PhD Scientific Days 2026

Poster Session 2.G - Pharmaceutical Sciences and Health Technologies

Separation of Plasma Extracellular Vesicles from Lipoproteins Using Density Gradient Ultracentrifugation and Size Exclusion Chromatography for Diagnostic Applications

Name of the presenter

Kapui, Dóra

Institute/workplace of the presenter

Semmelweis University, Department of Pharmacology and Pharmacotherapy

Authors

Dóra Kapui¹, Szabolcs Hambalkó¹, Yavuz Artuner¹, Orsolya Szalai¹, Sándor Zsidai¹, Richárd Rácz¹, Péter Ferdinandy², Csenger Kovácsházi¹, Zoltán Giricz²
1: Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest; Center for Pharmacology and Drug Research & Development, Semmelweis University, Budapest
2: Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest; Center for Pharmacology and Drug Research & Development, Semmelweis University, Budapest; Pharmahungary Group, Szeged

Text of the abstract

Introduction: Biomarkers in circulating extracellular vesicles (EVs) hold great potential for diagnostic applications. However, lipoproteins (LPs) share overlapping physical properties and molecular cargo with EVs, which can obscure EV-related signals. Therefore, efficient separation of EVs and LPs is needed to increase the sensitivity and specificity of EV-based diagnostics.
Aim: Our aim was to develop a method which separates EVs from LPs from human blood samples with optimal purity, yield and time efficiency, and to apply it in samples of patients suffering from acute myocardial infarction (AMI).
Methods: EVs were isolated from 1 mL human platelet-free plasma using iodixanol density gradient ultracentrifugation (DGUC) followed by size exclusion chromatography (SEC) with Sepharose 4 Fast Flow (4FF) columns. Isolates were analysed using nanoparticle tracking analysis (NTA), Qubit protein assay and Western blot (WB).
Results: WB analysis of iodixanol DGUC fractions revealed significant overlap between LP and EV markers, demonstrating the need of further purification steps. After 4FF SEC of the EV-rich fractions, WB analysis revealed the presence of EV markers, while mild LP contamination also presented. Application of the total workflow needed two experimental days for 6 samples. NTA revealed the presence of EV-sized nanoparticles in the isolates, with a significantly larger size than LP samples. NTA and Qubit protein assay revealed no significant difference in EV sample concentration between AMI and non-AMI patients.
Conclusion: Combined DGUC followed by 4FF SEC demonstrated acceptable EV-LP separation and processing time for plasma EV isolation. No significant difference was observed in plasma EV numbers of AMI and non-AMI patients. Molecular analysis of the EV samples may highlight EV-related differences, that can be used for the development of future diagnostic-prognostic applications.
Funding: The Ministry for Innovation and Technology of Hungary from the source of National Research, Development and Innovation Fund supported CK (EKÖP-2024-226 New National Excellence Program), ZG(K139105) and the project (2020-1.1.5-GYORSÍTÓSÁV-2021-00011). The project was further supported by the European Union (RRF-2.3.1-21-2022-00003). KD was supported by SE 250+ Excellence PhD Scholarship.