Design and synthesis of a multivalent fluorescent folate-calix[4]arene conjugate: cancer cell penetration and intracellular localizationElectronic supplementary information (ESI) available: NMR, fluorescence, and DLS spectra. See DOI: 10.1039/c4ob02333a

A novel fluorescently labeled folate conjugate in which four folic acid units are covalently conjugated with a 7-nitro-benzofurazan fluorophore by means of a calix[4]arene platform was synthesized by using a Cu-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The synthesized construct (FA-C4-NBD) was characterized by mass spectrometry, NMR and fluorescence spectroscopy. Confocal fluorescence microscopy experiments were carried out to evaluate the cell penetration ability of FA-C4-NBD on normal and cancer cells. The cellular uptake of FA-C4-NBD proceeds via folate receptor-mediated endocytosis. FA-C4-NBD is internalized into HeLa cancer cells which express high levels of folate receptors, whereas the uptake into fibroblast NIH3T3 cells which have very low expression levels of folate receptors is negligible. The involvement of the folate receptor was corroborated by competition tests with free folic acid. Co-localization analysis with different organelle markers indicated that FA-C4-NBD is not eliminated by recycling towards the outside of the cell, but accumulates intracellularly in the endo-lysosomal system. Fluorescent multivalent folate-calix[4]arene-NBD selectively penetrates cancer cells via folate receptor-mediated endocytosis and localizes in endo-lysosomes.