The Role of Membrane Asymmetry in Vesicle-Mediated In Vitro Delivery of Nucleic Acid Cargo

Abstract Nucleic acid therapeutics typically require a delivery vector, such as lipid-based nanoparticles. However, limitations in e.g. specific targeting and biodistribution, pose challenges to their clinical applications, and alternative lipid-based delivery vectors are therefore highly desirable. Here, we investigated the effect of introducing membrane asymmetry to lipid vesicles (i.e., the inner and outer leaflets have different lipid compositions), which has previously been shown to alter their biophysical properties. In this study, we prepared asymmetric large unilamellar vesicles (aLUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) on the inner leaflet and either 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) or sphingomyelin (SM) on the outer leaflet, with the addition of cholesterol (Chol), using methyl-β-cyclodextrin-catalyzed lipid exchange. The extent of outer leaflet exchange was found to be lipid-dependent. Further, we probed intracellular uptake and nucleic acid cargo delivery of DOPC:SM:Chol aLUVs. Widefield fluorescence imaging using HeLa_Luc705 cells and confocal imaging using SH-SY5Y-miRFP670 cells revealed cellular internalization and nuclear localization of aLUVs as early as 4 h, with analogous symmetric vesicles showing lower uptake and notable localization in the perinuclear region. However, HeLa_Luc705 cells treated with aLUVs and symmetric LUVs loaded with a splice-switching oligonucleotide showed similar levels of luciferase production after 24 h, indicating similar levels of functional oligonucleotide cargo delivery. Overall, the data presented indicates that asymmetric vesicles are able to deliver nucleic acid cargo and that the asymmetric distribution of lipids across membrane leaflets can influence the cellular uptake and intracellular distribution of vesicle nanocarriers.