Fluorescent hydrophobic ion pairs: A powerful tool to investigate cellular uptake of hydrophobic drug complexes via lipid-based nanocarriers

Hypothesis: Hydrophobic ion pairs (HIPs) between two fluorescent components and incorporation into nano emulsions (NE) allows tracking in cellular uptake studies. Experiments: HIPs were formed between propidium iodide and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), azure A chloride and NBD-PE or coumarin 343 and 4-(4dihexadecylaminostyryl)-N-methylpyridinium iodide) (DiA). Fluorescence spectra of the resulting complexes were recorded. HIPs were loaded into zwitterionic NE and their size, stability in different media, haemolytic properties and cytotoxicity were evaluated. Furthermore, cellular uptake at 37 degrees C and 4 degrees C was investigated via flow cytometry and confocal microscopy. Findings: HIP-formation increased lipophilicity of the hydrophilic model drugs. NE exhibited a size between 80 and 150 nm and were not toxic in concentrations up to 0.1 % but showed high haemolytic properties. Cellular uptake of propidium, azure A and coumarin 343 were 8-fold, 115-fold and 1.3-fold improved by the formation of HIPs and up to 59-fold, 120-fold and 50-fold by incorporating these HIPs in NE, respectively. Lower uptake was observed at 4 degrees C. In case of propidium/NBD-PE and azure A/NBD-PE HIPs, propidium and azure A were delivered into the cytosol, whereas NBD-PE was unable to enter cells. In case of coumarin 343/DiA HIPs, both components accumulated in the cell membrane. Therefore, HIPs between two fluorescent compounds are a powerful tool to investigate cellular uptake of hydrophobic complexes via nanocarriers by visualization of their cellular distribution.