Acidic Conditions Promote Clustering of Cancer Cell Derived Extracellular Vesicles and Enhance their Fusion with Synthetic Liposomes

Abstract

Extracellular vesicles (EVs) are endogenous vesicles secreted by cells. Exosomes, a subset of EVs ranging from 30 to 150 nanometers in diameter, contain cytosolic proteins, gene-silencing miRNA, and gene-encoding mRNA and have roles in intercellular communication. Exosomes show promise as cancer chemotherapeutic drug delivery vehicles given their low immunogenicity and cell-specific delivery of luminal contents to the cytosol of target cells. However, loading exosomes with cancer chemotherapeutic drugs is inefficient, which limits their therapeutic application. To overcome this barrier, methods have been developed which allow fusion of EVs with synthetic liposomes preloaded with therapeutic drug. While these methods show more efficient fusion than passive incubation of EVs with liposomes, they risk either damage to the membrane proteins of the EVs or contamination of the final EV-liposome hybrid with residual depletant molecules, which can cause side effects or hinder content delivery. Here, we present a new, weakly perturbative method which uses acidic conditions (pH 5) to significantly enhance the fusion of EVs and synthetic, neutral liposomes (NLs) compared to passive incubation in pH 7.4 at 37 {degrees}C. An adapted Forster resonance energy transfer (FRET) based lipid mixing assay confirms that fusion is enhanced with this method. This significant finding implies that lipid-only synthetic liposomes are able to fuse with EVs, creating EV-liposome hybrids under relevant temperature and pH conditions, with no other non-lipidic component, such as fusogenic amphipathic peptides, added to the synthetic liposomes. Remarkably, differential interference contrast (DIC) and fluorescence microscopy show that this enhancement of fusion corresponds with the onset of clustering of mixtures of EVs and NLs, or EVs alone, in acidic but not neutral pH conditions. The findings support a hypothesis that content release from EVs in early to late endocytic environment may be a combination of protein-protein clustering interactions and a lipidic component. Further, this study provides a novel method for enhanced fusion of EVs and liposomes which is expected to preserve EV membrane proteins and functionality towards the development of therapeutic hybrid drug delivery vehicles in nanomedicine applications.