Determination of the upper size limit for uptake and processing of ligandsby the asialoglycoprotein receptor on hepatocytes in vitro and in vivo

The asialoglycoprotein receptor (ASGPr) on hepatocytes plays a role in the clearance of desialylated proteins from the serum. Although its sugar prefe rence (N-acetylgalactosamine (GalNAc) >> galactose) and the effects of liga nd valency (tetraantennary > triantennary >> diantennary >> monoantennary) and sugar spacing (20 Angstrom >> 10 Angstrom >> 4 Angstrom) are well docum ented, the effect of particle size on recognition and uptake of ligands by the receptor is poorly defined. In the present study, we assessed the maxim um ligand size that still allows effective processing by the ASGPr of mouse hepatocytes in vivo and in vitro. Hereto, we synthesized a novel glycolipi d, which possesses a highly hydrophobic steroid moiety for stable incorpora tion into liposomes, and a triantennary GalNAc(3)-terminated cluster glycos ide with a high nanomolar affinity (2 nm) for the ASGPr. Incorporation of t he glycolipid into small (30 nm) [H-3]cholesteryl oleate-labeled long circu lating liposomes (1-50%, w/w) caused a concentration-dependent increase in particle clearance that was liver-specific (reaching 85 +/- 7% of the injec ted dose at 30 min after injection) and mediated by the ASGPr on hepatocyte s, as shown by competition studies with asialoorosomucoid in vivo. By using glycolipid-laden liposomes of various sizes between 30 and 90 nm, it was d emonstrated that particles with a diameter of >70 nm could no longer be rec ognized and processed by the ASGPr in vivo. This threshold size for effecti ve uptake was not related to the physical barrier raised by the fenestrated sinusoidal endothelium, which shields hepatocytes from the circulation, be cause similar results were obtained by studying the uptake of liposomes on isolated mouse hepatocytes in vitro. From these data we conclude that in ad dition to the species, valency, and orientation of sugar residues, size is also an important determinant for effective recognition and processing of s ubstrates by the ASGPr. Therefore, these data have important implications f or the design of ASGPr-specific carriers that are aimed at hepatocyte-direc ted delivery of drugs and genes.