Monitoring the subcellular localization of doxorubicin in CHO-K1 using MEKC-LIF: liposomal carrier for enhanced drug delivery

Abstract

Doxorubicin (DOX) is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and finally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modified MEKC-LIF (Micellar electrokinetic chromatography-Laser-induced fluorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an efficient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3-725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400 g, 1400-14000 g, and >14000 g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 μM (equivalent to 14.5 μg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to confirm the enhanced efficiency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid-liquid extraction and MEKC-LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs.