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Research Articles: Targets

Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors

Experimental Oncology Laboratory, Shaare Zedek Medical Center, and Hebrew University-Hadassah Medical School, Jerusalem, Israel

Requests for reprints: Alberto Gabizon, Oncology Institute, Shaare Zedek MC, 11 Shmuel Bayt Street, P.O. Box 3235, Jerusalem 91031, Israel. Phone: 972-2-655-5036; Fax: 972-2-652-1431. E-mail: alberto{at}md.huji.ac.il

Abstract

The folate receptor is overexpressed in a broad spectrum of malignant tumors and represents an attractive target for selective delivery of anticancer agents to folate receptor–expressing tumors. This study examines folate-lipid conjugates as a means of enhancing the tumor selectivity of liposome-encapsulated drugs in a mouse lymphoma model. Folate-derivatized polyethylene glycol (PEG₃₃₅₀)-distearoyl-phosphatidylethanolamine was post-loaded at various concentrations into the following preparations: radiolabeled PEGylated liposomes, PEGylated liposomes labeled in the aqueous compartment with dextran fluorescein, and PEGylated liposomal doxorubicin (PLD, Doxil). We incubated folate-targeted radiolabeled or fluorescent liposomes with mouse J6456 lymphoma cells up-regulated for their folate receptors (J6456-FR) to determine the optimal ligand concentration required in the lipid bilayer for liposomal cell association, and to examine whether folate-targeted liposomes are internalized by J6456-FR cells in suspension. Liposomal association with cells was quantified based on radioactivity and fluorescence-activated cell sorting analysis, and internalization was assessed by confocal fluorescence microscopy. We found an optimal ligand molar concentration of 0.5% using our ligand. A substantial lipid dose-dependent increase in cell-associated fluorescence was found in folate-targeted liposomes compared with nontargeted liposomes. Confocal depth scanning showed that a substantial amount of the folate-targeted liposomes are internalized by J6456-FR cells. Binding and uptake of folate-targeted PLD by J6456-FR cells were also observed in vivo after i.p. injection of folate-targeted PLD in mice bearing ascitic J6456-FR tumors. The drug levels in ascitic tumor cells were increased by 17-fold, whereas those in plasma were decreased by 14-fold when folate-targeted PLD were compared with nontargeted PLD in the i.p. model. Folate-targeted liposomes represent an attractive approach for the intracellular delivery of drugs to folate receptor–expressing lymphoma cells and seem to be a promising tool for in vivo intracavitary drug targeting. [Mol Cancer Ther 2006;5(4):818–24]

Grant support: ALZA Corporation, the Arnall Family Fund, and the Posnansky Trust.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Dr. Chris Leamon, Endocyte, personal communication.

² H. Shmeeda and A. Gabizon, unpublished data.

Received 12/28/05; accepted 2/16/06.