Kinetic analysis of the initial steps involved in lipoplex--cell interactions: effect of various factors that influence transfection activity.
We investigated the mode of interaction of lipoplexes (DOTAP:DOPE/DNA) with HeLa cells, focusing on the analysis of the initial steps involved in the process of gene delivery. We evaluated the effect of different factors, namely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin into the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were performed upon cell treatment with inhibitors of endocytosis. Our results indicate that a decrease of the net charge of the complexes (upon addition of DNA) generally leads to a decrease in the extent of binding, cell association and fusion, except for the neutral complexes. Association of transferrin to the lipoplexes resulted in a significant enhancement of the interaction processes referred to above, which correlates well with the promotion of transfection observed under the same conditions. Besides triggering internalization of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was reduced upon their incubation with cells in the presence of serum, suggesting that serum components limit the transferrin fusogenic properties. Results were analyzed by using a theoretical model which allowed to estimate the kinetic parameters involved in lipoplex--cell interactions. The deduced fusion and endocytosis rate constants are discussed and compared with those obtained for other biological systems. From the kinetic studies we found a twofold enhancement of the fusion rate constant (f) for the ternary lipoplexes. We also concluded that HeLa cells yield a relatively low rate of endocytosis. Overall, our results estimate the relative contribution of fusion of lipoplexes with the plasma membrane, endocytosis and fusion with the endosomal membrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies.
da Cruz
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MT
Department of Biochemistry, Faculty of Sciences and Technology, University of Coimbra, Portugal.
Simões
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Pires
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PP
Nir
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de Lima
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MC
D001769:Blood; D002462:Cell Membrane / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004705:Endocytosis; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D020404:Glycerophospholipids / Q000737:chemistry; D006367:HeLa Cells / Q000737:chemistry*; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D008561:Membrane Fusion; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014168:Transferrin / Q000737:chemistry
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D020404:Glycerophospholipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D014168:Transferrin; D004247:DNA; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane