Liposomes containing copper and the copper ionophore neocuproine were prepared and
characterized for in vitro and in vivo anticancer activity. Thermosensitive PEGylated
liposomes were prepared with different molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine
(DPPC) and hydrogenated soybean phosphatidylcholine (HSPC) in the presence of copper(II)
ions. Optimal, temperature dependent drug release was obtained at 70:30 DPPC to HSPC
weight ratio. Neocuproine (applied at 0.2 mol to 1 mol phospholipid) was encapsulated
through a pH gradient while using unbuffered solution at pH 4.5 inside the liposomes,
and 100 mM HEPES buffer pH 7.8 outside the liposomes. Copper ions were present in
excess, yielding 0.5 mM copper-(neocuproine)2 complex and 0.5 mM free copper. Pre-heating
to 45 °C increased the toxicity of the heat-sensitive liposomes in short-term in vitro
experiments, whereas at 72 h all investigated liposomes exhibited similar in vitro
toxicity to the copper(II)-neocuproine complex (1:1 ratio). Thermosensitive liposomes
were found to be more effective in reducing tumor growth in BALB/c mice engrafted
with C26 cancer cells, regardless of the mild hyperthermic treatment. Copper uptake
of the tumor was verified by PET/CT imaging following treatment with [64Cu]Cu-neocuproine
liposomes. Taken together, our results demonstrate the feasibility of targeting a
copper nanotoxin that was encapsulated in thermosensitive liposomes containing an
excess of copper.
