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

Extracellular vesicles (EVs) are endogenous vesicles secreted by cells. Exosomes (30-150 nm), are a subset of EVs playing key roles in intercellular communication. Exosomes show promise as cancer chemotherapeutic drug delivery vehicles given their low immunogenicity and cell-specific cytosolic delivery of their contents. However, inefficient drug loading limits their therapeutic application. To address this, methods for the fusion of EVs with therapeutic drug-loaded synthetic liposomes have been developed. While more efficient than passive incubation of EVs with liposomes, these risk either damage to EV membrane proteins or contamination of the EV-liposome hybrids 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 enhance the fusion of EVs and synthetic, neutral liposomes (NLs) compared to passive incubation in pH 7.4 at 37 °C. An adapted Forster resonance energy transfer (FRET) based lipid mixing assay confirms that fusion is enhanced with this method, albeit less efficiently than with depletant-induced fusion. This significant finding implies that lipid-only synthetic liposomes can fuse with EVs, creating EV-liposome hybrids under relevant temperature and pH conditions, without nonlipidic components, such as fusogenic amphipathic peptides, added to the synthetic liposomes. Remarkably, differential interference contrast (DIC) and fluorescence microscopy show that this enhanced fusion corresponds with the clustering of mixtures of EVs and NLs, or EVs alone, in acidic <i>but not neutral</i> pH conditions. The findings support a hypothesis that content release from EVs in early to late endocytic environments 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 toward the development of therapeutic hybrid drug delivery vehicles in nanomedicine applications.