Cellular uptake of self-assembled cationic peptide-DNA complexes: multifunctional role of the enhancer chloroquine.
A small library of carriers consisting of various combinations of the cell penetrating peptide TAT, the SV40 Large T protein nuclear localisation signal (NLS) and a cationic dendrimer of 7 lysine residues (DEN) was synthesised and each member was tested for its ability to deliver exogenous DNA to human HeLa cells. We found that the TAT peptide was essential, but not sufficient for efficient uptake of exogenous DNA. The addition of either NLS or DEN significantly enhanced uptake and expression with the most active carrier consisting of the TAT, NLS and DEN peptides. For those peptides that facilitated DNA uptake, the complexes were targeted to intracellular compartments that required incubation with a fusogenic agent such as chloroquine before gene expression was observed. However, our data suggest that chloroquine did not enhance expression solely by promoting endosomal release since a fusogenic peptide derived from the influenza virus haemagglutinin protein did not improve gene expression. Chloroquine was found to protect DNA from degradation and enhance transcription of DNA bound to the respective carriers. Our results demonstrate that multi-component peptide-based gene carriers can be designed to deliver exogenous DNA. The actions of lysosomotropic agents such as chloroquine reveal the multifactorial properties required for carriers used in non-viral gene delivery.
Yang
Shu
S
School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Coles
Daniel J
DJ
Esposito
Anna
A
Mitchell
Deanne J
DJ
Toth
Istvan
I
Minchin
Rodney F
RF
D001583:Benzoxazoles / Q000378:metabolism; D002412:Cations / Q000378:metabolism*; D002477:Cells / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002738:Chloroquine / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D050091:Dendrimers / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008561:Membrane Fusion / Q000502:physiology; D015394:Molecular Structure; D019913:Nuclear Localization Signals / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011806:Quinolinium Compounds / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
D001583:Benzoxazoles; D002412:Cations; D002614:Chelating Agents; D050091:Dendrimers; D005456:Fluorescent Dyes; D019913:Nuclear Localization Signals; D010455:Peptides; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D002738:Chloroquine; D004247:DNA; D004492:Edetic Acid; D049410:Luciferases, Renilla; D014357:Trypsin; D008239:Lysine