Characterization and investigation of redox-sensitive liposomes for gene delivery

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redoxsensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nanosized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.