Targeting Efficiency of Galactosylated Liposomes to Hepatocytes in Vivo: Effect of Lipid Composition Aki Murao, 1 Makiya Nishikawa, 1 Chittima Managit, 1 Joseph Wong, 2 Shigeru Kawakami, 1 Fumiyoshi Yamashita, 1 and Mitsuru Hashida 1,3 Received August 15, 2002; accepted August 29, 2002 Purpose. To investigate the effects of the lipid composition of galac- tosylated liposomes on their targeted delivery to hepatocytes. Methods. Several types of liposomes with a particle size of about 90 nm were prepared using distearoyl-L-phosphatidylcholine (DSPC), cholesterol (Chol) and cholesten-5-yloxy-N-(4-((1-imino-2-D- thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol), and la- beled with [ 3 H]cholesterol hexadecyl ether. Their tissue disposition was investigated in mice following intravenous injection. The binding and internalization characteristics were also studied in HepG2 cells. Results. Compared with [ 3 H]DSPC/Chol (60:40) liposomes, [ 3 H]D- SPC/Chol/Gal-C4-Chol (60:35:5) liposomes exhibit extensive hepatic uptake. Separation of the liver cells showed that galactosylated lipo- somes are preferentially taken up by hepatocytes, whereas those lack- ing Gal-C4-Chol distribute equally to hepatocytes and nonparenchy- mal cells (NPC). Increasing the molar ratio of DSPC to 90% resulted in enhanced NPC uptake of both liposomes, suggesting their uptake via a mechanism other than asialoglycoprotein receptors. DSPC/ Chol/Gal-C4-Chol (60:35:5) and DSPC/Chol/Gal-C4-Chol (90:5:5) li- posomes exhibited similar binding to the surface of HepG2 cells, but the former were taken up faster by the cells. Conclusions. The recognition of galactosylated liposomes by the asia- loglycoprotein receptors is dependent on the lipid composition. Cho- lesterol-rich galactosylated liposomes, exhibiting less non-specific in- teraction and greater receptor-mediated uptake, are better for tar- geting drugs to hepatocytes in vivo. KEY WORDS: liposome; asialoglycoprotein receptor; hepatocytes; drug targeting; internalization. INTRODUCTION Drug carriers with specific ligands for the corresponding receptors on the cell surface are useful for targeted drug de- livery. Among various ligands investigated so far, galactose has been shown to be a promising targeting ligand to hepa- tocytes (liver parenchymal cells) because the cells possess a large number of the asialoglycoprotein receptors that recog- nize the galactose units on the oligosaccharide chains of gly- coproteins or on chemically galactosylated drug carriers (1). The receptor-ligand interaction is known to show a significant “cluster effect” in which a multivalent interaction results in extremely strong binding of ligand to the receptors (2). We have already demonstrated that the in vivo recognition of galactosylated macromolecules by asialoglycoprotein recep- tors correlates with the degree of galactose modification (3– 5). A pharmacokinetic analysis of the tissue disposition pat- terns of galactosylated proteins in mice has clearly shown that the density of galactose units on the protein surface deter- mines the affinity of galactosylated proteins for asialoglyco- protein receptor-mediated hepatic uptake (4). These results indicate that the drug targeting efficiency to hepatocytes us- ing galactosylated macromolecular carriers is dependent on the degree of galactose modification. Liposomes are another class of drug carriers that have several advantages such as ease of preparation and a large capacity for drug loading (6). In previous papers, we synthe- sized a galactosylated cholesterol derivative and formulated it into neutral or cationic liposomes to obtain the galactosylated counterparts (7–11). These galactosylated liposomes were able to effectively deliver prostaglandin E 1 , probucol and plasmid DNA to hepatocytes in vivo, indicating that the ga- lactose units on the liposome surface can increase the affinity of the liposome for asialoglycoprotein receptors on hepato- cytes. To ensure hepatocyte-specific targeting of liposomes by galactosylation, however, properties other than galactose density should also be controlled, such as the size and electric charge. The recognition of liposomes by the mononuclear phagocyte system (MPS) is known to be dependent on its lipid composition (12,13), which is an important factor in de- termining the surface properties of liposomes. Therefore, the clearance of liposomes from the circulation in vivo is highly dependent on the lipid composition (14). The surface prop- erties of liposomes, such as the rigidity of the membrane and co-existence of two or more phases, is determined by their composition, which might affect the clustering of galactose units incorporated into a liposome formulation. To understand the effects of the lipid composition of galactosylated liposomes on their targeted delivery to hepa- tocytes in vivo, various liposomes with or without galactose units were prepared involving different lipid mixing ratios: distearoyl-L-phosphatidylcholine (DSPC), cholesterol (Chol) a and galactosylated cholesterol derivative, and cholesten-5- yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)amino)bu- tyl)formamide (Gal-C4-Chol). The liposomes were adjusted to a size of about 90 nm in diameter and radiolabeled with [ 3 H]cholesteryl hexadecyl ether (CHE). The tissue disposi- tion of each type of liposome was studied in mice after intra- venous injection. The distribution of liposomes within the liver (i.e., to hepatocytes or liver nonparenchymal cells (NPC)), was examined after digestion of the liver by collage- nase. In addition, the internalization of liposomes was inves- tigated in HepG2 cells in vitro. We report here that the lipid composition of the galactosylated liposomes is important for their recognition by asialoglycoprotein receptors. 1 Department of Drug Delivery Research, Graduate School of Phar- maceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. 2 I.V. Systems Division, Baxter Healthcare Corporation, Round Lake, Illinois 60073. 3 To whom correspondence should be addressed. (e-mail: hashidam@ pharm.kyoto-u.ac.jp) ABBREVIATIONS: DSPC, distearoyl-L-phosphatidylcholine; Chol, cholesterol; Gal-C4-Chol, cholesten-5-yloxy-N-(4-((1-imino-2-D- thiogalactosylethyl)amino)butyl)formamide; NPC, nonparenchymal cells; MPS, mononuclear phagocyte system; CHE, cholesteryl hexa- decyl ether; IME-thiogalactoside, 2-imino-2-methoxyethyl-1- thiogalactoside; Gal-BSA, galactosylated bovine serum albumin; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; CL liver , liver uptake clearance; PC, phospholipid. Pharmaceutical Research, Vol. 19, No. 12, December 2002 (© 2002) Research Paper 1808 0724-8741/02/1200-1808/0 © 2002 Plenum Publishing Corporation