Elucidating the pre- and post-nuclear intracellular processing of 1,4-dihydropyridine based gene delivery carriers.
The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre- and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre- and the post-nuclear intracellular processes is equally essential for successful transgene expression.
School of Pharmacy, Department of Health Sciences, University of Eastern Finland, PO Box 1627 FIN-70211 Kuopio, Finland. zanna.hyvonen@uef.fi
Zanna
Z
Hyvönen
Vesa
V
Hämäläinen
Marika
M
Ruponen
Bart
B
Lucas
Joanna
J
Rejman
Dries
D
Vercauteren
Jo
J
Demeester
Stefaan
S
De Smedt
Kevin
K
Braeckmans
D001692:Biological Transport; D002460:Cell Line; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics; D004095:Dihydropyridines / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D014162:Transfection; D019076:Transgenes
D004095:Dihydropyridines; D012333:RNA, Messenger; C038806:1,4-dihydropyridine; D004247:DNA