A cell fractionation approach for the quantitative analysis of subcellular drug disposition.

Abstract

The purpose of this work was to develop and validate a method that can be used to quantify drugs associated with intracellular compartments. The human leukemic cell line U-937 was used to evaluate the distribution of model compounds with known and different subcellular distribution profiles. Lysotracker Red is a lysosomal vital stain and doxorubicin is an anticancer agent with a strong propensity for nuclear accumulation in U-937 cells. After incubation with compounds, cells were separated into fractions containing nuclei, cytosol, and cytoplasmic organelles (lysosomes, mitochondria, Golgi apparatus). Compounds contained within isolated fractions were subsequently extracted and analyzed by high-performance liquid chromatography. Diffusion of compounds from isolated organelles was also investigated. Using this approach we have shown that the model compounds Lysotracker Red and doxorubicin preferentially accumulated within lysosomes and nuclei, respectively. We have reproducibly determined concentrations of these compounds in each of the cellular fractions. We have also shown that diffusion of these compounds from isolated cellular compartments was minimal during the time required to complete the experimental procedure. The analytical approach described in this manuscript yielded reproducible quantitative data regarding the intracellular distribution of model compounds in U-937 cells. With the aid of a relatively sensitive analytical assay, this technique should be useful for most drugs that have a specific concentrative mechanism for organelle accumulation similar to Dox and LTR.