Saccharide Determinants in Selective Drug Delivery

Mannose-lactoperoxidase, a neoglycoprotein prepared by reaction of lactoperoxidase with cyanomethyl 1-thiomannoside, bound to alveolar macrophages at 4 degrees C (Kd = 5.8 X 10(-8) M) and was rapidly internalized at 37 degrees C (K uptake = 2 X 10(-8) M). Mannose-lactoperoxidase binding and uptake were blocked by yeast mannan, and mannose-lactoperoxidase inhibited uptake of 125I-labeled mannose-BSA (bovine serum albumin). Radioiodination of cells with surface-bound mannose-lactoperoxidase was carried out in the presence of glucose and glucose oxidase. A major polypeptide (175 kDa) was radioiodinated by this procedure. Iodination of the 175-kDa polypeptide appeared to be receptor-mediated, since it was blocked by the presence of yeast mannan. Specific iodination was absent from receptor-negative cells. To demonstrate that the 175-kDa species is a ligand-binding protein, cells were iodinated by the standard lactoperoxidase method. Washed cells were then allowed to bind mannose-BSA. Receptor-ligand complexes, prepared by detergent extraction, were passed over anti-BSA IgG affinity columns. Mannose, but not mannose 6-phosphate or galactose, eluted a radioactive protein from the column that migrated with an apparent molecular mass of 175 kDa on NaDodSO4/PAGE. Detergent extracts of crude membranes prepared from macrophage-enriched whole rabbit lung were adsorbed to mannose-Sepharose; the fraction obtained by elution with mannose contained two protein components of 175 and 55 kDa. Subsequent chromatography on N-acetylglucosamine-agarose yielded a single protein of 175 kDa. The 175-kDa polypeptide was shown to bind 125I-labeled mannose-BSA in a precipitation assay. This binding could be blocked with mannan or mannose-BSA. The results indicate that the cell-surface mannose receptor is a 175-kDa protein.

A plasmid containing the rat preproinsulin I gene was entrapped in large liposomes and intravenously administered to rats. Four hours after inoculation, the livers were processed for the isolation of hepatocytes. Kupffer cells, and endothelial cells, DNA was purified, and the exogenous DNA was detected in the different cell DNA preparations by Southern blotting. By using liposomes consisting of phospholipids and cholesterol, Kupffer cells were shown to be, on a per cell basis, the primary target for gene incorporation. In an attempt to target the liposomes to other liver cells, a glycolipid, lactosylceramide, was included in the lipid bilayer of the liposomes; this resulted in a substantial increase in the proportion of the exogenous gene in the hepatocytes and mainly in the endothelial cells, with a simultaneous decrease of this proportion in the Kupffer cells. Thus, it is shown that inclusion of a specific glycolipid within the bilayer of the liposomes may direct the DNA-containing vesicles to specific cell types in the liver.