Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytesin Vitro and in Vivo

Abstract

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 rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development

“…Ligand binding was assessed using flow cytometry to monitor the fraction of cells bound to biotinylatedGalNAc as a function of ligand concentration The calculated Kd values for the bi-and triantennary GalNAc were 28.3 and 2.3 nM respectively, consistent with binding affinities obtained for radiolabeled ligand-receptor assays previously reported where calculated Kd values were 4.3 nM for GalNAc 3 and 25 nM for a bi-antennary ligand (GalNAc 2 ) (Connolly et al 1982;Connolly et al 1983;Biessen et al 1995;Lee et al 1983;Rensen et al 2001) (Fig. 6).…”

“…In addition, we developed a receptor-ligand binding assay to assess hepatocyte function. This assay (Connolly et al 1982: Connolly et al 1983Biessen et al 1995;Lee et al 1983;Rensen et al 2001). In addition, we demonstrated that the binding of the GalNAc ligand led to cellular uptake and gene silencing when attached to an ApoB-targeting siRNA.…”

“…The binding affinity of GalNAc ligands to ASGPR depends on the number of GalNAc clusters present in the molecule with the tetraantennary (GalNAc 4 ) showing higher affinity followed by triantennary (GalNAc 3 ) and biantennary (GalNAc 2 ) ligands. This ligandreceptor binding characteristic was observed in primary hepatocytes, but not when the isolated receptor was used in cell-free based assays (Connolly et al 1982;Rensen et al 2001). We demonstrate that the ASGPR is present, active, and functional after using the isolation protocol described in this article.…”

Galactosylated polymeric carriers for liver targeting of sorafenib

“…Many efforts have been dedicated to the targeted delivery of drugs and genes in particular to hepatocytes by the development of galactosylated polymeric or lipid carriers in order to enhance the therapeutic effects (in case of hepatocellular carcinoma, viral hepatitis or gene-based therapies) or to reduce side-effects of drugs (in case of antiviral or anticancer drugs) (Poelska et al, 2012). Galactosylation is a speculated strategy in this field because targeting via galactosylated carriers exploit highly specific interactions of galactose ligands with endogenous lectin receptors such as asialoglycoprotein receptor (ASGPR), which is specifically and abundantly present on hepatocytes (Jain et al, 2012;Rensen et al, 2001;Wu et al, 2004). This receptor has been used to deliver to hepatocytes all kinds of therapeutic compounds ranging from therapeutic proteins, antiviral agents (Di Stefano et al, 1997) to anticancer drugs (Di Stefano et al, 2006;Fiume et al, 2005).…”

Herbal and polymeric approaches for liver-targeting drug delivery: novel strategies and their significance

“…Liquorice is having two main bioactive chemical constituents named as glycyrrhizin (GL) and glycyrrhetinic acid (GA) which are widely used in medicine for the treatment of many disorders (Rensen et al, 2001;Wu et al, 2002). These are mainly used as anti-inflammatory, antiallergic, antigastric, antihepatitis and antihepatotoxic effects.…”