Single‐Particle Characterization Reveals Heterogeneous Extracellular Vesicle Fusion with Liposomes

Fusion of extracellular vesicles (EVs) with liposomes can be used to alter the properties of EVs to enhance their drug delivery capabilities. However, metrics for assessing fusion are not well established. Fusion efficiency, the most frequently provided metric, is often characterized in bulk, clouding distribution of fusion across heterogeneous EV populations, and lacking assessment of more precise physical effects of fusion. Here we applied orthogonal single-particle techniques including nanoparticle-tracking analysis (NTA), resistive-pulse sensing (RPS), nanoscale flow cytometry, interferometric fluorescence imaging, and laser trapping Raman spectroscopy (LTRS), each with different limitations, to examine the effects of fusion. All techniques reduced particle number, while single-particle fluorescence analyses revealed substantial differences in fused-particle yield. Nanoscale flow cytometry and interferometric fluorescence imaging consistently identified freeze-thaw and sonication as producing the highest numbers of fused vesicles, with freeze-thaw generating the lowest proportion of non-fused EVs and liposomes. Interferometric fluorescence imaging further showed that fused vesicles retained native EV membrane proteins, but that fusion also reduced the abundance of these proteins, indicating membrane perturbation. We introduce here a multi-metric framework to evaluate fusion efficiency, purity, and physical alterations to vesicles, as a basis for comparing techniques and to support future optimization of engineered EV formulations.