To date, preclinical studies have addressed drug accumulation and intracellular distribution of
cisplatin by determination of the total Pt content. In this work, the use of liquid chromatography in combination with inductively coupled plasma mass spectrometry (LC-ICP-MS) enabled accurate intact
cisplatin quantification in cell model experiments. Hence, for the first time, intracellular drug degradation, drug accumulation and drug efflux were studied by actually quantifying the intact drug, along with the total Pt content of the cell nucleus, the cytosol and the low molecular weight fraction of the cytosol. The latter fraction was obtained by centrifugal filtration (cut-off filter of 10 kDa). Flow injection (FI)-ICP-MS was implemented for
platinum quantification. In a first step, kinetics of intracellular
cisplatin degradation was addressed by incubating
cell extracts with sub-μM drug concentration levels. A half-life of 2 hours was observed in
cell extracts of two different
cancer cell lines (colon
carcinoma and human
mesothelioma), which was significantly shorter than that observed in
sodium chloride. Hence, it was suggested that intact and nonaquated
cisplatin was reacting with cellular components. Due to the large excess of potential binding partners pseudo first order kinetics were observed. The drug accumulation experiments revealed rapid uptake of the drug into the cytosol and the nucleus. Moreover, a significant fraction of Pt was bound to intracellular high molecular weight biomolecules after one hour of exposure. With ongoing time, the intracellular Pt concentration was increasing. However, the
cisplatin concentration remained constant during 5 hours of continuous exposure. Assuming a cell volume of 10(-12) L, an intracellular concentration corresponding to the
cisplatin concentration in the cell culture medium (5 μM) was estimated. At any time of investigation, intact
cisplatin was the predominant species in the low molecular weight fraction of the cytosol. These findings support the hypothesis of passive diffusion as an uptake mechanism. Finally, a model experiment was designed resembling the situation of limited drug exposure time. Human
mesothelioma cells were incubated with 5 μM
cisplatin for 3 hours. Then the culture medium was replaced and the drug efflux was studied. The observed efflux was biphasic, with the intact
cisplatin being removed within the first hour of investigation, while the Pt-
protein adduct fraction was removed only partially (30% were still found in the cytosol after 24 hours). No net transfer of Pt from the cytosol to the nucleus fraction was observed after medium replacement.