Determining Subcellular Drug Distributions

APPLICANT'S DESCRIPTION: The long-range objective of this research is to establish pharmacological profiles based on individual organelle and single cell analyses as a means of defining subcellular drug distributions. These distributions will contribute to improved drug screening methods, more accurate prediction of the side effects of drug treatment, and ultimately, the development of personalized drug treatments. Doxorubicin, a fluorescent anti-cancer agent, is used as a model. It is hypothesized that the subcellular localization of this drug in a few drug-treated single cells will accurately represent drug localization in the original cell culture. Furthermore, the analysis of individual organelles released from a doxorubicin-treated single cell will reveal differences in subcellular drug distributions and organelle properties that are related to drug resistance and drug sensitivity. Current analysis of subcellular drug distributions is based on confocal microscopy or on chromatographic separation of drug-containing cell extracts. The proposed studies will determine subcellular drug distributions in single cells based on the analysis of individual organelles by capillary electrophoresis with laser-induced fluorescence detection. For the first time, this technique will be used to directly measure the drug content in individual intact organelles. This approach will also reveal an additional biochemical property of the individual organelle that may be involved in doxorubicin subcellular distribution. Such properties include mitochondial membrane potential, lysosomal pH, the presence of P.glycoprotein, and cell cycle.