In Vivo Fate of Folate-Targeted Polyethylene-Glycol Liposomes in Tumor-Bearing Mice Alberto Gabizon, 1 Aviva T. Horowitz, 2 Dorit Goren, 2 Dina Tzemach, 1 Hilary Shmeeda, 1 and Samuel Zalipsky 3 1 Shaare Zedek Medical Center and 2 Hadassah Medical Center, Jerusalem, Israel and 3 ALZA Corporation, Mountain View, California ABSTRACT Purpose: To compare the in vivo tissue distribution of folate-targeted liposomes (FTLs) injected i.v. in mice bear- ing folate receptor (FR)-overexpressing tumors (mouse M109 and human KB carcinomas, and mouse J6456 lym- phoma) to that of nontargeted liposomes (NTLs) of similar composition. Experimental Design: A small fraction of a folate-poly- ethylene-glycol (PEG)-distearoyl-phosphatidylethanolamine conjugate was incorporated in FTLs. Both FTLs and NTLs were PEGylated with a PEG-distearoyl-phosphatidyleth- anolamine conjugate to prolong circulation time. Liposomes were labeled with [ 3 H]cholesterol hexadecyl ether with or without doxorubicin loading. Liposome levels in plasma, tissues, or ascites were assessed by the number of [ 3 H] counts. For doxorubicin-loaded formulations, we also deter- mined the tissue doxorubicin levels by fluorimetry. To esti- mate the amount of liposomes directly associated with tumor cells in vivo, we determined the [ 3 H]radiolabel counts in washed pellets of ascitic tumor cells using the ascitic J6456 lymphoma Results: FTLs retained the folate ligand in vivo, as demonstrated by their ability to bind ex vivo to FR-express- ing cells after prolonged circulation and extravasation into malignant ascitic fluid. In comparison with NTLs, FTLs were cleared faster from circulation as a result of greater liver uptake. Despite the lower plasma levels, tumor levels of FTL-injected mice were not significantly different from those of NTL-injected mice. When NTLs and FTLs were loaded with doxorubicin, liver uptake decreased because of liver blockade, and uptake by spleen and tumor increased. When tumor-to-tissue liposome uptake ratios were ana- lyzed, the targeting profile of FTLs was characterized by higher tumor:skin, and tumor:kidney ratios but lower tu- mor:liver ratio than NTLs. After a concomitant dose of free folic acid, FTLs (but not NTLs) plasma clearance and liver uptake were inhibited, indicating that accelerated clearance was mediated by the folate ligand. Surprisingly tumor up- take was not significantly affected by a codose of folic acid. In the J6456 ascitic tumor model, tumor cell-associated liposome levels were significantly greater for FTL-injected mice than for NTL-injected mice, despite slightly higher levels of the latter in whole ascites. Conclusions: Whereas folate targeting does not enhance overall liposome deposition in tumors, the targeting profile of tumor versus other tissues is substantially different and intratumor liposome distribution in ascitic tumors is af- fected favorably with a selective shift toward liposome asso- ciation with FR-expressing cells. INTRODUCTION Receptor-mediated endocytosis pathways can be exploited for specific targeting of liposomes and intracellular delivery of liposome contents (1, 2). Coupling liposomes to a ligand, that is directed to an over-expressed receptor in cancer cells and that normally undergoes endocytosis, is a strategy that can improve selectivity and facilitate access of liposomes to the intracellular compartment. Folic acid (FA) is one of the well-studied target- ing ligands used for this strategy. Macromolecules and particu- late carriers, conjugated to FA are successfully recognized by folate receptors (FRs) and internalized into cells via folate- receptor mediated endocytosis (3–5). Cell surface receptors for FA are overexpressed across a broad spectrum of human tumors (6, 7). The FR is a glycosyl-phosphatidylinositol-anchored gly- coprotein with high affinity for the FA vitamin (K d  10 -10 M; see Refs. 8, 9). It is located in caveolae and participates in the cellular accumulation of folates through the process of potocy- tosis. In this process, receptor-bound ligand is sequestered in caveolae, internalized into postcaveolar plasma vesicles, re- leased from the receptor via an intravesicular reduction in pH, and subsequently transported into cytoplasm for polyglutama- tion (10, 11). The receptor is then recycled to the cell surface. The lack of immunogenicity and relatively simple chemistry of FA make folate-receptor mediated endocytosis a very useful tool in specific drug targeting. The relevance of FR as a useful target for tumor-specific drug delivery is supported by findings indicating up-regulation (higher expression) in many human cancers including those of the ovary, brain, kidney, lung, breast, and myeloid cells (12). In addition, aggressive or undifferentiated tumors with advanced stage or grade appear to have an increased FR density (13, suggesting that FR-mediated delivery may be a broad approach in cancer treatment. In previous studies, we have investigated the in vitro bind- ing of folate-targeted liposomes (FTLs) to tumor cells express- Received 6/17/03; revised 8/28/03; accepted 8/28/03. Grant support: This work was supported by research grants from the Israel Science Foundation (Jerusalem, Israel) and ALZA Corporation (Mountain View, CA). 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. Requests for reprints: Alberto Gabizon, Oncology Institute, Shaare Zedek Medical Ctr., POB 3235, Jerusalem, il-91031, Israel. Phone: 972-2-6555-036; Fax: 972-2-652-1431; E-mail: alberto@md.huji.ac.il. 6551 Vol. 9, 6551– 6559, December 15, 2003 Clinical Cancer Research Cancer Research. on October 23, 2021. © 2003 American Association for clincancerres.aacrjournals.org Downloaded from