Isolation of Citrus lemon extracellular vesicles: Development and process control using capillary electrophoresis

Aleksandra Steć; Martyna Chodkowska; Joanna Kasprzyk-Pochopień; Przemyslaw Mielczarek; Wojciech Piekoszewski; Bogdan Lewczuk; Agata Płoska; Leszek Kalinowski; Bartosz Wielgomas; Szymon Dziomba

doi:10.1016/j.foodchem.2023.136333

Isolation of Citrus lemon extracellular vesicles: Development and process control using capillary electrophoresis

Food Chem. 2023 Oct 30:424:136333. doi: 10.1016/j.foodchem.2023.136333. Epub 2023 May 12.

Affiliations

¹ Department of Toxicology, Faculty of Pharmacy, Medical University of Gdansk, 107 Hallera Street, 80-416 Gdansk, Poland.
² Laboratory of High-Resolution Mass Spectrometry, Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland.
³ AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Analytical Chemistry and Biochemistry, 30 Mickiewicza Avenue, 30-059 Krakow, Poland.
⁴ Laboratory of High-Resolution Mass Spectrometry, Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland; Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, 2 Gronostajowa Street, 30-387 Krakow, Poland.
⁵ University of Warmia and Mazury in Olsztyn, Department of Histology and Embryology, 13 Oczapowskiego Street, 10-713 Olsztyn, Poland.
⁶ Department of Medical Laboratory Diagnostics - Fahrenheit Biobank BBMRI.pl, Faculty of Pharmacy, Medical University of Gdansk, 7 Debinki Street, 80-211 Gdansk, Poland.
⁷ Department of Medical Laboratory Diagnostics - Fahrenheit Biobank BBMRI.pl, Faculty of Pharmacy, Medical University of Gdansk, 7 Debinki Street, 80-211 Gdansk, Poland; BioTechMed Centre, Department of Mechanics of Materials and Structures, Gdansk University of Technology, 11/12 Narutowicza Street, 80-233 Gdansk, Poland.
⁸ Department of Toxicology, Faculty of Pharmacy, Medical University of Gdansk, 107 Hallera Street, 80-416 Gdansk, Poland. Electronic address: szymon.dziomba@gumed.edu.pl.

PMID: 37201469
DOI: 10.1016/j.foodchem.2023.136333

Abstract

A new and scalable method for the isolation of extracellular vesicles (EV) from Citrus lemon juice samples was developed. The methodology included preliminary preconcentration of the sample using ultrafiltration (UF) followed by size-exclusion chromatography (SEC) purification and final preconcentration of the eluates. Transmission electron microscopy and proteomic analysis showed that isolates contained exosome-like vesicles, exocyst-positive organelle (EXPO), and microvesicles. The efficiency of certain isolation steps was evaluated with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A good correlation between CE, BCA, and NTA results was shown. The application of CE enabled the detection of soluble contaminants, macromolecular aggregates, and vesicles' heterogeneity. The fluorescent staining of encapsulated nucleic acids was proposed for the identity confirmation of EV detected in CE. The study demonstrates the CE as a comprehensive tool for monitoring of the EV isolation process.

Keywords: Capillary electrophoresis; Exosome-like nanovesicles; Nanocarriers; Precipitation; Size-exclusion chromatography; Ultrafiltration.

MeSH terms

Citrus*
Electrophoresis, Capillary
Exosomes*
Extracellular Vesicles*
Proteomics