Kinetic analysis of the initial steps involved in lipoplex–cell interactions: effect of various factors that influence transfection activity

Abstract

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.

Higly fusogenic cationic liposomes transiently permeabilize the plasma membrane of HeLa cells

“…1A and B indicates diC14-amidine liposomes have higher lipid-mixing efficiencies than those prepared from DOTAP, which concurs with the results of others [13,22]. They also show a higher efficiency at interacting with HeLa cells, also demonstrated previously by others [23]. Our experiments were performed at different temperatures to evaluate how the increase in lipid fluidity (a set of highly temperature-dependent features of the lipid bilayer) affects the lipid-mixing process (Fig.…”

Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

“…Coating of lipoplexes with Tf has been demonstrated to promote both the cellular uptake of lipoplexes (da Cruz et al, 2001) and to trigger cytoplasmic delivery of the carried DNA through destabilization of the endosomal membrane under acidic conditions (da Cruz et al, 2001;Simoes et al, 1998). Our results clearly indicate that such a strategy resulted in a significant enhancement of transfection in both neuronal primary cultures and cell line, this effect being dependent on the type of cells, net charge, and composition of the complexes.…”

“…A large amount of effort has also been devoted to the development of cationic liposomes (Imaoka et al, 1998;Murray et al, 1999;Roessler and Davidson, 1994;Wu et al, 2000;Zou et al, 1999), the most widely used nonviral vectors, to improve transfection efficiency and duration of gene expression as well as to confer adequate features for their in vivo use. The ability of this system to mediate transfection was attributed to recognition of certain properties, namely, (1) a spontaneous electrostatic interaction between the positively charged liposomes and the negatively charged DNA, which results in an efficient condensation of the nucleic acids, and (2) the fact that the resulting cationic liposome/DNA complexes (lipoplexes) could exhibit a net positive charge that promotes their association with the negatively charged cell surface (da Cruz et al, 2001;Pires et al, 1999). Following internalization by endocytosis (Gao and Huang, 1995;Zabner et al, 1995), the lipoplexes promote the cytoplasmic delivery of DNA (Felgner, 1996;Felgner et al, 1987;Gao and Huang, 1995;Roessler and Davidson, 1994;Zhu et al, 1993).…”

γ-Polyglutamic acid-coated vectors for effective and safe gene therapy

“…In cationic systems, strong electrostatic interaction with a negatively charged cellular membrane can contribute to high gene expression through the endocytotic pathway [21]. We compared the transfection efficiency of each complex in mouse melanoma cell lines, B16-F10 cells, which are often used to examine the basic gene expression of non-viral vectors [22][23][24].…”