Bypassing Endocytic Barriers: Visualizing Membrane Fusion and Endosomal Escape of Cubic Phase Lipid Nanoparticles (Cubosomes)

Abstract Lipid nanoparticles (LNPs) are versatile platforms for drug delivery, offering solutions for targeted therapeutics, gene therapy, and vaccines. Cubosomes ‐ cubic phase lipid nanoparticles ‐ exhibit unique structural properties that may allow direct cytosolic delivery, bypassing endocytic pathways that often limit intracellular drug delivery. Despite their potential, the mechanisms of cubosome interactions with mammalian cells, particularly their membrane fusion behavior, remain unclear. This study employs a multimodal microscopy‐based approach to investigate cubosome interactions with cells, focusing on membrane fusion, lipid exchange, and endosomal escape. Advanced imaging techniques, including transmission, scanning, and cryogenic scanning electron microscopy, and live‐cell fluorescence imaging, are used alongside tailored cubosome variants to examine interactions at micro‐ and nanoscale dimensions. Cubosome behaviors are compared to other nanoparticleslike liposomes and gold nanoparticles. For the first time, membrane fusion with mammalian plasma and endosomal membranes is visualized at the nanoscale, revealing how cubosomes bypass conventional endocytic pathways to deliver cargo directly into the cytosol. This work provides critical insights into LNP–cell interactions, establishing cubosomes as promising candidates for overcoming endosomal escape limitations in drug delivery. These findings will aid in developing next‐generation lipid‐based nanocarriers, particularly for RNA therapeutics, where efficient cytosolic delivery is essential for therapeutic efficacy.