20.500.12556/RUL-189719 Proteomic and physicochemical analysis of erythrocyte membrane vesicles from fresh and frozen blood 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. membrane vesicles erythrocyte membrane vesicles blood storage proteomics electron microscopy nano-flow cytometry membranes vesicles peptides proteins cells anatomy membranski vezikli kri eritrociti true false true Angleški jezik Slovenski jezik Članek v reviji 2026-10-13 16:00:29 2026-10-13 16:00:37 2026-10-14 04:14:07 0000-00-00 00:00:00 2026 0 0 Str. 5534-5548 iss. 10 Vol. 25 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577 1535-3907 10.1021/acs.jproteome.6c00535 291636483 acs.jproteome.6c00535.pdf acs.jproteome.6c00535.pdf 1 EFEFD4C82C3173426EDAE927DF2BE8FD d64eb97aa48fb498542d1096619c8bbeca894eaa12d69415ace573cc96948c66 e9274c5e-c70e-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=250474 https://pubs.acs.org/jprobs/article/doi/10.1021/acs.jproteome.6c00535/5426715/Proteomic-and-Physicochemical-Analysis-of 1 504f3354-c70e-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=250473 Fakulteta za kemijo in kemijsko tehnologijo Medicinska fakulteta 0 0 0