PURPOSE: 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.
PURPOSE: 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: Maria Manunta; Peng Hong Tan; Pervinder Sagoo; Kirk Kashefi; Andrew J T George Journal: Nucleic Acids Res Date: 2004-05-17 Impact factor: 16.971
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Authors: Dries Vercauteren; Roosmarijn E Vandenbroucke; Arwyn T Jones; Joanna Rejman; Joseph Demeester; Stefaan C De Smedt; Niek N Sanders; Kevin Braeckmans Journal: Mol Ther Date: 2009-12-15 Impact factor: 11.454
Authors: Raman Deep Singh; Vishwajeet Puri; Jacob T Valiyaveettil; David L Marks; Robert Bittman; Richard E Pagano Journal: Mol Biol Cell Date: 2003-05-18 Impact factor: 4.138
Authors: M A E M van der Aa; U S Huth; S Y Häfele; R Schubert; R S Oosting; E Mastrobattista; W E Hennink; R Peschka-Süss; G A Koning; D J A Crommelin Journal: Pharm Res Date: 2007-03-24 Impact factor: 4.200