title: Coupling of metal containing homing devices to liposomes via a maleimide linker: use of TCEP to stabilize thiol-groups without scavenging metals.
abstract: Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe(3+)), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe(3+)). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2-3 h coupling to the liposomes, Fe(3+) is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes. Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe(3+) from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself. In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe(3+) from Tf. This method can be applied to other metal-containing homing devices as well.
authors.affiliation: Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University, Division of Pharmacology, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Corine C
authors.initials: CC
authors.lastname: Visser
authors.forename: L Heleen
authors.initials: LH
authors.lastname: Voorwinden
authors.forename: Liesbeth R
authors.initials: LR
authors.lastname: Harders
authors.forename: Mohamed
authors.initials: M
authors.lastname: Eloualid
authors.forename: Louis
authors.initials: L
authors.lastname: van Bloois
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; D002417:Cattle; D016503:Drug Delivery Systems / Q000379:methods; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry* / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D010720:Phosphines / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008081:Liposomes; D008301:Maleimides; D008670:Metals; D010720:Phosphines; D013438:Sulfhydryl Compounds; C080938:tris(2-carboxyethyl)phosphine; C043592:maleimide