Abstract
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.
Highlights
Cancer is the second most frequent cause of death [1,2]
Concentrated nitric acid (65% w/w) and H2O2 (30% w/w) of Suprapur quality needed for the sample preparation of cell lines for Total-Reflection X-ray Fluorescence (TXRF) measurement were supplied by Merck (Darmstadt, Germany)
The IR measurements were carried out using a Varian FTS-2000 (Scimitar Series) FTIR spectrometer (Varian Inc., Agilent, Santa Clara, CA, USA) that was equipped with a ‘Golden Gate’ single reflection diamond attenuated total reflection (ATR) accessory (Specac Ltd., Orpington, UK). 3 μL of preformed copper-neocuproine complex (1:1) or liposome samples were spread on the surface of the diamond ATR element and a thin dry film was obtained by slowly evaporation of the buffer solvent under ambient conditions
Summary
Cancer is the second most frequent cause of death [1,2]. Treatment of advanced stage diseases is still a major clinical problem despite the availability of a broad range of therapeutic agents with different mechanisms of anticancer activity. Common examples of copper chelating ligands include derivates of dithiocarbamate [11], 8-hydroxyquinoline [12], thiosemicarbazone [13,14], and phenantroline structures [15] These ligands form stable and highly toxic copper complexes with persistent toxicity, even in multidrug resistant cell lines. The toxicity of copper chelating ionophores is usually accompanied by the increased cellular accumulation of copper [18], which is primarily responsible for cell death [15]. This is in contrast to the mechanism of toxicity of another group of chelators including tetrathiomolybdate, which kill cells by copper depletion [10,19] Compounds belonging to this second group do not cross cell membranes and are ionic in their chemical nature. We describe the development and biological characterization of a drug delivery system consisting of liposomes loaded with excess copper and the water-soluble active ingredient neocuproine
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