Gene therapy is a technique utilized to treat diseases caused by missing, defective or overexpressing genes. Although viral vectors transfect cells efficiently, risks associated with their use limit their clinical applications. Nonviral delivery systems are safer, easier to manufacture, more versatile and cost effective. However, their transfection efficiency lags behind that of viral vectors. Many groups have dedicated considerable effort to improve the efficiency of nonviral gene delivery systems and are investigating complexes composed of DNA and soft materials such as lipids, polymers, peptides, dendrimers and gemini surfactants. The bottom-up approach in the design of these nanoparticles combines components essential for high levels of transfection, biocompatibility and tissue-targeting ability. This article provides an overview of the strategies employed to improve in vitro and in vivo transfection, focusing on the use of cationic lipids and surfactants as building blocks for nonviral gene delivery systems.

The incorporation of a pH-active imino group within the spacer of the gemini surfactant results in a significant increase in transfection efficiency that can be related to both pH-induced changes in nanoparticle structure and the formation of multiple phases that more readily allow for membrane fusion that may facilitate DNA release.

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 pHsensitive 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-1,9nonanediammonium dibromide surfactants, m-7NH-m, where m ¼ 12, 16, 18 and 18:1, are hypothesized to stage endosomal release of DNA. The m ¼ 18:1 chain, i.e., mono-unsaturated oleyl chain, is an alkenyl chain analogue for comparison with the saturated m ¼ 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 (r AE ) 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 ¼ 12 < m ¼ 16 < m ¼ 18 (p > 0.01), although the difference between m ¼ 16 and m ¼ 18 was insignificant. Morphological studies of the nanoparticles by transmission electron microscopy showed fusogenic changes at pH ¼ 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.

The MS(n) analyses of these novel gemini surfactants, reported here for the first time, revealed diagnostic ions for each compound, with a universal fragmentation pattern for each compound series. The diagnostic ions will be employed within liquid chromatography (LC)/MS/MS methods for screening, identification, and quantification of these compounds within biological samples.

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