Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99
Quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine Red DND-99
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DOI：
10.1016/j.ab.2004.01.010
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发表时间：
2004-04-15
影响因子：
2.9
通讯作者：
Falgueyret, JP
中科院分区：
文献类型：
--
作者：
Lemieux, B;Percival, MD;Falgueyret, JP
含有碱性基团的阳离子两亲性药物（CAD）通常积聚在溶酶体或其他酸性亚细胞区室中。这种溶酶体向性是由于酸性细胞器内CAD的质子化导致膜不渗透形式的形成。与亲脂性较低的cad相比，高亲脂性cad具有更大的积聚倾向（Log P）。在这里，我们描述了一种快速的方法来量化溶酶体内CAD的摄取。该方法的原理涉及到CAD和荧光碱胺探针Red DND-99 （LysoTracker）在HeLa细胞中的竞争。使用共聚焦荧光显微镜进行可视化。用10-150 nM的Red ddn -99加载HeLa细胞，在细胞核周围出现间断的荧光图案。在加入探针之前或之后，用10-100 mM NH4Cl孵育细胞，这种荧光会发生位移，这与探针在溶酶体中的可逆分配一致。氯丙嗪、丙咪嗪等CAD对探针荧光的位移呈剂量依赖性。低浓度的高logp的CAD比低logp的更有效地使探针移位。该试验代表了一种快速和半定量的方法，用于在体外系统中测量溶酶体向性，而不使用放射性和细胞分离。此外，显微镜证实了溶酶体内的碱性化合物的靶向性。(C) 2004爱思唯尔公司版权所有。
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 nM Red DND-99 gives a punctuated fluorescent pattern around the cell nucleus. This fluorescence is displaced by incubating the cells with 10-100 mM NH4Cl, 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. (C) 2004 Elsevier Inc. All rights reserved.