Stealth monensin liposomes as a potentiator of adriamycin in cancer treatment

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Abstract

Small unilamellar stealth monensin liposomes (SMLs) were prepared from multilamellar liposomes (MLVs). The MLVs were prepared by using dipalmitoyl phosphatidylcholine (DPPC), cholesterol, distearoyl glycerophosphoethanolamine coupled to poly(ethylene glycol) (DSPE–PEG) and stearylamine in the molar ratio of 10:5:1.4:1.4 (32.8 mM total lipid). The encapsulation efficiencies of monensin in MLVs and small unilamellar vesicles (SUVs) was 6×10−6 and 10−7 M, respectively. The stability of SMLs was studied at 4°C. The amount of leakage of monensin from SMLs was less than 20% after four weeks of storage. The in vitro release of monensin from SMLs in human serum was determined, and t1/2 was found to be 10 h. Pharmacokinetic studies on SMLs were carried out in BALB/c mice. More than 20% of SMLs remained in blood circulation after 24 h. SMLs increased the uptake of adriamycin (AM) in HL-60-resistant cells by more than two fold, compared to monensin in solution. SMLs potentiated the effect of AM against both sensitive and resistant HL-60 cells (six- and tenfold potentiation, respectively) and human LOVO tumor cells (four- and 200-fold potentiation, respectively). However, the highest potentiation was observed against resistant human breast tumor MCF7 cells, and was found to be 2400 times in comparison to AM alone. Transmission electron microscopic (TEM) studies carried out with HL-60-resistant tumor cells incubated with SMLs showed that SMLs caused dilation of the golgi of tumor cells within 10 min. The dilation of golgi was reversible after reincubation of the cells in fresh medium. SMLs showed considerable potential as a potentiator in combination with AM in overcoming drug resistance.

Introduction

The development of drug resistance to cancer chemotherapy is one of the biggest obstacles to the successful treatment of cancer disease. Various cellular factors are involved in drug resistance, including: (a) defective drug transport through the cell membrane; (b) enhanced drug inactivation or decreased drug activation; (c) altered affinity or levels of a target enzyme and (d) an enhanced level of repair of DNA damage. In most cases, more than one of the above-listed cellular factors are responsible for clinical drug resistance 1, 2.

Adriamycin (AM), an anthracycline anticancer agent, is currently being used for a variety of cancers, like breast, leukemia, ovarian, etc. However, during repeated courses of AM treatment, various tumors develop resistance to AM. Cancer cells, which are resistant to AM, often show cross-resistance to other types of anticancer drugs, even though these drugs were never used in the therapy (pleiotropic resistance). This phenomenon of multiple drug resistance (MDR) is associated with reduced drug accumulation, coupled with increased drug efflux mechanisms 3, 4, 5, 6. Multidrug resistance is mediated through overexpression of the membrane transport pump known as p-glycoprotein (Pgp, Mr∼130,000). Tumor cells that exhibit MDR, express the Pgp gene, known as mdrl. Several studies have indicated that increased Pgp expression could be relevant in the development of drug resistance in cancer cells. Recently, another drug transporter (MRP) was shown to be overexpressed in several MDR cell lines that do not overexpress Pgp 2, 7, 8.

It is known that acidic intracellular compartments are involved in governing pleiotropic resistance. It has been reported that the carboxylic ionophores monensin and nigericin disrupt the acidic vesicular traffic, leading to elevated intravesicular pH 6, 9. Monensin in solution has been reported to modulate AM resistance to Ehrlich ascites tumor cells [6]. However, monensin is very lipophilic in nature and has to be formulated in a drug delivery system for it to be bioavailable both in vitro and in vivo. In our laboratory, monensin has been encapsulated in conventional liposomes and these liposomes showed significant potentiation of AM and immunotoxins against a variety of human tumors 10, 11, 12. However, the relatively short half-life of these liposomes was a major drawback for their limited use in vivo. One of best possible ways of increasing the circulation time of liposomes is by the addition of synthetic lipid derivatives of poly(ethyleneglycol) (PEG). Several reports have been cited in the literature for the preparation of pegylated or `stealth' liposomes 13, 14, 15, 16.

In the present article, we report the formulation of sterically stabilized or `stealth' monensin liposomes (SMLs), their stability on storage and their potentiating effect with AM against different resistant and sensitive human tumor cell lines. The possible mechanism involved in potentiating the cytotoxic effect of AM has also been discussed.

Section snippets

Materials

Cholesterol (CH), stearylamine (SA), dipalmitoyl phosphatidylcholine (DPPC) and monensin were obtained from Sigma (St. Louis, MO, USA). Disteroyl glycerophosphoethanolamine coupled to poly(ethylene glycol) (DSPE–PEG) was obtained from Shearwater Polymers (Huntsville, AL, USA). Radioactive 3H-monensin was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). 14C-AM was purchased from Amersham Life Science (Arlington Heights, IL, USA). Buffers and other chemicals were of reagent

Results and discussion

SMLs were prepared using varying concentrations of lipids. Several formulations were made and tested for the amount of monensin entrapped, particle size, leakage and stability. The representative SML formulations discussed in this report consisted of varying concentrations of DPPC, cholesterol, DSPE–PEG, stearylamine and 7.5 mg of monensin. For the purpose of analysis, 50 μCi of radioactive 3H-monensin were added to the formulation. The purpose of these formulations was to prepare SMLs that had

Abbreviations

SUVSmall unilamellar vesicles
MLVsmultilamellar vesicles
SMLsstealth monensin liposomes
AMadriamycin
SML–AMcombination of SML and AM
CHcholesterol
SAstearylamine
DPPCdipalmitoyl phosphatidylcholine
DSPE–PEGdistearoyl glycerophosphoethanolamine coupled to poly(ethylene glycol)
TEMtransmission electron microscopy
MDRmultiple drug resistance
Pgpp-glycoprotein
PBSphosphate-buffered saline

Acknowledgements

This research was supported by RCMI award G12RR03020-1 1, and MBRS award GMO81 11-24.

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