title: Spacer Length: A Determining Factor in the Design of Galactosyl Ligands for Hepatoma Cell-Specific Liposomal Gene Delivery.
abstract: 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 &lt;0.05-0.001).
  
  
  CONCLUSIONS
  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.
authors.forename: Londiwe
authors.initials: L
authors.lastname: Mbatha
authors.forename: Santanu
authors.initials: S
authors.lastname: Chakravorty
authors.forename: Charles B
authors.initials: CB
authors.lastname: de Koning
authors.forename: Willem A L
authors.initials: WA
authors.lastname: van Otterlo
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
authors.affiliation: Non-viral Gene Delivery Laboratory, Discipline of Biochemistry, Westville campus, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000 South Africa. ariattim@ukzn.ac.za.
authors.forename: Mario
authors.initials: M
authors.lastname: Ariatti
authors.forename: Moganavelli
authors.initials: M
authors.lastname: Singh
mesh_terms: D037263:Asialoglycoprotein Receptor / Q000378:metabolism*; D045744:Cell Line, Tumor; D005690:Galactose / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008081:Liposomes; D014162:Transfection / Q000379:methods*
chemical_list: D037263:Asialoglycoprotein Receptor; D008024:Ligands; D008081:Liposomes; D005690:Galactose