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

• Published in: Food chemistry 2023-10, Vol.424, p.136333-136333, Article 136333
• Main Authors: Steć, Aleksandra, Chodkowska, Martyna, Kasprzyk-Pochopień, Joanna, Mielczarek, Przemyslaw, Piekoszewski, Wojciech, Lewczuk, Bogdan, Płoska, Agata, Kalinowski, Leszek, Wielgomas, Bartosz, Dziomba, Szymon
• Format: Article
• Language: English
• Subjects: Capillary electrophoresis; Citrus; Electrophoresis, Capillary; Exosome-like nanovesicles; Exosomes; Extracellular Vesicles; Nanocarriers; Precipitation; Proteomics; Size-exclusion chromatography; Ultrafiltration
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2023.136333

[Display omitted] •The extracellular vesicles (EV) were isolated from Citrus lemon juice.•The method combines ultrafiltration and size-exclusion chromatography.•Capillary electrophoresis (CE) was employed for the isolation process monitoring.•The CE was found complementary to protein content assay and particles counting.•The CE was able to detect the impurities and the heterogeneity of EV. 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.