title: Remote Loading of (64)Cu(2+) into Liposomes without the Use of Ion Transport Enhancers.
abstract: Due to low ion permeability of lipid bilayers, it has been and still is common practice to use transporter molecules such as ionophores or lipophilic chelators to increase transmembrane diffusion rates and loading efficiencies of radionuclides into liposomes. Here, we report a novel and very simple method for loading the positron emitter (64)Cu(2+) into liposomes, which is important for in vivo positron emission tomography (PET) imaging. By this approach, copper is added to liposomes entrapping a chelator, which causes spontaneous diffusion of copper across the lipid bilayer where it is trapped. Using this method, we achieve highly efficient (64)Cu(2+) loading (>95%), high radionuclide retention (>95%), and favorable loading kinetics, excluding the use of transporter molecule additives. Therefore, clinically relevant activities of 200-400 MBq/patient can be loaded fast (60-75 min) and efficiently into preformed stealth liposomes avoiding subsequent purification steps. We investigate the molecular coordination of entrapped copper using X-ray absorption spectroscopy and demonstrate high adaptability of the loading method to pegylated, nonpegylated, gel- or fluid-like, cholesterol rich or cholesterol depleted, cationic, anionic, and zwitterionic lipid compositions. We demonstrate high in vivo stability of (64)Cu-liposomes in a large canine model observing a blood circulation half-life of 24 h and show a tumor accumulation of 6% ID/g in FaDu xenograft mice using PET imaging. With this work, it is demonstrated that copper ions are capable of crossing a lipid membrane unassisted. This method is highly valuable for characterizing the in vivo performance of liposome-based nanomedicine with great potential in diagnostic imaging applications.
authors.affiliation: Department of Chemistry, Technical University of Denmark , Building 206, 2800 Lyngby, Denmark.|Center for Nanomedicine and Theranostics, Technical University of Denmark , 2800 Lyngby, Denmark.
authors.forename: Jonas R
authors.initials: JR
authors.lastname: Henriksen
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark , Building 423, 2800 Lyngby, Denmark.|Center for Nanomedicine and Theranostics, Technical University of Denmark , 2800 Lyngby, Denmark.
authors.forename: Anncatrine L
authors.initials: AL
authors.lastname: Petersen
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark , Building 423, 2800 Lyngby, Denmark.|Department of Clinical Physiology, Nuclear Medicine & PET, Faculty of Health Sciences and Cluster for Molecular Imaging, Rigshospitalet, University of Copenhagen , Blegdamsvej 3, 2200 Copenhagen N, Denmark.|Center for Nanomedicine and Theranostics, Technical University of Denmark , 2800 Lyngby, Denmark.
authors.forename: Anders E
authors.initials: AE
authors.lastname: Hansen
authors.affiliation: Department of Chemistry, Technical University of Denmark , Building 206, 2800 Lyngby, Denmark.
authors.forename: Christian G
authors.initials: CG
authors.lastname: Frankær
authors.affiliation: Department of Chemistry, Technical University of Denmark , Building 206, 2800 Lyngby, Denmark.
authors.forename: Pernille
authors.initials: P
authors.lastname: Harris
authors.affiliation: DTU Nutech, Hevesy Laboratory, Technical University of Denmark , Frederiksborgvej 399, 4000 Roskilde, Denmark.|Center for Nanomedicine and Theranostics, Technical University of Denmark , 2800 Lyngby, Denmark.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Elema
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen , Dyrlægevej 16, 1870 Frederiksberg C, Denmark.
authors.forename: Annemarie T
authors.initials: AT
authors.lastname: Kristensen
authors.affiliation: Department of Clinical Physiology, Nuclear Medicine & PET, Faculty of Health Sciences and Cluster for Molecular Imaging, Rigshospitalet, University of Copenhagen , Blegdamsvej 3, 2200 Copenhagen N, Denmark.
authors.forename: Andreas
authors.initials: A
authors.lastname: Kjær
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark , Building 423, 2800 Lyngby, Denmark.|Center for Nanomedicine and Theranostics, Technical University of Denmark , 2800 Lyngby, Denmark.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Andresen
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D003301:Copper Radioisotopes / Q000737:chemistry*; D004285:Dogs; D006801:Humans; D017136:Ion Transport; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D008297:Male; D008819:Mice, Nude; D009942:Organometallic Compounds / Q000737:chemistry; D049268:Positron-Emission Tomography; D014018:Tissue Distribution; D014057:Tomography, X-Ray Computed; D056928:X-Ray Absorption Spectroscopy; D023041:Xenograft Model Antitumor Assays
chemical_list: D003301:Copper Radioisotopes; D008055:Lipids; D008081:Liposomes; D009942:Organometallic Compounds
keywords: diagnostic; ion permeability; molecular imaging; nanoparticles; positron emission tomography; remote loading