Nuclear Delivery of Doxorubicin via Folate-targeted Liposomes with Bypass of Multidrug-resistance Efflux Pump 1 Dorit Goren, Aviva T. Horowitz, Dina Tzemach, Mark Tarshish, Samuel Zalipsky, and Alberto Gabizon 2 Sharet Institute of Oncology [D. G., A. T. H., D. T., A. G.] and Interdepartmental Unit [M. T.], Hadassah Hebrew University Medical Center, Jerusalem 91120, Israel, and ALZA Corporation, Mountain View, California 94039 [S. Z.] ABSTRACT Folic acid, attached to polyethyleneglycol-derivatized, distearoyl-phosphatidylethanolamine, was used to target in vitro liposomes to folate receptor (FR)-overexpressing tumor cells. Confocal fluorescence microscopic observations dem- onstrated binding and subsequent internalization of rho- damine-labeled liposomes by a high FR-expressing, murine lung carcinoma line (M109-HiFR cells), with inhibition by free folic acid. Additional experiments tracking doxorubicin (DOX) fluorescence with DOX-loaded, folate-targeted lipo- somes (FTLs) indicate that liposomal DOX is rapidly inter- nalized, released in the cytoplasmic compartment, and, shortly thereafter, detected in the nucleus, the entire process lasting 1–2 h. FR-mediated cell uptake of targeted liposomal DOX into a multidrug-resistant subline of M109-HiFR cells (M109R-HiFR) was unaffected by P-glycoprotein-mediated drug efflux, in sharp contrast to uptake of free DOX, based on verapamil-blockade experiments with quantitation of cell-associated DOX and flow cytometry analysis. Delivery of DOX by FTLs to M109R-HiFR cells increased continu- ously with time of exposure, reaching higher drug concen- trations in whole cells and nuclei compared with exposure to free DOX. The in vitro cytotoxic activity obtained with DOX- loaded FTLs was 10-fold greater than that of the nontar- geted liposome formulation, but was not improved over that of free DOX despite the higher cellular drug levels obtained with the targeted liposomes in M109R-HiFR cells. However, if M109R-HiFR cells were exposed to drugs in vitro and tested in an in vivo adoptive assay for tumor growth in syngeneic mice along a 5-week time span, FTL DOX was significantly more tumor inhibitory than free DOX. It is suggested that the biological activity of liposomal DOX re- leased inside the cellular compartment is reduced in vitro due to the aggregated state of DOX, resulting from the liposome drug-loading process, and requires a long period of time and/or an in vivo environment for full expression. INTRODUCTION FR 3 , a GPI membrane-anchored glycoprotein of 38 kDa (1), with extremely high affinity for folate (kDa  10 -10 M for -isoform and kDa  10 -9 M for -isoform; Ref. 2), is over- expressed in a wide variety of epithelial tumors (2–5). The FR participates in the cellular accumulation of folates in a number of epithelial cells through a process of endocytosis (6). In this process, ligand-bound receptor is internalized and released from the receptor through intravesicular reduction in pH. Ligand-free receptor is then recycled to the cell surface (6, 7). The receptor- mediated uptake of folic acid has been proposed as a potentially useful target in cancer treatment (7, 8) and as a route to promote entry of attached macromolecules or liposomes into cells (9). Liposomes with folate residues conjugated through a lipo- some-grafted PEG spacer are taken up avidly by KB cells (human nasopharyngeal cancer cell line; Refs. 10 and 11). The binding of these FTLs to KB cells is mediated by cell-surface FR, as demonstrated by competitive inhibition with excess free folate or with antiserum against the FR (10, 11). By providing a different pathway of tumor cell drug uptake, the use of targeted liposomes for chemotherapeutic delivery may conceivably cir- cumvent the MDR drug efflux mechanism, leading to resistance (12, 13). The Pgp, located in the plasma membrane, is an active efflux pump of cytotoxic agents conferring multidrug resistance to cancer cells (12, 13). Intracellular entry of drug-loaded lipo- somes via endocytosis, followed by release of entrapped agent in cytoplasm (11), is an alternative route of drug entry that may enable bypassing Pgp-mediated efflux. Here, we investigate the mechanism of drug delivery and activity of DOX-loaded FTLs as compared with free drug and drug encapsulated in nontargeted liposomes, and we report on drug uptake studies and tumor cell growth assays with DOX- sensitive and -resistant, high-FR-expressing murine tumor cells. MATERIALS AND METHODS Liposome Preparation. Preparation of liposomes and the sources of liposome components, including DSPE- PEG(3350)-Folate, were as described previously (14). DPPE- rhodamine was obtained from Avanti Polar Lipids (Birming- ham, AL). The following formulations were prepared: (a) Received 11/12/99; revised 2/11/00; accepted 2/14/00. 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 Supported by the Israel Science Foundation (Jerusalem, Israel) and by ALZA Corporation (Mountain View, CA). 2 To whom requests for reprints should be addressed, at Sharet Institute of Oncology, Hadassah Medical Center, Kiryat Hadassah, P.O. Box 12000, Jerusalem 91120, Israel. Fax: 972-2-6430622; E-mail: alberto@ md2.huij.ac.il. 3 The abbreviations used are: FR, folate receptor; GPI, glycosyl-phos- phatidylinositol; PEG, polyethyleneglycol; FTL, folate-targeted lipo- somes; Pgp, P-170 glycoprotein; MDR, multidrug-resistance; DOX, doxorubicin; DSPE, distearoyl-phosphatidylethanolamine; DPPE, di- palmitoyl-PE; HiFR, high expression of FR in M109 cells. 1949 Vol. 6, 1949 –1957, May 2000 Clinical Cancer Research Research. on October 23, 2021. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from