2.5. Liposome Characterization and Stability Measurement

The size expressed as the hydrodynamic diameter (d_h) and the polydispersity index (PDI) were measured via dynamic light scattering (DLS, ZetaPals, Brookhaven Instruments Corporation, Holtsville, NY, USA). Liposomal samples were diluted to 1–5 mM (total lipid) in filtered SH buffer for size and polydispersity analyses.

The Cu(DDC)₂ to lipid ratio (drug to lipid ratio, D/L) was calculated with Equation (2), where n_{entrapped drug} is the amount of substance of complexed DDC⁻ with Cu²⁺ and n_{total lipid} the amount of substance of lipid:

For storage stability studies the liposomes were kept at 4–6 °C for 6 months (183 days) and at RT (22 ± 2 °C) and −20 °C for 2 months (61 days). Therefore, aliquots of non-PEGylated and PEGylated Cu(DDC)₂ liposomes were prepared and characterized via DLS at the indicated time points. Stability studies included the determination of the release rate of Cu(DDC)₂ from liposomes after storage of samples at 4–6 °C for 6 months using surface area filtration (Section 2.3).