Proteomic and physicochemical analysis of erythrocyte membrane vesicles from fresh and frozen blood

Tomić, Katarina (author), Ivanovski, Sara (author), Šribar, Jernej (author), Čebulj, Daša (author), Žvanut, Samuel (author), Lenassi, Metka (author), Fonović, Marko (author), Križaj, Igor (author), Kostevšek, Nina (author)

Erythrocyte membrane vesicles (EMVs) have emerged as promising nanocarriers for drug delivery due to their biocompatibility and immune evasion through retention of erythrocyte surface proteins. The main limitation is their dependence on freshly drawn blood, which limits batch production. Long-term storage of EDTA-anticoagulated blood at −80°C without cryoprotectant represents an accessible alternative, but its effect on EMV physicochemical properties and protein composition has not been investigated. We prepared EMVs from fresh and frozen blood and characterized the resulting fractions by dynamic light scattering, nano-flow cytometry, cryo-transmission electron microscopy, negative staining transmission electron microscopy, and quantitative mass spectrometry-based proteomics. EMV size, surface charge, and morphology did not differ significantly between fresh and frozen preparations. Extrusion effectively normalized particle size despite increased heterogeneity in frozen ghost cell fractions, and both preparations yielded structurally intact vesicles after ultracentrifugation. At the proteomic level, freezing reduced proteomic complexity in membrane fractions and selectively depleted cytosol-associated proteins. The core membrane proteome, including Band 3, remained stable. However, glycophorin A showed the largest reduction in frozen preparations, and CD47 levels were reduced in the ultracentrifuged fraction. These differences may be critical for applications where preservation of specific surface proteins is required, such as immune evasion or targeted drug delivery. In some cases, frozen blood is potentially a viable source for EMV production when immediate processing is not feasible.

• Language: English
• Keywords: membrane vesicles, erythrocyte membrane vesicles, blood storage, proteomics, electron microscopy, nano-flow cytometry, membranes, vesicles, peptides, proteins, cells, anatomy
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology; MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: Str. 5534-5548
• Numbering: Vol. 25, iss. 10
• PID: 20.500.12556/RUL-189719
• UDC: 577
• ISSN on article: 1535-3907
• DOI: 10.1021/acs.jproteome.6c00535
• COBISS.SI-ID: 291636483
• Publication date in RUL: 13.10.2026

Record is a part of a journal

• Title: Journal of proteome research
• Shortened title: J. proteome res.
• Publisher: American Chemical Society
• ISSN: 1535-3907
• COBISS.SI-ID: 513576473

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: membranski vezikli, kri, eritrociti