Nonbilayer phase of lipoplex–membrane mixture determines endosomal escape of genetic cargo and transfection efficiency

Abstract

Cationic lipids are widely used for gene delivery, and inclusion of dioleoylphosphatidylethanolamine (DOPE) as a helper lipid in cationic lipid-DNA formulations often promotes transfection efficacy. To investigate the significance of DOPE's preference to adopt a hexagonal phase in the mechanism of transfection, the properties and transfection efficiencies of SAINT-2/DOPE lipoplexes were compared to those of lipoplexes containing lamellar-phase-forming dipalmitoylphosphatidylethanolamine (DPPE). After interaction with anionic vesicles, to simulate lipoplex-endosomal membrane interaction, SAINT-2/DOPE lipoplexes show a perfect hexagonal phase, whereas SAINT-2/DPPE lipoplexes form a mixed lamellar-hexagonal phase. The transition to the hexagonal phase is crucial for dissociation of DNA or oligonucleotides (ODN) from the lipoplexes. However, while the efficiencies of nucleic acid release from either complex were similar, SAINT-2/DOPE lipoplexes displayed a two- to threefold higher transfection efficiency or nuclear ODN delivery. Interestingly, rupture of endosomes following a cellular incubation with ODN-containing SAINT-2/DPPE complexes dramatically improved nuclear ODN delivery to a level that was similar to that observed for SAINT-2/DOPE complexes. Our data demonstrate that although hexagonal phase formation in lipoplexes is a prerequisite for nucleic acid release from the complex, it appears highly critical for accomplishing efficient translocation of nucleic acids across the endosomal membrane into the cytosol for transport to the nucleus.

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

“…Further, at our assay concentrations, lipid conjugation of bPEI 1.8kDa has not increased the inherent cytotoxicity of bPEI 1.8kDa (Fig. 1) in spite of DOPE having high membrane affinity and fusogenic capability [43,44]. However, comparing polyplexes of bPEI 25kDa and DOPE-PEI at the optimal N/P ratios ( Fig.…”

Cell division responsive peptides for optimized plasmid DNA delivery: The mitotic window of opportunity?

“…Interestingly, also precomplexation of pDNA with lipofectamine2000® is not beneficial. It is indeed know that lipid based carriers need endocytosis and endosomal escape to efficiently release the complexed DNA into the cytoplasm of the cells [24][25][26]. When ternary (pDNA/peptide)/RNAimax® complexes were used to deliver the pDNA/peptide complexes into the cytoplasm of the cells, again precondensation of pDNA with peptides improved the transfection efficiency compared to pDNA delivery by RNAimax®.…”

Factors Determining the Superior Performance of Lipid/DNA/Protammine Nanoparticles over Lipoplexes

“…Complexes appeared almost devoid of cytoplasmic plasmid DNA, suggesting that such binary formulation is defective in facilitating endosomal escape of nucleic acids, resulting in entrapment of plasmid DNA in endosomes. Since there is a possibility that lipoplexes exhibit DNA release with different kinetics than LPD complexes, the distribution was followed at various time points (4,6,8,10,12,24,36, and 48 h). Over such time scale, significant cytosolic DNA release from lipoplexes as that observed for LPD nanoparticles was not detected.…”