Interference of poly(ethylene glycol)-lipid analogues with cationic-lipid-mediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions.
Cationic liposomes are applied to transfer oligonucleotides (ODNs) into cells to regulate gene expression for gene therapeutic or cell biological purposes. In vivo, poly(ethylene glycol) (PEG)-lipid derivatives are employed to stabilize and prolong the circulation lifetime of nucleic acid-containing particles, and to improve targeting strategies. In this study, we have studied the effects of PEG-lipid analogues, i.e. PEG coupled to either phosphatidylethanolamine (PE) or ceramide, on cationic-lipid-DNA complex ('lipoplex') assembly and the mechanism of cationic-lipid-mediated delivery of ODNs in vitro. Inclusion of 10 mol% PEG-PE in ODN lipoplexes inhibited their internalization in Chinese hamster ovary cells by more than 70%. The intracellular fraction remained entrapped in the endosomal/lysosomal pathway, and no release of ODNs was apparent. Similar observations were made for complexes prepared from liposomes that contained PEG-ceramides. Interestingly, delivery resumed when lipoplexes had been externally coated with PEG-ceramides. In this case, the kinetics of delivery were dependent on the length of the ceramide acyl chain, consistent with a requirement for the PEG-lipid to dissociate from the complex. Moreover, although the chemical nature of the PEG-ceramides distinctly affected the net internalization of the complexes, impediment of delivery was largely related to an inhibitory effect of the PEG-lipid on the release of ODNs from the endosomal compartment. Cryo-electron microscopy and small-angle X-ray scattering revealed that the PEG-lipids stabilize the lamellar phase of the lipoplexes, while their acyl-chain-length-dependent transfer from the complex enables adaptation of the hexagonal phase. Within the endosomal compartment, this transition appears to be instrumental in causing the dissociation and cytosolic release of the ODNs for their nuclear homing.
Shi
Fuxin
F
Department of Membrane Cell Biology, Faculty of Medical Sciences, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Wasungu
Luc
L
Nomden
Anita
A
Stuart
Marc C A
MC
Polushkin
Evgeny
E
Engberts
Jan B F N
JB
Hoekstra
Dick
D
D000818:Animals; D016466:CHO Cells; D002412:Cations / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D020285:Cryoelectron Microscopy; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008051:Lipid Bilayers; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D009841:Oligonucleotides / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010957:Plasmids; D011092:Polyethylene Glycols / Q000378:metabolism*; D011485:Protein Binding; D012542:Scattering, Radiation; D013997:Time Factors; D014965:X-Rays
D002412:Cations; D008051:Lipid Bilayers; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011092:Polyethylene Glycols; D004247:DNA