Document Type

Data

Publication Date

7-8-2026

Abstract

Small extracellular vesicles (sEVs) are promising biomarkers and therapeutic delivery vehicles for disease diagnosis and treatment. However, standardizing isolation and characterization techniques is essential, as different isolation methods can alter the biophysical properties of the sEVs. In this study, we compared the physicochemical properties of HEK-293-derived sEVs isolated by polymer precipitation (PP) and sucrose cushion ultracentrifugation (UC). Using atomic force microscopy (AFM) and chemical force microscopy (CFM), we measured the morphology, adhesion, Young’s modulus, and lipid membrane tethering events to understand how the choice of isolation method influences the sEV properties. AFM topographic images revealed the presence of polymeric residues and co-precipitated proteins in PP-sEVs, which are absent in UC-sEVs. These findings were supported by other size-based analyses. UC-sEVs appeared stiffer than PP-sEVs, as measured by AFM, and lipid membrane tethering events occurred more frequently in UC-sEVs. Overall, our findings suggest that polymeric residues in PP-sEVs may influence membrane mechanical properties and limit membrane tethering interactions between a functionalized AFM tip and the vesicle membrane, resulting in an apparent reduction in stiffness. These findings demonstrate that the choice of isolation method affects the mechanical properties of sEVs and should be carefully considered when interpreting downstream analysis of isolated sEVs.

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