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J. Biol. Chem., Vol. 276, Issue 40, 37577-37584, October 5, 2001

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

Patrick C. N. Rensen, Leo A. J. M. Sliedregt, Michiel Ferns, Erwin Kieviet, Sabine M. W. van Rossenberg, Steven H. van Leeuwen, Theo J. C. van Berkel, and Erik A. L. Biessen

From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, Sylvius Laboratory, 2300 RA Leiden, The Netherlands

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 GalNAc₃-terminated cluster glycoside with a high nanomolar affinity (2 nM) for the ASGPr. Incorporation of the glycolipid into small (30 nm) [³H]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.

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