Background: Use of nucleic acids to treat acquired or inherited hepatic diseases has
considerable potential. Although recombinant viruses are popular vectors, interest in cheaper, often
less immunogenic, non-viral modalities, is increasing. Thus hepatotropic, galactosylated lipoplexes directed to the hepatic
asialoglycoprotein receptor (ASGP-R) are promising candidates.
Objective: Here we examine the effect that galactosyl ligand spacer length has on the transfection activity of ASGP-Rtargeted
lipoplexes in the human hepatoma cell line HepG2.
Methods: Galactosyl ligands with spacer lengths in the range 2.4-24.1 Å were prepared and formulated into lipoplexes
that were characterized by cryo-TEM, band shift, dye displacement and nuclease digestion assays. Cytotoxicity and
transfection profiles were determined in liver-derived HepG2 cells and the renal ASGP-R-negative HEK293 line.
Results: Lipoplexes, which formed at endpoint +/- charge ratios in the range 1:1-3:1, accorded cargo DNA good
protection from serum nuclease digestion and were well-tolerated by both cell lines. Transfection activities in the
hepatoma cell line decreased markedly in the presence of a competing ASGP-R cognate ligand and also as the ligand
spacer length increased, while activities in HEK293 cells were significantly lower (P <0.05-0.001).
Conclusion: Targeted lipoplexes enter HepG2 cells by receptor mediation and the uptake of transfecting complexes and
those displaying more rigid short and medium length spacers is more efficient. This observation will inform the design of
hepatotropic lipoplexes that are suitable for applications in vivo.