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.
Hovorka
Ondřej
O
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic. hovorka@biomed.cas.cz
Subr
Vladimír
V
Větvička
David
D
Kovář
Lubomír
L
Strohalm
Jiří
J
Strohalm
Martin
M
Benda
Aleš
A
Hof
Martin
M
Ulbrich
Karel
K
Ríhová
Blanka
B
D016475:3T3 Cells; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism* / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D016503:Drug Delivery Systems; D005434:Flow Cytometry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016399:Lymphoma, T-Cell / Q000188:drug therapy / Q000378:metabolism; D051379:Mice; D011108:Polymers / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D000903:Antibiotics, Antineoplastic; D011108:Polymers; C079835:7,8-dehydro-9,10-desacetyldoxorubicinone; D004317:Doxorubicin; D004247:DNA