Design and mechanistic evaluation of charge-converting fusogenic liposomes for efficient nucleic acid delivery

Efficient cytosolic delivery of nucleic acids is critically limited by poor endosomal escape. While the cationic surface of nanocarriers can enhance endosomal destabilization, these systems suffer from rapid clearance and protein corona formation, a long-standing challenge known as the polycation dilemma. Charge-converting nanocarriers can mitigate this issue by masking positive charges during circulation; however, they remain constrained by inefficient endosomal escape. To overcome this limiting step, we designed enzyme-responsive, charge-converting fusogenic liposomes that combine polyphosphate coatings with DOPE/DOTAP-based fusogenic liposomes. The polyphosphate suppresses cationic charge during circulation, while alkaline-phosphatase-mediated dephosphorylation restores the cationic charge required for membrane fusion. Liposomes were systematically characterized for size, zeta-potential, and phosphate-release-induced charge conversion. Their interaction with Caco-2 cells was mechanistically investigated using confocal microscopy, flow cytometry, and Förster resonance energy transfer, demonstrating the existence of a fusion-driven uptake. Upon loading with plasmid DNA, the most fusogenic formulations enabled a rapid onset of GFP expression consistent with direct cytosolic access. Overall, this work provides a mechanistic investigation of polyphosphate-coated fusogenic liposomes and demonstrates that enzyme-triggered charge conversion can be coupled to membrane fusion to facilitate cytosolic delivery.