Pharmaceutical Nanotechnology
Vascular targeting of doxorubicin using cationic liposomes

https://doi.org/10.1016/j.ijpharm.2007.01.003Get rights and content

Abstract

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p < 0.05), DOX uptake of these liposomes at 24 h post-injection was similar to that of PEGylated liposomal DOX (p > 0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p < 0.01), neutral liposome control (p < 0.01), as well as PEGylated liposomes (p < 0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.

Introduction

Physiological barriers have been shown to hinder effective delivery of drugs to tumors (Jain, 1998, Pluen et al., 2001). In order to reach cancer cells, a therapeutic agent must first cross the vasculature and then travel through the interstitium. Vascular targeting avoids these barriers by attacking the blood supply instead of cancer cells, either suppressing vessel formation (antiangiogenic therapy) or abolishing established vascular networks (antivascular therapy) (Denekamp, 1982, Thurston et al., 1998). Subsequently, tumor cells undergo apoptosis as a consequence of impaired nutrient and oxygen supply. Due to their high rate of proliferation, tumor endothelial cells have increased susceptibility to cytotoxic drugs (Denekamp, 1999). Furthermore, these cells are more genetically stable and are thus less prone to drug resistance compared to tumor cells. Selective delivery of cytotoxic drugs to tumor endothelium is, therefore, a promising therapeutic strategy.

Liposomes have been evaluated as a carrier of anticancer chemotherapeutic agents, such as doxorubicin (Mayer et al., 1989). Neutral liposomes exhibit preferential localization in solid tumors based on enhance permeation and retention (EPR) effect (Papahadjopoulos et al., 1991, Huang et al., 1992, Yuan et al., 1994), which relies on gradual passive accumulation of liposomes in the tumor (Huang et al., 1992, Yuan et al., 1994, Wu et al., 1993). In contrast, cationic liposomes are rapidly cleared from circulation by liver, spleen, and lung, and primarily have been used in gene delivery (McLean et al., 1997, Mahato et al., 1995, Litzinger et al., 1996). The unique properties of cationic liposomes for targeting tumor endothelium suggest that they may have utility as drug carriers. Strieth et al. (2004) and Schmitt-Sody et al. (2003) reported that paclitaxel encapsulated in cationic liposomes exhibited improved antitumor efficacy of, which was associated with impaired function of tumor microvasculature. The study of Thurston et al. (1998) showed that uptake of cationic liposomes by endothelial cells in angiogenic blood vessels was considerably greater than that of extravasation from these vessels. Furthermore, this uptake was selective to cationic liposomes and that anionic, neutral, or sterically stabilized neutral liposomes were not taken up by these cells.

The aim of our study was to evaluate in vitro cytotoxicity, in vivo biodistribution, pharmacokinetics and efficacy of doxorubicin delivered in cationic liposomes.

Section snippets

Materials

Egg phosphatidylcholine (EPC), cholesterol (CHOL), and methoxy-polyethylene glycol (M.W. 2000) distearoyl phosphatidylethanolamine (mPEG-DSPE) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), doxorubicin (DOX), and dimethyl dioctadecyl ammonium bromide (DDAB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were analytical or HPLC grade.

Cell culture

KB, a human oral carcinoma cell line,

Properties of cationic L-DOX

Cationic liposomes containing 20% DDAB and control liposomes were prepared by repeated extrusion through polycarbonate membranes with a pore size of 100 nm and DOX were incorporated by remote loading. Properties of the liposomes are summarized in Table 1, including particle size, DOX loading efficiency and zeta potential.

We found that increasing the mol% of cationic lipid resulted in increased accumulation in the lung and reduced plasma half-life of the liposomal formulation. Percentage (20%) of

Discussion

The vascular network is a highly accessible target for tumor therapy. Cationic liposomes have been shown to accumulate selectively in angiogenic tumor endothelial cells. Therefore, they are promising as a drug carrier for delivery to the tumor endothelium.

This study reports the characterization of cationic liposomal formulations of DOX. Enhancement in both cellular uptake and in vitro cytotoxicity was demonstrated. This might be due to the ability of cationic liposomes to interact with cells

Conclusion

A cationic liposomal DOX formulation was synthesized and evaluated. The formulation was efficiently taken up by and showed enhanced cytotoxicity in KB and L1210 cells. Pharmacokinetic and biodistribution studies indicated that cationic L-DOX showed rapid clearance from the blood, rapid accumulation in the tumor, and increased therapeutic efficacy. Further studies will focus on understanding of mechanisms of the observed antitumor activity by drugs delivered via cationic liposomes.

Acknowledgement

This work was supported in part by NSF grant EEC-0425626.

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