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

© The Author(s), under exclusive licence to Huazhong University of Science and Technology 2025

Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s11596-025-00045-z.

Funding National Natural Science Foundation of China (No. 82495171, 92268202, 82270485); National Key R&D Program of China (No. 2021YFA0805100); Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (No. 2023- PT320-03); Guangdong Basic and Applied Basic Research Foundation, China (No. 2024A1515030041); The Science and Technology Planning Project of Guangdong Province, (No. 2023B1212060018); Sun Yat-sen University Basic Research Funds ( No. 24qnpy354); Clinical Research 5010 Program (No. 2014002); Program of National Key Clinical Specialties.

Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Conflict of interests On behalf of all authors, the corresponding author states that no competing interests exist.

Ethical Approval and Consent to Participate This study was reviewed and approved by the Ethics Review Board of the First Affiliated Hospital, Sun Yat-sen University and Fuwai Central China Cardiovascular Hospital (#2020-8). Prior informed consent was obtained from each subject enrolled in this study.

Human and animal rights This study had been approved by a suitably constituted Ethics Committee of the institution, and conformed to the provisions of the Declaration of Helsinki (as revised in Edinburgh 2000).

Consent for Publication All the authors agreed to submit the manuscript to Current Medical Science with the signature of the corresponding authors.

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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