title: Mechanisms of polyethylenimine-mediated DNA delivery: free carrier helps to overcome the barrier of cell-surface glycosaminoglycans.
abstract: BACKGROUND
  Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.
  
  
  METHODS
  Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays.
  
  
  RESULTS
  Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells.
  
  
  CONCLUSIONS
  Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.
authors: Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland.
authors: Martina
authors: M
authors: Hanzlíková
authors: Marika
authors: M
authors: Ruponen
authors: Emilia
authors: E
authors: Galli
authors: Atso
authors: A
authors: Raasmaja
authors: Vladimir
authors: V
authors: Aseyev
authors: Heikki
authors: H
authors: Tenhu
authors: Arto
authors: A
authors: Urtti
authors: Marjo
authors: M
authors: Yliperttula
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006025:Glycosaminoglycans / Q000378:metabolism*; D042541:Intracellular Space / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D014162:Transfection; D019076:Transgenes
chemical_list: D006025:Glycosaminoglycans; D011094:Polyethyleneimine; D004247:DNA