Fusion of liposomes incorporating α-linolenic acid with the cell plasma membrane is site-restricted

This study assesses the interactions of liposomes incorporating unsaturated cis-9,12,15-octadecatrienoic acid (α-linolenic acid) with model and cell membranes. The liposome-cell membrane interactions that initiate a membrane fusion process may enable a direct cytoplasmic delivery of liposomal cargo. Experimental results confirm the incorporation of α-linolenic acid (αLA) into, and a consequent concentration-dependent increase in the fluidity of, the liposomal lipid bilayer, as demonstrated by ¹H-NMR spectroscopy and a laurdan emission assay, respectively. On mixing with simple membrane-model liposomes, Förster Resonance Energy Transfer (FRET) analysis of embedded donor-acceptor pairs reveals a reduction in the FRET ratio, indicative of structural alterations in the lipid bilayer and a membrane fusion of αLA containing liposomes, not observed for their non-αLA counterparts. Following application to cells in vitro a reduction in the FRET ratio was seen for both αLA-containing and non-αLA liposomes, implying changes in the liposomal lipid bilayer in both systems. However, confocal microscopy and Pearson's correlation coefficient analysis reveal a crucial difference: αLA containing liposomes preferentially localize at the cell plasma membrane, whereas their non-αLA counterparts predominantly exhibit intracellular localisation. Notably, cell membrane-associated fluorescence appears punctate and heterogeneously distributed, suggesting that αLA-liposome fusion with the cell membrane does not lead to a homogeneous mixing of lipid membrane/dye lateral diffusion but that, for the system and conditions tested, the liposome fusion is a site-restricted process. Observations in this study are critical in the design of drug delivery systems capable of achieving direct cytoplasmic delivery of active compounds to potentially overcome a current bottleneck in effective cytosolic drug delivery.