Mechanism of DNA Release from Cationic Liposome/DNA Complexes Used in Cell Transfection^,

Abstract

To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.

Novel dextran–spermine conjugates as transfecting agents: comparing water-soluble and micellar polymers

“…The process of plasmid dissociation from complexes was attributed to anionic molecules in the cell cytoplasm capable of replacing the anionic DNA. 10,26 Other work suggested that the mechanism for complex dissociation involves components in the endosome or prelysosomal membrane. 27 In some cases, the plasmid dissociation takes place in the nucleus.…”

Stabilized plasmid-lipid particles: construction and characterization

“…This is consistent with the ability of the PEG-CerC 14 coating to diffuse away from the SPLP surface. The SPLP containing either PEG-CerC 14 or PEG-CerC 20 exerted no apparent toxic effects on the cells as evaluated by monitoring protein content in the cell extract.…”

“…In order to determine whether the improved transfection properties of SPLP containing PEG-CerC 14 as compared with SPLP containing PEG-CerC 20 could be related to a faster dissociation rate from the SPLP surface, the dissociation rates at 37°C of radiolabeled PEG-CerC 14 and PEG-CerC 20 from 100 nm diameter large unilamellar vesicles (LUV) composed of egg phosphatidylcholine (EPC) were measured as indicated in Materials and methods. It should be noted that it is difficult to measure the dissociation rate of PEG-Cer from the surface of SPLP containing DOPE as the stability of these SPLP is dependent on the presence of the PEG-Cer coating.…”

“…The LUV were prepared containing 10 mol% PEG-Cer and a trace of 14 C-CHE ( 3 H/ 14 C ratio approximately 5) by detergent dialysis as described above for SPLP. MLV were prepared by hydration of EPC in HBS (250 mg/ml) at 65°C.…”

Cationic lipid-mediated transfection of cells in culture requires mitotic activity

“…[1][2][3][4][5][6] Despite these clear advantages, nonviral transfection systems result in substantially less gene expression than that observed with viral systems. Most cationic lipid/DNA-based systems are taken up efficiently though an endocytosis pathway, [7][8][9][10] but the exact mechanisms of DNA release from the endosomes and the subsequent translocation to the nucleus are not clear.…”