title: Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer.
abstract: BACKGROUND
  Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable.
  
  
  METHODS
  Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice.
  
  
  RESULTS
  SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI.
  
  
  CONCLUSIONS
  This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.
authors.affiliation: Pharmaceutical Biology-Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, D-81377 Munich, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Boeckle
authors.forename: Katharina
authors.initials: K
authors.lastname: von Gersdorff
authors.forename: Silke
authors.initials: S
authors.lastname: van der Piepen
authors.forename: Carsten
authors.initials: C
authors.lastname: Culmsee
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
authors.forename: Manfred
authors.initials: M
authors.lastname: Ogris
mesh_terms: D000818:Animals; D002412:Cations / Q000737:chemistry*; D002460:Cell Line; D002845:Chromatography; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005821:Genetic Techniques; D015316:Genetic Therapy / Q000379:methods*; D007834:Lasers; D051379:Mice; D008807:Mice, Inbred BALB C; D009584:Nitrogen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry / Q000302:isolation & purification*; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
chemical_list: D002412:Cations; D011094:Polyethyleneimine; D004247:DNA; D009584:Nitrogen