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, nam ely the stoichiometry of cationic lipids and DNA, the presence of serum in the cell culture medium, and the incorporation of the ligand transferrin in to the lipoplexes, on the extent of binding, association and fusion (lipid mixing) of the lipoplexes with the cells. Parallel experiments were perform ed 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 an d fusion, except for the neutral complexes. Association of transferrin to t he lipoplexes resulted in a significant enhancement of the interaction proc esses referred to above, which correlates well with the promotion of transf ection observed under the same conditions. Besides triggering internalizati on of the complexes, transferrin was also shown to mediate fusion with the endosomal membrane. The extent of fusion of this type of complexes was redu ced upon their incubation with cells in the presence of serum, suggesting t hat serum components limit the transferrin fusogenic properties. Results we re analyzed by using a theoretical model which allowed to estimate the kine tic parameters involved in lipoplex-cell interactions. The deduced fusion a nd endocytosis rate constants are discussed and compared with those obtaine d 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 lipop lexes with the plasma membrane, endocytosis and fusion with the endosomal m embrane to their interactions with cells, this information being of crucial importance for the development of gene therapy strategies. (C) 2001 Elsevi er Science B.V. All rights reserved.