Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red...

Cationic amphiphilic drugs (CAD) containing a basic moiety often accumulate in lysosomes or other acidic subcellular compartments. This lysosomotropism is due to the protonation of the CAD within acidic organelles leading to the formation of a membrane-impermeable form. Highly lipophilic CADs show a greater propensity to accumulate than those with a lower lipophilicity (Log P). Here we describe a rapid method to quantitate the uptake of CAD within lysosomes. The principle of the method involves a competition between the CAD and the fluorescent basic amine probe Red DND-99 (LysoTracker) in HeLa cells. Visualization is performed using confocal fluorescence microscopy. Loading HeLa cells with 10-150 nMRed DND-99 gives a punctuated fluorescent pattern around the cell nucleus. This fluorescence is displaced by incubating the cells with 10-100mM NH(4)Cl, either before or after the addition of the probe, consistent with the reversible partitioning of the probe in the lysosome. The displacement of probe fluorescence by CAD such as chlorpromazine and imipramine was observed in a dose-dependent manner. Lower concentrations of CAD with high Log P displaced the probe more efficiently than those with a lower Log P. This assay represents a rapid and semiquantitative method for the measurement of lysosomotropism in an in vitro system without the use of radioactivity and cell fractionation. Furthermore, the microscopy confirms the targeting of the basic compounds to within the lysosomes.