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

Abstract

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 pK_a. 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.