Designing pH-sensitive gemini nanoparticles for non-viral gene delivery into keratinocytes

This study aimed to develop a more effective non-viral gene delivery system for keratinocyte transfection. To this end, gemini nanoparticles were formulated from plasmid DNA, the lipid DOPE (dioleoylphosphatidylethanolamine) and surfactants, where the surfactant components are novel pH-sensitive gemini surfactant derivatives based on the m-7-m (alkyl chain–spacer–alkyl chain, m-s-m) unsubstituted base structure. The resultant 1,9-bis(alkyl)-1,1,9,9-tetramethyl-5-amino-1,9-nonanediammonium dibromide surfactants, m-7NH-m, where m = 12, 16, 18 and 18:1, are hypothesized to stage endosomal release of DNA. The m = 18:1 chain, i.e., mono-unsaturated oleyl chain, is an alkenyl chain analogue for comparison with the saturated m = 18 alkyl chain based on the m-7NH-m frame. Analytical and physicochemical characterization of the gemini surfactants included purity, aggregation properties under pH 2.5–10.5, critical micellar concentration and pKa. Gemini nanoparticles were characterized by dynamic light scattering, zeta potential, small-angle X-ray scattering and transmission electron microscopic studies. Keratinocyte transfection efficiency and cytotoxicity were evaluated using the luciferase reporter assay and luminescent cell viability assay, respectively. Gemini nanoparticles formulated from the m-7NH-m gemini surfactants at a surfactant:DNA charge ratio (ρ±) 10:1 showed higher transfection efficiency compared to the unsubstituted compounds (m-7-m, m-3-m series) (p < 0.01). Transfection efficiency increased with increasing tail length, i.e., m = 12 < m = 16 < m = 18 (p > 0.01), although the difference between m = 16 and m = 18 was insignificant. Morphological studies of the nanoparticles by transmission electron microscopy showed fusogenic changes at pH = 5. The incorporation of a pH-active amine group within the spacer of the gemini surfactants significantly enhanced transfection efficiency in keratinocytes. This may be attributed to optimal interactions between DNA phosphate groups and the m-7NH-m gemini surfactants owing to their –NH– groups, trimethylene spacing between nitrogen centers and the acidic pH-induced polymorphic changes, leading to endosomal release of plasmid. Such results highlight the amino-substituted gemini surfactants as potential components for developing non-viral nanoparticles with enhanced gene delivery for targeting diseases affecting the skin.