Organometallic complexes are one of the most promising types of anticancer drugs. Studies of the last few years show that copper coordination compounds are particularly promising as antitumor therapeutic agents. Many copper complexes are characterized by hydrophobicity and, as a consequence, low bioavailability, which significantly complicates their practical use. Such complexes include Medation, an antitumor drug, which is a coordination compound of copper based on 2-alkylthioimidazolone. According to the results of preclinical trials, the drug is recommended for triple-negative breast cancer treatment.
One of the ways to improve the parameters of solubility and bioavailability of hydrophobic active molecules is their encapsulation into nanocontainers for drug delivery. The most studied and used system for the delivery of biologically active substances are liposomes. The hydrophobicity of the copper complex does not allow the use of usual methods to hydrophilic substances for its loading into liposomes, therefore, the purpose of this work is to find the optimal method for encapsulation of copper coordination compound into liposomes.
We have developed an approach for copper complex encapsulation into liposomes, which is realized by injection of organic solvents mixture into copper salt solution. Liposomes were prepared from dipalmitoyl phosphatidylcholine (DPPC), cholesterol (Chol) and PEG-distearoyl phosphoethanolamine (DSPE-PEG(2000)) in the ratio DPPC:Chol:DSPE-PEG(2000) 55:40:5. Quantitative determination of the ligand and complex in liposomal suspension was carried out using HPLC. This new approach allowed us to obtain a suspension resistant to aggregation and sedimentation for up to two weeks, the loading efficiency was 1.22% (molar ratio of the complex to lipids). Ionic strength of copper solution and cholesterol content in lipid membrane affected the loading capacity. The developed technique has been successfully applied to obtain liposomal forms of similar copper coordination compounds based on 2-thioxoimidazolone, which indicates the possibility of using the technique for encapsulation of hydrophobic copper complexes in liposomes.
The stability of the resulting suspension was evaluated in NaCl solution at 4^oС and in RPMI cell medium at 37^oС. The suspension retained loading for several days in both environments, which indicates the advantage of the developed technique. It was shown by derivative spectroscopy that the complex in liposomes is stable when stored at a temperature of 4^oС and dissociates within 24 hours at temperatures of 37^oС and 45^oС. Incubation at 37^oС leads to the release of 10% of copper loaded into liposomes. Quenching constants for fluorescently labeled liposomes were calculated using spectrofluorimetry, which made it possible to analyze the behavior of the complex in the lipid bilayer. The cytotoxicity of the liposomal formulation was evaluated using an MTT test on MCF-7 cells – the test showed that the formulation has greater cytotoxicity due to increased bioavailability. In addition to antitumor activity, copper complexes have also demonstrated antibacterial properties, which can be used for joint therapy in the future.