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

Thermal undulations of quasi-spherical vesicles stabilized by gravity

The classical treatment of quasi-spherical vesicle undulations has, in the present work, been reviewed and extended to systems, which are affected by a gravitational field caused by a density difference across the membrane. The effects have been studied by the use of perturbation theory leading to corrections to the mean shape and the fluctuation correlation matrix. These corrections have been included in an analytical expression for the flicker spectrum to probe how the experimentally accessible spectrum changes with gravity. The results are represented in terms of the gravitational parameter, $g_{0}=\Delta\rho \mathrm{g} R^{4}/\kappa$. The contributions from gravity are in most experimental situations small and thus negligible, but for values of $g_{0}$ above a certain limit, the perturbational corrections must be included. Expressions for the relative error on the flicker spectrum have been worked out, so that it is possible to define the regime where gravity is negligible. An upper limit of $g_{0}$ has also been identified, where the error in all modes of the flicker spectrum is significant due to distortion of the mean shape