Unraveling the Heterogeneity of Cargo Distribution in the Exogenous Association of Proteins With Extracellular...

Extracellular vesicles (EVs) are emerging as promising nanocarriers for delivering molecules, including proteins. Various exogenous methods are proposed for loading EVs with specific cargo proteins. While the loading yield and the heterogeneity of cargo distribution are crucial quality attributes, a comprehensive quantification of these properties is still lacking. Here, we characterize the heterogeneity of EVs loaded with a model cargo protein, GFP, using various exogenous methods. A combination of biophysical methods is applied to quantify the overall yield and cargo distribution at both the ensemble and single-particle levels. Among the loading methods evaluated, electroporation is most effective for associating GFP with EVs. However, the GFP molecules per vesicle is fewer than 100, representing approximately 4% of the maximum protein capacity that EVs can potentially accommodate. Across all loading methods, the distribution of protein content per vesicle displays significant heterogeneity and follows an exponential decay function, with a higher prevalence of vesicles featuring lower protein content and fewer with higher content. Moreover, loading efficiency increases with EV size. This study shows that overall yields of exogenous loading methods to associate proteins with EVs remain modest and the resulting distribution of cargo proteins associated with EVs is highly heterogeneous.