Novel pyridinium surfactants for efficient, nontoxic in vitro gene delivery

Abstract

Novel, double-chained pyridinium compounds have been developed that display highly efficient DNA transfection properties. The transfection efficiency of several of these compounds is enhanced by an order of magnitude, when compared with the transfection efficiency accomplished with the widely used cationic lipid system, lipofectin. Most importantly, the pyridinium compounds were found to be essentially nontoxic toward cells. Using various reporter genes, such as ␤-galactosidase and pNEO (a gene construct that renders cells resistent to antibiotic derivatives of neomycin like G418), we demonstrate that the enhanced efficiency relates to the fact that a relative higher number of cells in the population is transfected (Ϸ50% in the case of COS cells) by the pyridinium derivatives, whereas the delivery of DNA per cell is also enhanced. Furthermore, application of the pyridinium derivatives shows little cellular preference in their ability to transfect cells. By systematically modifying the structure of the pyridinium amphiphile, i.e., by changing either the headgroup structure or the alkyl chains, some insight was obtained that may lead to unraveling the mechanism of amphiphile-mediated transfection, and thus to protocols that further optimize the carrier properties of the amphiphile. Our results reveal that unsaturated alkyl chains enhance the transfection properties of the pyridinium-based amphiphiles. Preliminary experiments suggest that the structure-dependent improvement of transfection efficiency, when comparing pyridinium derivatives with lipofectin, likely relates to the mechanism of delivery rather than the packaging of the amphiphile͞DNA complex.

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

“…Canine cav-1 was cloned in the PCDNA3 plasmid. Cells were transfected with the cationic lipid SAINT-2, according to a procedure previously described by van der Woude et al (1997). Briefly, cells were seeded in 35-mm dishes at low density, and the transfection was performed the following day.…”

Effect of Headgroup on DNA−Cationic Surfactant Interactions

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] There has been considerable interest in characterizing the nature of the fundamental interactions between cationic agents and DNA, and several general approaches have been designed to unravel such interactions. [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] One of the important driving forces in all of these systems is the long-range electrostatic interaction between molecules of opposite charge.…”

Lipoplex-mediated Transfection of Mammalian Cells Occurs through the Cholesterol-dependent Clathrin-mediated Pathway of Endocytosis

“…As has been recently shown, cholesterol depletion, as accomplished in this manner, results in the inhibition of clathrin-mediated endocytosis, whereas nonclathrin-mediated endocytosis is much less affected. It was demonstrated that the decrease in clathrin-mediated endocytosis is caused by the disability of coated pits to invaginate and detach from the plasma membrane (17,18).Here, we have determined the transfection efficiency of COS7 cells, using lipoplexes composed of the cationic amphiphile SAINT-2 and dioleoylphosphatidylethanolamine (DOPE) (5,19,20), following depletion of plasma membrane cholesterol with M␤CD. Because M␤CD treatment of cells, next to an interference of coated pit organization, may potentially interfere with the internalization via caveolae as well, the effect of two additional drugs, filipin III and chlorpromazine, were tested to differentiate between the involvement of either type of endocytosis (non-coated pit (caveolae) versus coated pit (clathrindependent)).…”

“…Here, we have determined the transfection efficiency of COS7 cells, using lipoplexes composed of the cationic amphiphile SAINT-2 and dioleoylphosphatidylethanolamine (DOPE) (5,19,20), following depletion of plasma membrane cholesterol with M␤CD. Because M␤CD treatment of cells, next to an interference of coated pit organization, may potentially interfere with the internalization via caveolae as well, the effect of two additional drugs, filipin III and chlorpromazine, were tested to differentiate between the involvement of either type of endocytosis (non-coated pit (caveolae) versus coated pit (clathrindependent)).…”