Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine

In order to develop the non-viral Bioplex vector system for targeted delivery of genes to hepatocytes, we have evaluated the structure-function relationship for a number of synthetic ligands designed for specific interaction with the hepatic lectin ASGPr. Biotinylated ligand derivatives containing two, three or six beta-linked N-acetylgalactosamine (GalNAc) residues were synthesized, bound to fluorescent-labeled streptavidin and tested for binding and uptake to HepG2 cells using flow cytometry analysis (FACS). Uptake efficiency increased with number of displayed GalNAc units per ligand, in a receptor dependent manner. Thus, a derivative displaying six GalNAc units showed the highest uptake efficacy both in terms of number of internalizing cells and increased amount of material taken up per each cell. However, this higher efficiency was shown to be due not so much to higher number of sugar units, but to higher accessibility of the sugar units for interaction with the receptor (longer spacer). Improving the flexibility and accessibility of a trimeric GalNAc ligand through use of a longer spacer markedly influenced the uptake efficiency, while increasing the number of GalNAc units per ligand above three only provided a minor contribution to the overall affinity. We hereby report the details of the chemical synthesis of the ligands and the structure-function studies in vitro. Published in 2003.