Targeting of liposomes by covalent coupling with ecto-NAD+-glycohydrolase ligands

We have tested the feasibility of targeting liposomes via interaction with specific ecto-enzymes, i.e., enzymes which have their active site oriented to the external surface of the cell. 3,4-Dimethylpyridine adenine dinucleotide, a competitive inhibitor of ecto-NAD+-glycohydrolase, was substituted at N6 with a hydrophilic spacer arm, functionalized with a sulfhydryl group, and covalently linked to performed liposomes containing 4-(p-maleimidophenyl)butyryl phosphatidylethanolamine. We show that compared to control vesicles, the binding of the conjugated liposomes was greatly increased (up to 5-fold) to cells presenting ecto-NAD+-glycohydrolase activity (Swiss 3T3 fibroblasts, mouse peritoneal macrophages); in contrast, no specific binding was detected with hepatoma tissue culture cells, which lack this enzyme. Specific binding was found to depend on the ligand/lipid molar ratio of the vesicles and on the length of the arm. High concentrations of free 3,4-dimethylpyridine adenine dinucleotide virtually abolished the specific binding to cells of the targeted liposomes. Analysis of binding revealed that the ligand conjugated to the liposomes presented a functional affinity for 3T3 fibroblasts 15-fold superior to that of the free ligand.