Confocal laser scanning microscopy (CLSM) based evidence for cell permeation by...

Beta-cyclodextrin (β-CyD), amantadine and glucose were fluorescently tagged with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD chloride) to afford NBD-β-CyD, NBD-amantadine and NBD-glucose, respectively. NBD-β-CyD/amantadine and β-CyD/NBD-amantadine inclusion complexes were prepared. Fluorescence emission maxima (λ(max) 544nm) and relative fluorescence intensities for NBD-β-CyD and NBD-β-CyD/amantadine were virtually identical, precluding the use of emission spectrum shifts for distinguishing free NBD-β-CyD from the complex. Intracellular accumulation of NBD-β-CyD was studied in HepG2 and SK-MEL-24 cells using confocal laser scanning microscopy (CLSM). No major differences were observed between uptake of NBD-β-CyD and NBD-β-CyD/amantadine. Serum proteins did not perturb uptake, whereas temperature-dependent uptake, indicative of cell entry via diffusion, was observed. Intracellular distribution favoured mitochondria, with less fluorescent material present in cytoplasm and none in cell nuclei. No experimental evidence of NBD-β-CyD breakdown to NBD-glucose was found upon chromatographic analysis of incubation mixtures, providing additional evidence of intact NBD-β-CyD entry into these cells. Endocytosis and/or cholesterol-independent membrane modulation are discussed as possible mechanisms for the transmembrane passage of NBD-β-CyD.