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 <1400g, 1400–14000g, and >14000g 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.

Introduction

Anthracyclines are among the most commonly used anticancer drugs, used for more than 30 years and are still considered among the most useful anticancer agents developed. Doxorubicin (DOX) (structure shown in Scheme 1) is a clinically important anthracycline [1], offering therapeutic effectiveness against a variety of solid tumors, such as leukemia, ovarian cancer, breast cancer, prostate cancer and cervix cancer [2], [3], [4], [5], [6], [7], [8]. Despite its extensive clinical utilization, the action mechanisms of anthracycline on cancer cells remain a matter of controversy. The proposed mechanisms are considered as follows [9], [10], [11]: (1) inhibition on synthesis of macromolecules due to its intercalation into DNA; (2) DNA damage or lipid peroxidation resulted from the generation of free radicals; (3) DNA binding and alkylation; (4) DNA cross-linking; (5) interference with DNA unwinding or DNA strand separation and helicase activity; (6) direct membrane effects; (7) initiation of DNA damage via inhibition of topoisomerase II; and lastly (8) induction of apoptosis in response to topoisomerase II inhibition.

Aside from its therapeutic effect, regrettably DOX treatment also often comes with incidents of cardiac/liver toxicity and drug resistance [2] that may result from cellular processes involving the parent compound or drug metabolites. The oxidative activity of DOX aglycone metabolites, which often leads to the release of mitochondrial Ca²⁺, the swelling of mitochondria, a change of the mitochondrial membrane potential, and the production of superoxide (O₂⁻), may be the factor contributing to acute cardiac and liver toxicity [12]. Needless to say, exploration of possible causes and research into the action mechanism on biological processes (such as drug resistance) and cytotoxicity induced by DOX treatment are considered significant. Moreover, it is also important to study the subcellular localization of DOX, which helps to identify metabolic pathway that DOX takes upon entry of the cell. An efficient and rapid quantification procedure for measurement of DOX or other anthracycline in biological samples is thus in urgent need. Previous studies have reported various analytical approaches for studying DOX, daunorubicin as well as other anthracyclines and their metabolites, including confocal microscopy [13], polarography [14], high-performance liquid chromatography (HPLC) [15], [16], [17], [18], displacement chromatography [19], laser flow cytometry [20], and capillary electrophoresis (CE) [1], [2], [21]. Though chromatographic methods remain the principal analytical approaches for in vitro and in vivo analysis of anthracyclines due to its superior sensitivity [22], [23], the requirement of larger sample size and expensive equipment, complication of sample pretreatment, and consumption of large amounts of solvents have made them less ideal for high-throughput and environmental friendly analytical techniques. CE, on the other hand, is easier to be home-built and capable of analyzing minute quantities of samples. CE related analytical techniques have been widely used as a simple, rapid means to investigate anthracyclines and their metabolites at the single-cell level with no degradation [2], [21], [24], [25], [26].

Drug delivery systems (DDS) are exploited to circumvent some of the non-ideal properties of free drug or conventional formulation and provide better control over the pharmacokinetics (PK) and pharmacodynamics (PD) of the encapsulated drugs relative to free drugs. Liposome has drawn increasing interest from various branches of medicine for its ability to deliver drugs in the optimum dosage range, resulting in improved therapeutic efficacy of the drug and a decline in toxic side effects [27], [28], and some examples of success on non-targeted liposomes used in clinical practices have already been demonstrated (i.e., Doxil, which is the trade name for the generic chemotherapy drug—doxorubicin HCl liposome injection; DaunoXome, which is a chemotherapy drug that is given to treat AIDS-related Kaposi's sarcoma). Doctors in Taiwan prescribe Lipo-dox^®, a pegylated liposomal doxorubicin HCl (TTY Biopharm, Taiwan) for curing ovarian cancer, breast cancer, AIDS-related Kaposi's sarcoma, as this medication is recognized by and can be given full reimbursement by the Taiwan National Health Insurance. It was also found that lower dosage of liposomal DOX was able to efficiently kill more cancer cells in vitro and in vivo as compared to free DOX [29], [30]. However, the quantification of exact amounts of drugs being delivered via liposomal carriers to cells or subcellular fractions, comparing to free drug, has seldom been investigated. In this study, we extend the utilization of a modified MEKC–LIF with reliable separation and good reproducibility to monitor subcellular distribution of DOX, which enters cells via different routes (free form vs. liposomal form). The developed method was used to determine the subcellular accumulation of DOX in cultured Chinese hamster ovary CHO-K1 cells at clinically relevant concentrations, making the method potentially useful for mechanistic studies.