Fusion Between Bare Liposomes and Exosomes is Enhanced Through Induced Depletion Attraction or in Acidic Conditions.

Exosomes are cell-secreted, nanometer scale extracellular vesicles (EVs) involved in functional miRNA and mRNA transfer from donor to acceptor cells and alteration of gene expression in acceptor cells. In line with their biological function, existing evidence points to reduced immunogenicity, cell-specific binding in vivo, and endosomal escape upon entry into target cells, properties which strongly support the use of exosomes as drug delivery vehicles. Previously, we isolated EVs, defined as a mixture of exosomes and microvesicles, from human prostate (PC3) and melanoma (M21) cancer cells using serial velocity ultracentrifugation. We confirmed these EVs were within the expected 30 - 200 nm diameter range for exosomes and contained the EV marker protein CD63 with nanoparticle tracking analysis and western blot assays [W.S. Fisher, C. Tchounwou, S. Wei, L. Roberts, K.K. Ewert, C.R. Safinya, Exosomes are secreted at similar densities by M21 and PC3 human cancer cells and show paclitaxel solubility, Biochimica et Biophysica Acta (BBA) - Biomembranes, 1864 (2022) 183841]. To enable EVs to serve as drug delivery vehicles, fusion between synthetic liposomes and EVs must be optimized to allow for transfer of desired hydrophobic drugs or nucleic acids from the liposomes to the EVs. We explored two different pathways leading to enhanced fusion. We induced depletion attraction, in reaction mixtures containing polyethylene oxide (PEO) as the depletant, to crowd labeled EVs and liposomes into micron-scale clusters observable in DIC and fluorescence microscopy. The crowding of EVs and synthetic liposomes enhances fusion verified via a Förster resonance energy transfer (FRET) based lipid-mixing assay. With this FRET assay, we also identified a statistically significant enhancement of EV-liposome fusion in acidic (pH 5) compared to neutral (pH 7.4) buffer conditions in mixtures containing no crowding depletants.