Bis-quaternary ammonium gemini surfactants for gene therapy: Effects of the spacer hydrophobicity on the DNA complexation and biological activity

Abstract

Gemini surfactants (GS) have been highlighted as attractive gene carriers for a few years now; however, key aspects of the role of the GS chemical structure on the DNA-GS complexation and subsequent biological activity remain to be determined. Aiming to elucidate the effects of the GS spacer hydrophobicity, this work was focused on the biophysical characterization of the self-assembly, DNA complexation, cytocompatibility, and DNA transfection of a series of bis-quaternary ammonium GS with fixed side alkyl chains of 14 carbons and varying head-to-head alkyl chain spacers of 4, 6, and 14 carbons (referred to as GS4, GS6, and GS14, respectively). The characterization was carried out by a battery of experimental techniques including UV-vis and fluorescence sprectroscopies, ζ potential, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and flow cytometry, among others. Overall, the spectroscopic results showed that the self-assembly of the GS was favored with the spacer hydrophobicity since lower values of critical micelle concentration (CMC) were observed for samples with longer spacer chains. On the other hand, the ITC results revealed that the DNA-GS complexation was driven by an initial electrostatic attraction between DNA and GS monomers/micelles followed by complementary hydrophobic interactions which strengthen the DNA-GS binding, the latter being more pronounced for GS with longer spacers. Finally, the biological tests demonstrated that while GS with moderate hydrophobicity (GS4 and GS6) yielded outstanding levels of cytocompatibility and DNA transfection over a range of concentrations, the most hydrophobic sample (GS14) proved to be cytotoxic upon administration to cultured HeLa cells (p < 0.05). In our opinion, the fundamental information here presented might be pivotal not only for understanding the DNA-GS complexation mechanism, but also for developing efficient GS-based carriers for gene therapy.

Keywords: Cytocompatibility; DNA complexation; DNA transfection; Gemini surfactants; Gene therapy; Surfactant spacer.