Intracellular delivery of doxorubicin with RGD-modified sterically stabilized liposomes for an improved antitumor efficacy: In vitro and in vivo

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Abstract

Passive targeting by sterically stabilized liposomes (SSL), once combined with efficient intracellular delivery, may be a very useful strategy to improve the antitumor efficacy for the anticancer agents. The arginine–glycine–aspartic acid tripeptide (RGD) is known to serve as a recognition motif for several different integrins located on cell surface. In this study, the RGD tripeptide was coupled to the distal end of the poly (ethylene glycol)-coated liposomes (RGD-SSL) aimed to achieve increased tumor accumulation and enhanced intracellular uptake. DOX-loaded RGD-SSL (RGD-SSL-DOX), DOX-loaded SSL (SSL-DOX), and free DOX were compared with respect to their in vitro uptake and cytotoxicity and their in vivo biodistribution and therapeutic efficacy in tumor-bearing mice. Flow cytometry and confocal microscopy studies revealed that RGD-SSL could facilitate the DOX uptake into melanoma cells by integrin-mediated endocytosis. RGD-SSL-DOX displayed higher cytotoxicity on melanoma cells than SSL-DOX. While RGD-SSL-DOX demonstrated prolonged circulation time and increased tumor accumulation as SSL-DOX did, it showed remarkably higher splenic uptake than SSL-DOX. Mice receiving RGD-SSL-DOX (5 mg DOX/kg) showed effective retardation in tumor growth compared with those receiving same dose of SSL-DOX, free DOX solution, or saline. These results suggest that RGD-modified SSL may be a feasible intracellular targeting carrier for efficient delivery of chemotherapeutic agents into tumor cells.

Section snippets

Abbreviations:

    DOX

    doxorubicin

    Chol

    cholesterol

    SPC

    soya phosphatidylcholine

    DSPE-PEG

    methoxypolyetheleneglycol (Mw 2000)-distearylphosphatidylethanolamine

    DSPE-PEG-BTC

    1,2-dioleyol-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-N-benzotriazole carbonate, PEG Mw 3400

    RGD

    arginine–glycine–aspartic acid

    SSL

    sterically stabilized liposomes

    SSL-DOX

    DOX-loaded SSL

    RGD-SSL

    RGD-modified SSL

    DOX-RGD-SSL

    DOX-loaded RGD-SSL

    IC50

    50% inhibitory concentration

    DMEM

    Dulbecco's modification of Eagle's medium

    PBS

    phosphate-buffered

INTRODUCTION

Liposomes have been extensively investigated as targeting carriers for a variety of anticancer agents. The drug-loaded liposomes are usually injected intravenously for systemic application. However, there is usually rapid clearance of them from the circulation by the reticuloendothelial system (RES). To avoid this disadvantage, “Stealth® technology” (or called sterically stabilized liposomes, SSL) has been developed.1 Growing solid tumors have capillaries with increased permeability and

Materials

Methoxypolyetheleneglycol (Mw 2000)-distearylphosphatidylethanolamine (DSPE-PEG) was purchased from NOF Co. (Tokyo, Japan). DSPE-PEG-BTC [1,2-dioleyol-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-N-benzotriazole carbonate, PEG Mw 3400] was purchased from Shearwater Polymers, Inc. (Huntsville, AL), and soya phosphatidylcholine (SPC) from Lucas Meyer (Hamburg, Germany). Cholesterol (Chol) and Sephadex G50 were obtained from Pharmacia Biotech (Piscataway, NJ).

Preparation of RGD-Modified SSL

An activated DSPE-PEG (DSPE-PEG-BTC) was used to conjugate RGD to DSPE-PEG. The reaction takes place between α-amines of the RGD and BTC group of the lipid, which acts as the linking agent. RGD was successfully covalently conjugated to the DSPE-PEG-BTC under the conditions of reaction (4 h at 4°C, pH 7.5, gentle stirring and 1:2 molar ratio). The disappearance of N-benzotriazole identified by NMR (data not shown) suggests that there was almost no existence of DSPE-PEG-BTC or uncoupled RGD in the

DISCUSSION

Drugs with intracellular targets or therapeutic genes are required to cross the cell membrane for pharmacological activity. Delivery of these agents into tumor tissue only represents part of the problems for tumor targeting, and other challenges for successful tumor targeting include drug release from the carrier and drug passage across the biomembranes. Rigid lipid was frequently used in the liposomes preparation to gain stability advantage. In this case the entrapped drugs were tightly

Acknowledgements

This work was supported in part by the Doctoral Foundation from Chinese Minister of Education (20040001141).

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