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

Abstract

Objective

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.

Methods

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.

Results

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.

Conclusion

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.

Keywords

Extracellular vesicles / Plasma / Ultracentrifugation / Filtration / Dynamic light scattering

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Funding

National Natural Science Foundation of China(82495171)

National Key R&D Program of China(2021YFA0805100)

Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2023-PT320-03)

Guangdong Basic and Applied Basic Research Foundation, China(2024A1515030041)

The Science and Technology Planning Project of Guangdong Province(2023B1212060018)

Sun Yat-sen University Basic Research Funds(24qnpy354)

Clinical Research 5010 Program(2014002)

RIGHTS & PERMISSIONS

The Author(s), under exclusive licence to Huazhong University of Science and Technology