Intracellular processing of poly(ethylene imine)/ribozyme complexes can be observed in living cells by using confocal laser scanning microscopy and inhibitor experiments.
OBJECTIVE
Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism.


METHODS
Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin Al, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions.


RESULTS
Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1.


CONCLUSIONS
Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.
Merdan
Thomas
T
Department of Pharmaceutics and Biopharmacy, Philipps University, Marburg, Germany.
Kunath
Klaus
K
Fischer
Dagmar
D
Kopecek
Jindrich
J
Kissel
Thomas
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002264:Carboxylic Acids; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008156:Luciferases / Q000737:chemistry / Q000235:genetics; D018942:Macrolides; D018613:Microscopy, Confocal; D008970:Molecular Weight; D010957:Plasmids / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D002264:Carboxylic Acids; D004791:Enzyme Inhibitors; D018942:Macrolides; C436864:Oregon Green 488 carboxylic acid; D016337:RNA, Catalytic; C040929:bafilomycin A1; D011094:Polyethyleneimine; D008156:Luciferases; D006180:Proton-Translocating ATPases