Cellular entry pathway and gene transfer capacity of TAT-modified lipoplexes.
Several reports have shown a fast and efficient translocation of TAT-modified lipoplexes and particles into the cell cytoplasm. However, neither the uptake mechanism nor the biological effect of TAT-modified lipoplexes has been studied in detail. In this report we show that the increase in gene transfer of TAT-modified lipoplexes depends on the amount of cationic lipid in the lipoplexes and on the way TAT was coupled to the lipoplexes. We demonstrate that the cellular uptake of both TAT-modified and unmodified lipoplexes is very fast and, in contrast to previous publications, temperature-dependent. Additionally, after internalization TAT-modified as well as unmodified lipoplexes end up in lysosomal vesicles, indicating the involvement of clathrin-mediated endocytosis. Furthermore, chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, strongly inhibits the cellular uptake and biological activity of both the TAT-modified and unmodified lipoplexes. We also found that the uptake and biological activity of these lipoplexes are diminished when cholesterol in the cell membrane was bound by filipin, an inhibitor of the lipid-raft mediated pathway. Considering these data, we conclude that TAT-modified and unmodified lipoplexes are mainly internalized via a cholesterol-dependent clathrin-mediated pathway.
Vandenbroucke
Roosmarijn E
RE
Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
De Smedt
Stefaan C
SC
Demeester
Joseph
J
Sanders
Niek N
NN
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005372:Filipin / Q000378:metabolism / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015696:Gene Products, tat / Q000737:chemistry*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D010714:Phosphatidylethanolamines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D002966:Clathrin; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; C038089:1,2-distearoylphosphatidylethanolamine; D011092:Polyethylene Glycols; D005372:Filipin; D002784:Cholesterol; D016650:Fluorescein-5-isothiocyanate; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane