Fusogenic LipidCubosomes as Nanocarriers for Next-GenerationNanomedicine

Conspectus The further development of lipid nanocarriers remains central to advancing next-generation nanomedicine and gene delivery. Although clinically approved lipid nanoparticles (LNPs) have achieved remarkable clinical impact, especially during the COVID-19 pandemic, their reliance on endocytosis often leads to endosomal degradation and low cytosolic delivery efficiency; e.g., typically less than 10% of mRNA in the clinical COVID-19 vaccines reaches the cytosol. Lyotropic liquid crystalline LNPs with internal inverse cubic phases, namely, cubosomes, present an attractive alternative owing to their intrinsic fusogenicity with biological membranes. Their lipid bilayer architecture promotes fusion with plasma or endosomal membranes, providing a pathway for efficient cytosolic release of therapeutic cargo. The fusion behavior of individual cubosomes remained elusive for many years due to limitations in sufficient spatiotemporal resolution and the complexity of disentangling multiple interactions occurring simultaneously. Another major barrier to exploiting cubosomes in nanomedicine has been the lack of control over particle size in the traditional homogenization preparation method, restricting both fundamental research and large-scale industrial translation. For nano–bio interactions, the connections between the LNP properties, including internal nanostructure and particle size, with their biological performance are still poorly defined. For more than two decades, our Molecular Assembly Lab has had extensive experience with amphiphile self-assembly, particularly in the preparation, characterization, and application of cubosomes. In this Account, we focus on introducing our progress on cubosomes since 2019, including the mechanistic understanding of their membrane fusion behavior, microfluidic-based formation with precise size control, phase- and size-dependent nano–bio interactions across multiple in vitro mammalian cells and bacteria systems, and proof-of-concept therapeutic applications. We have deciphered that the fusion behavior of lipid dye-labeled cubosomes with supported lipid bilayers (SLBs), mammalian cell membranes, and the outer membrane of Gram-negative bacteria follows a universal scaling law of lipid dye fluorescence intensity over time, I ∼ t–1/6, as characterized by advanced total internal reflection fluorescence microscopy (TIRF). Moreover, we have tailored cubosome sizes with precise control from 130 nm to over 300 nm using a commercial microfluidic platform following a quantitative scaling law with total flow rate (Q), Size ∼ Q–0.15. A systematic in vitro evaluation of how the mesophase and particle size of LNPs modulate biological performance revealed that lipid nanocarriers with cubic phases and smaller sizes displayed stronger cell association, compared to their corresponding counterparts. Finally, we demonstrated enhanced antibiotic penetration across bacterial envelopes and elevated antimicrobial efficiency via fusion-mediated transport when antimicrobials were carried by cubosomes as a proof-of-concept. We have also highlighted the therapeutic potential of cubosomes for the delivery of diverse cargos, such as anticancer drugs, thrombolytic agents, and nucleic acids, expanding the clinical impact to broader nanomedicine frontiers.