Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes.

We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K(+) depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC-poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene. Uptake of the complexes was assayed by fluorescence-activated cell sorting. Lipoplex internalization was inhibited by chlorpromazine and K(+) depletion but unaffected by filipin and genistein. In contrast, polyplex internalization was inhibited by all four inhibitors. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while polyplexes are taken up by two mechanisms, one involving caveolae and the other clathrin-coated pits. Transfection by lipoplexes was entirely abolished by blocking clathrin-mediated endocytosis, whereas inhibition of the caveolae pathway had no effect. By contrast, transfection mediated by polyplexes was completely blocked by genistein and filipin but was unaffected by inhibitors of clathrin-mediated endocytosis. Fluorescence colocalization studies with a lysosomal marker, AlexaFluor-dextran, revealed that polyplexes taken up by clathrin-mediated endocytosis are targeted to the lysosomal compartment for degradation, while the polyplexes internalized via caveolae escape this compartment, permitting efficient transfection.

[1]  G. Merlo,et al.  Polyethylenimine-based intravenous delivery of transgenes to mouse lung , 1998, Gene Therapy.

[2]  I. Zuhorn,et al.  Lipoplex-mediated Transfection of Mammalian Cells Occurs through the Cholesterol-dependent Clathrin-mediated Pathway of Endocytosis* , 2002, The Journal of Biological Chemistry.

[3]  D. Hoekstra,et al.  Molecular Shape of the Cationic Lipid Controls the Structure of Cationic Lipid/Dioleylphosphatidylethanolamine-DNA Complexes and the Efficiency of Gene Delivery* , 2001, The Journal of Biological Chemistry.

[4]  T. Bieber,et al.  Intracellular route and transcriptional competence of polyethylenimine-DNA complexes. , 2002, Journal of controlled release : official journal of the Controlled Release Society.

[5]  Leaf Huang,et al.  Nonviral gene therapy: promises and challenges , 2000, Gene Therapy.

[6]  F. Szoka,et al.  Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. , 1993, Bioconjugate chemistry.

[7]  M. Conese,et al.  Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs , 2000, Gene Therapy.

[8]  S. Abraham,et al.  Caveolae as portals of entry for microbes. , 2001, Microbes and infection.

[9]  P. Oh,et al.  Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules , 1994, The Journal of cell biology.

[10]  G. Levi,et al.  Rapid crossing of the pulmonary endothelial barrier by polyethylenimine/DNA complexes , 2000, Gene Therapy.

[11]  R. G. Anderson,et al.  Spatial organization of EGF receptor transmodulation by PDGF. , 1999, Biochemical and biophysical research communications.

[12]  T Salditt,et al.  An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. , 1998, Science.

[13]  M. Conese,et al.  Restoration of bacterial killing activity of human respiratory cystic fibrosis cells through cationic vector-mediated cystic fibrosis transmembrane conductance regulator gene transfer. , 1999, Human gene therapy.

[14]  E. Brambilla,et al.  An electron microscopy study into the mechanism of gene transfer with lipopolyamines. , 1996, Gene therapy.

[15]  D. W. Pack,et al.  On the kinetics of polyplex endocytic trafficking: implications for gene delivery vector design. , 2002, Molecular therapy : the journal of the American Society of Gene Therapy.

[16]  H. Kogo,et al.  Isoforms of caveolin-1 and caveolar structure. , 2000, Journal of cell science.

[17]  S. Carotta,et al.  Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo , 2001, The journal of gene medicine.

[18]  S. Ferrari,et al.  ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo , 1997, Gene Therapy.

[19]  Xiang Gao,et al.  Cationic liposome-mediated gene transfer. , 1995, Gene therapy.

[20]  D. Scherman,et al.  A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Behr,et al.  Systemic linear polyethylenimine (L‐PEI)‐mediated gene delivery in the mouse , 2000, The journal of gene medicine.

[22]  V. Puri,et al.  Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways , 2001, The Journal of cell biology.

[23]  F. Szoka,et al.  Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. , 1996, Biochemistry.

[24]  H. Farhood,et al.  The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. , 1995, Biochimica et biophysica acta.

[25]  T. Aoki,et al.  Tyrosine phosphorylation of caveolin-1 in the endothelium. , 1999, Experimental cell research.

[26]  D. Friend,et al.  Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. , 1996, Biochimica et biophysica acta.

[27]  P. Cullis,et al.  On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids , 2001, Gene Therapy.

[28]  Richard G. W. Anderson,et al.  Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts , 1983, Cell.

[29]  K. Joiner,et al.  Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. , 1990, Science.

[30]  P. Saggau,et al.  Poly(ethylenimine)-mediated transfection: a new paradigm for gene delivery. , 2000, Journal of biomedical materials research.

[31]  A. R. Klemm,et al.  Effects of polyethyleneimine on endocytosis and lysosome stability. , 1998, Biochemical pharmacology.

[32]  I. Zuhorn,et al.  Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. , 2004, The Biochemical journal.

[33]  P. Orlandi,et al.  Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains , 1998, The Journal of cell biology.

[34]  R. Wattiaux,et al.  Uptake and intracellular fate of polyethylenimine in vivo. , 2000, Biochemical and biophysical research communications.

[35]  S. Randell,et al.  Loss of Binding and Entry of Liposome-DNA Complexes Decreases Transfection Efficiency in Differentiated Airway Epithelial Cells* , 1997, The Journal of Biological Chemistry.

[36]  O. Danos,et al.  Polyethylenimine‐mediated gene delivery: a mechanistic study , 2001, The journal of gene medicine.

[37]  R. G. Anderson,et al.  Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation , 1993, The Journal of cell biology.

[38]  A. Boletta,et al.  Comparison between cationic polymers and lipids in mediating systemic gene delivery to the lungs , 1999, Gene Therapy.