A Simple Modification Results in a Significant Improvement in Measuring the Size of Extracellular Vesicles

Size distribution is an important biophysical property of extracellular vesicles (EVs). EVs include small EVs (s-EVs) and large EVs (l-EVs) by size. Differential ultracentrifugation (dUC) is widely used to separate EVs from biofluids, but it can precipitate large impurity particles. Dynamic light scattering (DLS) is a simple and fast method for analyzing the size distribution of EVs. However, this approach is nonideal for heterogeneous and polydisperse samples since a small quantity of large impurity particles can markedly disturb the DLS results. Here, we developed a simple method to improve the reliability of DLS measurements.

Plasma was obtained from 13 volunteers. The plasma was first processed by dUC to obtain crude l-EVs. The crude l-EVs were filtered with syringe filters (pore size of 1 μm and membrane material of hydrophilic polyvinylidene fluoride (PVDF)) to remove large impurity particles from l-EVs. The size distributions of the crude l-EVs and filtered l-EVs were measured via DLS.

After the samples were filtered, the coefficients of variation of the hydrodynamic radius and Peak 1 intensity of the filtered l-EVs decreased from 20.39% (12.76-28.96%) and 20.44% (14.58-28.32%) to 3.05% (1.79-4.72%) and 3.43% (1.76-5.88%), respectively, compared with those of the crude l-EVs.

These findings suggest that filtration can effectively separate circulating l-EVs in plasma to remove large impurity particles and make samples suitable for characterization by DLS. Our findings provide a simple method to improve precision via DLS to measure the size distribution of EVs.