Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy

Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4-10), osmolarity (50-1000 mOsm/L), temperature (15-60 °C), and surfactant Triton X-100 (10-500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (R_h), intensity of scattered light (I), and the shape parameter (ρ). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS-citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50-1000 mOsm/L, pH 4-10) had no significant effect on the R_h (=100-130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in I of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.