Anna Placcia, Stefano Giordania, Ghazal Narimanfarb, Barbara Rodaa,c, Pierluigi Reschigliana,c, Lucia Catanib,d, Valentina Marassia,c, Andrea Zattonia,c

aDepartment of Chemistry “G. Ciamician”, University of Bologna, 40129 Bologna, Italy;
bInstitute of Hematology "L. e A. Seràgnoli", Department of Surgical and Medical Sciences, University of Bologna, 40126 Bologna, Italy;
cbyFlow srl, 40129 Bologna, Italy; dIRCCS Azienda Ospedaliero-Universitaria di Bologna-UOC Ematologia, 40126 Bologna, Italy.

Extracellular vesicles (EVs) are spherical nanoparticles secreted by cells in the extracellular environment and are found in many body fluids. EVs are involved in many processes, including intracellular communication and immune responses. For these reasons they are attracting attention as both diagnostic and therapeutic tools. Isolating and characterizing EVs from body fluids is challenging due to their low concentration and high heterogeneity. The isolation techniques usually exploited (AUC, SEC, UF) present many drawbacks like the requirement of large amount of sample, and low efficiency in time and purity. They also risk compromising the integrity of the vesicles with consequent loss of biological activity hence not allowing a characterization in native conditions. We present here an approach to isolate EVs from small amounts of plasma (60 µl/subject) under physiological and native conditions using a multidetection Hollow-Fiber Flow Field-Flow Fractionation (HF5-MD) platform. We separated and analyzed plasma samples from healthy donors. Size-based separation occurs in less than 25 min, and analytes are simultaneously characterized for size, shape, and spectroscopic properties, allowing downstream collection of EV-enriched fractions with minimal dilution.  In addition, EVs were isolated from the same plasma samples by SEC with collection of EV-enriched fractions. 

To evaluate and compare the performance of the innovative HF5-MD system with the EV isolation protocol using SEC, the fractions resulting from both separations were ultrafiltered as a pre-concentration step and then reanalyzed by HF5-MD to estimate the isolation efficiency and their differences in content/size distribution. This allowed to demonstrate the superiority of the HF5-MD approach in terms of analysis time and low amount of plasma, and to demonstrate the efficient isolation of EVs with minimal pressure and damage to the vesicles. The results obtained suggest that the HF5-MD platform could be an excellent approach to isolate biologically active EVs from plasma and thus understand the role of circulating EVs in tumor and healthy microenvironments. Preliminary results of the chemometric analysis of the mass distribution of the obtained fractions are also presented.

Acknowledgements: Financed by the European Union – NextGeneration EU through the Italian Ministry of University and Research under PRIN 2022 RESOLVE, 202233FTW8 CUP B53D23013360006.

Keywords: extracellular vesicles, field-flow fractionation, size exclusion chromatography, chemometric analysis