title: Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways.
abstract: OBJECTIVE
  Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing.
  
  
  METHODS
  We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways.
  
  
  RESULTS
  PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms.
  
  
  CONCLUSIONS
  The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
authors.affiliation: Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, Illinois, 61801, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Hwang
authors.forename: Rahul K
authors.initials: RK
authors.lastname: Keswani
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Pack
mesh_terms: D022461:Caveolins / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D050091:Dendrimers / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011094:Polyethyleneimine / Q000737:chemistry*; D014162:Transfection / Q000379:methods*
chemical_list: D022461:Caveolins; D002966:Clathrin; D050091:Dendrimers; C104700:PAMAM Starburst; D011094:Polyethyleneimine; D049409:Luciferases, Firefly