Characterization, stability and in-vivo distribution of asialofetuin glycopeptide incorporating DSPC/CHOL liposomes prepared by mild cholate incubation

In this study, a small triantennary asialoglycopeptide of fetuin (A-F-2) wa s used as a ligand to direct liposomes to hepatocytes. A-F2 was cleaved fro m asialofetuin, purified, conjugated with fatty acids and incorporated into pre-formed sonicated DSPC/Chol (2:1) liposomes. A mild cholate incubation method for incorporating the A-F2 ligand on pre-formed vesicles was used. I n preliminary in vivo experiments In-111(3+) encapsulated in A-F-2/palmityl liposomes was seen to accumulate in the liver of mice significantly faster than when encapsulated in non-ligand bearing liposomes of the same lipid c omposition (studied before), justifying further investigation of this syste m. The presence of the A-F-2/fatty acid conjugate in a functional form on t he vesicle surface was confirmed by their reversible agglutination in the p resence of Ricinus communis agglutinin (RCA(120)). Effects of ligand incorp oration on the vesicle size distribution, z-potential, membrane integrity a nd stability were monitored. The results demonstrate that highest ligand in corporation was achieved when liposomes and ligand were co-incubated in the presence of 1mM sodium cholate. Incorporation increased with the length of the fatty acid used for A-F2 conjugation. Ligand-bearing liposomes were de monstrated to be smaller in diameter (about 30%) with a more positive z-pot ential in comparison to control vesicles while ligand incorporation did not influence the liposome membrane integrity. The size of the ligand-incorpor ating vesicles was maintained after 24 hours of incubation in isotonic buff er, proving that the vesicles do not aggregate. Although the preliminary bi odistribution results may suggest that ligand bearing liposomes are accumul ating in the liver, further cell culture in vivo distribution and especiall y liver fractionation studies are required in order to clarify the intrahep atic localization of these liposomes and the ability to target liver hepato cytes in vivo.