title: Glutathione conjugation dose-dependently increases brain-specific liposomal drug delivery in vitro and in vivo.
abstract: The blood-brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology®) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands; Blood-Brain Barrier Research Group, Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, 1007 MB Amsterdam, The Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Maussang
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: Rip
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Joan
authors.initials: J
authors.lastname: van Kregten
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Angelique
authors.initials: A
authors.lastname: van den Heuvel
authors.affiliation: Blood-Brain Barrier Research Group, Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, 1007 MB Amsterdam, The Netherlands.
authors.forename: Susanne
authors.initials: S
authors.lastname: van der Pol
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Burt
authors.initials: B
authors.lastname: van der Boom
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Arie
authors.initials: A
authors.lastname: Reijerkerk
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: Chen
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands.
authors.forename: Marco
authors.initials: M
authors.lastname: de Boer
authors.affiliation: to-BBB technologies BV (now 2-BBB Medicines BV), J.H. Oortweg 19, 2333 CH, Leiden, The Netherlands. Electronic address: PieterGaillard@2-BBB.com.
authors.forename: Pieter
authors.initials: P
authors.lastname: Gaillard
authors.affiliation: Blood-Brain Barrier Research Group, Department of Molecular Cell Biology and Immunology, Neuroscience Campus Amsterdam, VU University Medical Center, 1007 MB Amsterdam, The Netherlands.
authors.forename: Helga
authors.initials: H
authors.lastname: de Vries
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage* / Q000493:pharmacokinetics; D001692:Biological Transport; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism; D005978:Glutathione / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D057809:HEK293 Cells; D006801:Humans; D008081:Liposomes; D008297:Male; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D017208:Rats, Wistar; D012254:Ribavirin / Q000008:administration & dosage* / Q000493:pharmacokinetics; D013552:Swine
chemical_list: D000998:Antiviral Agents; D008081:Liposomes; D011092:Polyethylene Glycols; D012254:Ribavirin; D005978:Glutathione