Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation

There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.