors.lastname: Gaillard
authors.forename: Daan J A
authors.initials: DJ
authors.lastname: Crommelin
authors.forename: Meindert
authors.initials: M
authors.lastname: Danhof
authors.forename: Albertus G
authors.initials: AG
authors.lastname: De Boer
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000098:blood supply / Q000378:metabolism*; D002199:Capillary Permeability; D002417:Cattle; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D016503:Drug Delivery Systems; D004705:Endocytosis; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D007502:Iron Chelating Agents / Q000494:pharmacology; D011990:Receptors, Transferrin / Q000378:metabolism*; D013997:Time Factors; D014168:Transferrin / Q000737:chemistry / Q000493:pharmacokinetics
chemical_list: D007502:Iron Chelating Agents; D011990:Receptors, Transferrin; D014168:Transferrin; D006735:Horseradish Peroxidase; D003676:Deferoxamine