HPLC and Flow Cytometric Analyses of Uptake of Adriamycin and Menogaril by Monolayers and Multicell Spheroids

Abstract

We have used both HPLC and flow cytometry to measure and compare the uptake of two anthracyclines, menogaril (MEN) and Adriamycin (ADR), in V79 Chinese hamster lung fibroblasts grown as monolayers and as 650 microns multicell spheroids. In order to compare intracellular drug accumulation in spheroid cells measured by the two methods, we converted mean channel fluorescence of the flow cytometer to drug uptake expressed as ng/10(6) cells by using a standard curve. The standard curve related the flow cytometric mean channel fluorescence, of monolayer cells exposed to either drug, to the intracellular drug accumulation determined by HPLC. This standard curve was then used to convert the mean channel fluorescence of cells from drug-exposed spheroids to ng/10(6) cells. Our results show that equal intracellular drug accumulation (determined by HPLC) in spheroids and monolayers does not result in equal cellular fluorescence emission (determined by flow cytometry) by these 2 cell populations. For example, monolayer cells with an intracellular MEN accumulation of 650 ng/10(6) cells, emit 40 units of fluorescence as measured by flow cytometry. However, spheroid cells with the same intracellular accumulation emit about 80 units of fluorescence. This results in the intracellular MEN uptake in spheroids measured by flow cytometry being as much as 2- to 3-fold higher than that measured by HPLC. Intracellular ADR accumulation measured by flow cytometry was also higher than that obtained by HPLC. In spite of the quantitative difference between the two methods, qualitatively both methods gave similar results. Thus, both techniques showed that at equal drug concentration in medium drug uptake in monolayers was much greater than in spheroids.(ABSTRACT TRUNCATED AT 250 WORDS)