Macropinocytosis of polyplexes and recycling of plasmid via the clathrin-dependent pathway impair the transfection efficiency of human hepatocarcinoma cells.
Knowledge of the entry mechanism and intracellular routing of polyplexes is of major importance for designing efficient gene delivery systems. We therefore investigated the internalization and trafficking of polyplexes in HepG2 cells. pDNA encoding the luciferase was complexed with histidylated polylysine (His-pLK), a polymer that requires acidic pH for pDNA endosomal release. Fluoresceinylated polyplexes (F-His-pLK or F-pDNA) were internalized by clathrin-dependent and -independent pathways. The latter most likely occurred by macropinocytosis since it was stimulated by phorbol myristate and blocked by dimethylamiloride. Intracellular routing of the plasmid was analyzed by confocal microscopy and flow cytometry. These data revealed that: (i) one part of the plasmid was present in vesicles that were not labeled with any known organelle-specific marker, (ii) the other part was in transferrin receptor-positive vesicles, and (iii) the plasmid was not transferred to late endosomes/lysosomes. Using luciferase activity as a readout for gene expression, we found that it was strongly reduced when macropinocytosis was stimulated, whereas macropinocytosis inhibitors had no effect. However, blocking clathrin-dependent internalization by chlorpromazine completely prevented gene expression. These findings demonstrate that: (i) macropinocytosis of polyplexes and (ii) plasmid recycling impair the transfection efficiency and (iii) clathrin-dependent endocytosis is the most productive route for transfection of HepG2 cells.
Centre de Biophysique Moléculaire, UPR4301 CNRS, Rue Charles Sadron, 45071 Orléans, Cedex 2, France.
Christine
C
Gonçalves
Eric
E
Mennesson
Renate
R
Fuchs
Jean-Pierre
JP
Gorvel
Patrick
P
Midoux
Chantal
C
Pichon
D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D019793:Fluorescein / Q000032:analysis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D015739:Nocodazole / Q000494:pharmacology; D010873:Pinocytosis / Q000187:drug effects / Q000502:physiology*; D010957:Plasmids / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011107:Polylysine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D002966:Clathrin; D011107:Polylysine; D008156:Luciferases; D013755:Tetradecanoylphorbol Acetate; D015739:Nocodazole; D019793:Fluorescein; D002746:Chlorpromazine