Remote Loading of ⁶⁴Cu²⁺ into Liposomes without the Use of Ion Transport Enhancers

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 ⁶⁴Cu²⁺ into liposomes, which is important for <i>in vivo</i> 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 ⁶⁴Cu²⁺ 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 <i>in vivo</i> stability of ⁶⁴Cu-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 <i>in vivo</i> performance of liposome-based nanomedicine with great potential in diagnostic imaging applications

PET/CT Based In Vivo Evaluation of 64Cu Labelled Nanodiscs in Tumor Bearing Mice

64Cu radiolabelled nanodiscs based on the 11 α-helix MSP1E3D1 protein and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine lipids were, for the first time, followed in vivo by positron emission tomography for evaluating the biodistribution of nanodiscs. A cancer tumor bearing mouse model was used for the investigations, and it was found that the approximately 13 nm nanodiscs, due to their size, permeate deeply into cancer tissue. This makes them promising candidates for both drug delivery purposes and as advanced imaging agents. For the radiolabelling, a simple approach for 64Cu radiolabelling of proteins via a chelating agent, DOTA, was developed. The reaction was performed at sufficiently mild conditions to be compatible with labelling of the protein part of a lipid-protein particle while fully conserving the particle structure including the amphipathic protein fold

Novel Preparation Methods of ⁵²Mn for ImmunoPET Imaging

52Mn (t1/2 =5.59 d, ß+ = 29.6%, Eßave = 0.24 MeV) shows promise in positron emission tomography (PET) and in dual-modality manganese-enhanced magnetic resonance imaging (MEMRI) applications including neural tractography, stem cell tracking, and biological toxicity studies. The extension to bioconjugate application requires high specific activity 52Mn in a state suitable for macromolecule labeling. To that end a 52Mn production, purification, and labeling system is presented, and its applicability in preclinical, macromolecule PET is shown using the conjugate 52Mn-DOTA-TRC105. 52Mn is produced by 60 µA, 16 MeV proton irradiation of natural chromium metal pressed into a silver disc support. Radiochemical separation proceeds by strong anion exchange chromatography of the dissolved Cr target, employing a semi-organic mobile phase, 97:3 (v:v) ethanol: HCl (11M, aqueous). The method is 62 ± 14% efficient (n=7) in 52Mn recovery, leading to a separation factor from Cr of (1.6 ± 1.0) x106 (n = 4), and an average effective specific activity of 0.8 GBq/µmol (n = 4) in titration against DOTA. 52Mn-DOTA-TRC105 conjugation and labeling demonstrate the potential for chelation applications. In vivo images acquired using PET/CT in mice bearing 4T1 xenograft tumors are presented. Peak tumor uptake is 18.7 ± 2.7 %ID/g at 24 hours post injection and ex vivo 52Mn biodistribution validates the in vivo PET data. Free 52Mn2+(as chloride or acetate) is used as a control in additional mice to evaluate the non-targeted biodistribution in the tumor model