title: Genetic blockage of endocytic pathways reveals differences in the intracellular processing of non-viral gene delivery systems.
abstract: Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.
authors.affiliation: Division of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, P.O. Box 56, FI-00014, University of Helsinki, Finland. polina.ilina@helsinki.fi
authors.forename: Polina
authors.initials: P
authors.lastname: Ilina
authors.forename: Zanna
authors.initials: Z
authors.lastname: Hyvonen
authors.forename: Maeva
authors.initials: M
authors.lastname: Saura
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
authors.forename: Marjo
authors.initials: M
authors.lastname: Yliperttula
authors.forename: Marika
authors.initials: M
authors.lastname: Ruponen
mesh_terms: D002130:Calcium Phosphates / Q000008:administration & dosage / Q000737:chemistry; D045744:Cell Line, Tumor; D004247:DNA / Q000008:administration & dosage / Q000737:chemistry; D004705:Endocytosis / Q000502:physiology*; D005229:Fatty Acids, Monounsaturated / Q000008:administration & dosage / Q000737:chemistry; D018014:Gene Transfer Techniques; D006801:Humans; D010957:Plasmids; D011094:Polyethyleneimine / Q000008:administration & dosage / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D002130:Calcium Phosphates; D005229:Fatty Acids, Monounsaturated; D000644:Quaternary Ammonium Compounds; D011094:Polyethyleneimine; D004247:DNA; C020243:calcium phosphate; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane