The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications.
Keywords: CMV promoter; photosensitizer; polymeric gene delivery; reactive oxygen species; stimuli-responsive gene delivery.