Photoactivated bioinspired lipoplexes with a chalcone/flavylium photoswitch enhance siRNA delivery—towards precise spatiotemporal control in gene delivery

The efficient delivery of nucleic acids (NAs) remains a major challenge in gene therapy due to their poor stability and limited cellular uptake. Even though non-viral vectors have been pivotal to overcoming some of these challenges, significant barriers, such as intracellular digestion of NAs and limited endosomal escape, still remain. Here, we developed novel stimuli-responsive lipoplexes integrating a 2-hydroxychalcone-based cationic amphiphile (C_<i>n</i>NCh, with 4 or 6 carbons in their alkyl chains, <i>n</i> = 4 or 6) and monoolein (MO). This combination leverages the photoisomerization and pH-sensitivity of chalcone derivatives, along with the fusogenic capabilities of MO, to achieve enhanced transfection efficiency <i>via</i> light irradiation. To reach this goal, we first assessed the cytotoxicity of the cationic amphiphiles in healthy and tumor cells. We then prepared mixtures with varying C_<i>n</i>NCh/MO molar ratios, yielding net cationic vesicles with long-term colloidal stability. Subsequently, NAs were efficiently compacted into lipoplexes at N/P ratios (positively charged nitrogen/negatively charged phosphate) higher than 1, attaining near-complete compaction. Light and pH stimuli induce the formation of the expected products, but without compromising lipoplex stability or activating premature NA release. Vesicles with different C_<i>n</i>NCh/MO molar ratios do not induce the loss of viability of normal fibroblasts for concentrations up to 50 µM. Crucially, siRNA-lipoplex mixtures having C₄NCh/MO molar ratios of 1/1 and 2/1 (N/P = 6) achieve significant GFP knockdown after irradiation, indicative of successful siRNA delivery and biological effects. Using biomimicking endosomal membranes, we show that photoactivation enhances membrane fusion, suggesting a mechanism entailing light-mediated endosomal escape. Our study provides proof-of-concept for a "light-switch" mechanism offering precise spatiotemporal control over gene silencing, a highly desirable feature in therapeutic applications.