The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar preference (N-acetylgalactosamine (GalNAc) > > galactose) and the effects of ligand valency (tetraantennary > triantennary > > diantennary > > monoantennary) and sugar spacing (20 Å > > 10 Å > > 4 Å) are well documented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maximum ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Hereto, we synthesized a novel glycolipid, which possesses a highly hydrophobic steroid moiety for stable incorporation into liposomes, and a triantennary GalNAc 3 -terminated cluster glycoside with a high nanomolar affinity (2 nM) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [ 3 H]cholesteryl oleate-labeled long circulating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 ؎ 7% of the injected dose at 30 min after injection) and mediated by the ASGPr on hepatocytes, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was demonstrated that particles with a diameter of >70 nm could no longer be recognized and processed by the ASGPr in vivo. This threshold size for effective uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, because similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in addition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of substrates by the ASGPr. Therefore, these data have important implications for the design of ASGPr-specific carriers that are aimed at hepatocyte-directed delivery of drugs and genes.The hepatic asialoglycoprotein receptor (ASGPr) 1 is a C-type (Ca 2ϩ -dependent) lectin that is expressed on the surface of hepatocytes (1) and plays a role in the clearance (endocytosis and lysosomal degradation) of desialylated proteins from the serum (2, 3) as has been shown for cellular fibronectin (4) and all IgA2 allotypes (5). The human functional receptor is a noncovalent heterotetramer composed of two homologous type II membrane polypeptides with 55% sequence identity, generally called HL-1 (hepatic lectin 1) and HL-2, at a 2:2 stoichiometry (6). The ASGPr binds glycoproteins with either nonreducing terminal -D-galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, at which the affinity for GalNAc is approximately 50-fold higher than for Gal (7-9). From studies using mice that are deficient in either the subunit HL-1 (10) or HL-2 (11), it is evident that both polypeptides are necessary for efficient clearance of asialoglyco...
A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.
A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable incorporation into lipidic particles. The glycolipid spontaneously associated with low-density lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma, and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake, establishing that both HDL and LDL were selectively recognized and processed by the ASGPr on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-lowering effect. These results indicate that the glycolipid may be a promising alternative for the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-lowering therapies.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.