The use of anionic lipids (ALs) as non-viral gene vectors depicts a promising alternative to cationic lipids (CLs) since they are more biocompatible and present lower levels of phagocytosis by macrophages. Several experimental methods, such as electrophoretic mobility (ζ-potential), gel electrophoresis, small-angle X-ray scattering (SAXS), fluorescence and confocal fluorescence microscopies (FM and CFM), flow assisted cell sorting-flow cytometry (FACS-FCM), and cell viability/cytotoxicity assays can be used for a complete physicochemical and biochemical characterization of lipoplexes formed by an AL, a zwitterionic lipid (ZL), and a plasmid DNA (pDNA), their electrostatic interaction being necessarily mediated by divalent cations, such as Ca(2+). In the present chapter, we summarize the protocols optimized for the mentioned characterization techniques.
Keywords: Anionic lipids; Flow cytometry; Fluorescence microscopy; Gel electrophoresis; Lipoplexes; Liposomes; Non-viral gene vectors; SAXS; pDNA; ζ-potential.