Proteomic and Physicochemical Analysis of Erythrocyte Membrane Vesicles from Fresh and Frozen Blood.
6 affiliations
- 1Department of Nanostructured Materials, K7, Jožef Stefan Institute, Ljubljana1000, Slovenia.
- 2Jožef Stefan International Postgraduate School, Jožef Stefan Institute , Ljubljana1000, Slovenia.
- 3Department of Biochemistry and Molecular and Structural Biology, B1, Jožef Stefan Institute, Ljubljana1000, Slovenia.
- 4Department of Molecular and Biomedical Sciences, B2, Jožef Stefan Institute, Ljubljana1000, Slovenia.
- 5Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana1000, Slovenia.
- 6Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana1000, Slovenia.
Abstract
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.
Related
- Effect of Pure EPA on Erythrocyte Membrane Fatty Acids During Supplementation and Washout: A Randomized Controlled Trial
- Erythrocyte Membrane β-Adrenoreactivity as a Systemic Adrenergic Phenotype Associated with Ventricular Tachyarrhythmia in Heart Failure Patients Undergoing Cardiac Resynchronization Therapy
- A New Method for the Reconstitution of Membrane Proteins into Giant Unilamellar Vesicles
- Tissue‐Specific Isolation of Hepatocyte Extracellular Vesicles From Human Plasma Enables Tissue‐Resolved Proteomic and miRNA Profiling
- Tuberculous Meningitis Alters the Proteomic Landscape of Brain-Derived Extracellular Vesicles.
BibTeX
@article{tomic2026proteomic,
title = {{Proteomic and Physicochemical Analysis of Erythrocyte Membrane Vesicles from Fresh and Frozen Blood.}},
author = {Katarina Tomić and Sara Ivanovski and Jernej Šribar and Daša Čebulj and Samuel Žvanut and Metka Lenassi and Marko Fonović and Igor Križaj and Nina Kostevšek},
journal = {Journal of proteome research},
year = {2026},
month = oct,
doi = {10.1021/acs.jproteome.6c00535},
url = {https://europepmc.org/article/MED/42918115},
note = {CC BY},
}
RIS
TY - JOUR
TI - Proteomic and Physicochemical Analysis of Erythrocyte Membrane Vesicles from Fresh and Frozen Blood.
AU - Katarina Tomić
AU - Sara Ivanovski
AU - Jernej Šribar
AU - Daša Čebulj
AU - Samuel Žvanut
AU - Metka Lenassi
AU - Marko Fonović
AU - Igor Križaj
AU - Nina Kostevšek
PY - 2026
DA - 2026/10/01
JO - Journal of proteome research
DO - 10.1021/acs.jproteome.6c00535
UR - https://europepmc.org/article/MED/42918115
AB - 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.
SN - 1535-3893
SN - 1535-3907
ER -