title: Cellular uptake pathways of lipid-modified cationic polymers in gene delivery to primary cells.
abstract: Hydrophobic modifications have emerged as a promising approach to improve the efficiency of non-viral gene delivery vectors (GDV). Functional GDVs from non-toxic polymers have been created with this approach but the mechanism(s) behind lipid-mediated enhancement in transfection remains to be clarified. Using a linoleic acid-substituted 2 kDa polyethylenimine (PEI2LA), we aimed to define the cellular uptake pathways and intracellular trafficking of plasmid DNA in normal human foreskin fibroblast cells. Several pharmacological compounds were applied to selectively inhibit uptake by clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis (CvME) and macropinocytosis. We found that PEI2LA complexes were taken up predominantly through CME, and to a lesser extent by CvME. In contrast, its precursor molecule, PEI2 complexes was internalized primarily by CvME and macropinocytosis. The commonly used 25 kDa PEI 25 complexes utilized all endocytic pathways, suggesting its efficiency is derived from a different set of transfection pathways than PEI2LA. We further applied several endosome disruptive agents and found that hypertonic media enhanced the transfection of PEI2LA by 6.5-fold. We infer that lipid substitution changes the normal uptake pathways significantly and transfection with hydrophobically modified GDVs may be further enhanced by incorporating endosome disruptive elements into vector design.
authors.affiliation: Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Charlie Y M
authors.initials: CY
authors.lastname: Hsu
authors.forename: Hasan
authors.initials: H
authors.lastname: Uludağ
mesh_terms: D002412:Cations; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism*; D005453:Fluorescence; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D057446:Hydrodynamics; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D008297:Male; D018613:Microscopy, Confocal; D010316:Particle Size; D010957:Plasmids / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000378:metabolism*; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: D002412:Cations; D008055:Lipids; D011108:Polymers; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA