Red blood cell-derived extracellular vesicles as biomaterials: the opportunity of freezing-induced accelerated aging
Source abstract
Abstract
Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, owing to their intrinsic biocompatibility, immune-evasion properties, and minimal oncogenic risk. However, their broader application is currently limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage stability. By combining discontinuous sucrose density gradient separation with high-resolution interferometric nanoparticle tracking analysis, we identified a sharp bimodal size distribution of vesicles in freshly prepared samples. We then tracked how long-term storage at -80 °C drove their conversion into a monomodal distribution. To reproduce these conditions in a shorter time frame, we developed an "accelerated-ageing" protocol based on freeze-thaw cycles that generates RBC-EV samples with homogeneous density, size distribution, and biological activity, effectively replicating the properties of preparations stored for six months at -80 °C. This new vesicle population remains stable and retains membrane integrity and cellular internalization capacity, as confirmed by surface-associated enzymatic activity assays and uptake tests in cancer cell lines. These results suggest that freezing-induced "accelerated ageing" represents an effective method for the optimization and standardization of RBC-EVs as building blocks for biomaterial and bioengineering applications.
Source: openalex_inverted_index · Quality: candidate unverified · Source record ↗
Research context
OpenAlex topics: Extracellular vesicles in disease · Erythrocyte Function and Pathophysiology · Nanoplatforms for cancer theranostics
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
Affiliations
Dept. of Medical and Surgical Specialties, Radiological Sciences and Public Health (DSMC), University of Brescia, Brescia, Italy
Center for Colloid and Surface Science, (CSGI), Sesto Fiorentino (FI), Italy
ASST Spedali Civili di Brescia, Dept. of Emergency, Spedali Civili University Hospital, Brescia, Italy
Dept. of Molecular and Translational Medicine (DMMT), University of Brescia, Brescia, Italy
IRCCS Fondazione Don Carlo Gnocchi ONLUS, Milano, Italy
Max-Planck-Institut für die Physik des Lichts, 91058 Erlangen, Germany
Max-Planck-Zentrum für Physik und Medizin, 91058 Erlangen, Germany
“Angelo Nocivelli” Institute for Molecular Medicine, ASST Spedali Civili, 25123, Brescia, Italy
Istituto di Scienze e Tecnologie Chimiche “Giulio Natta” – National Research Council of Italy (SCITEC-CNR), Milan, Italy
Department Physik, Friedrich-Alexander Universität Erlangen, Nürnberg, 91058 Erlangen, Germany
Laboratory of Stem Cells, U.O.C. of Immunohaematology and Transfusion Medicine, Santo Spirito Hospital, Pescara, Italy
Section of Medical Genetics and Cytogenetics, ASST Spedali Civili of Brescia, 25123 Brescia, Italy
National Center for Gene Therapy and Drugs based on RNA Technology, Padua, Italy
References (89)
Show source-provided references
1. Théry · J. Extracell. Vesicles · 2018 · 7 · 153575010.1080/20013078.2018.1535750 ↗
2. Petraroia · Cell Death Dis. · 2023 · 14 · 68110.1038/s41419-023-06212-1 ↗
3. Raab-Traub · Nat. Rev. Microbiol. · 2017 · 15 · 55910.1038/nrmicro.2017.60 ↗
4. Kalluri · Science · 2020 · 367 · eaau697710.1126/science.aau6977 ↗
5. Kumar · Mol. Pharm. · 2023 · 20 · 382910.1021/acs.molpharmaceut.3c00363 ↗
6. Herrmann · Nat. Nanotechnol. · 2021 · 16 · 74810.1038/s41565-021-00931-2 ↗
7. Pham · Cell Stress · 2018 · 2 · 23910.15698/cst2018.09.155 ↗
8. Musicò · Nanoscale Adv. · 2023 · 5 · 470310.1039/D3NA00280B ↗
9. Pham · J. Extracell. Vesicles · 2021 · 10 · e1205710.1002/jev2.12057 ↗
10. Pham · Pharmacol. Res. · 2023 · 188 · 10666510.1016/j.phrs.2023.106665 ↗
11. Nguyen · ACS Nano · 2023 · 17 · 518710.1021/acsnano.2c11965 ↗
12. Repsold · BioMed Res. Int. · 2018 · 2018 · 940561710.1155/2018/9405617 ↗
13. Dodson · Blood Cells · 1987 · 12 · 555
14. Hasse · Biochem. Pharmacol. · 2021 · 192 · 11466710.1016/j.bcp.2021.114667 ↗
15. Nguyen · Cell. Physiol. Biochem. · 2016 · 38 · 108510.1159/000443059 ↗
16. Ebeyer-Masotta · Transfus. Apher. Sci. · 2024 · 63 · 10389110.1016/j.transci.2024.103891 ↗
17. Gamonet · Blood Adv. · 2020 · 4 · 552710.1182/bloodadvances.2020001658 ↗
18. Usman · Nat. Commun. · 2018 · 9 · 235910.1038/s41467-018-04791-8 ↗
19. Prudent · Transfus. Apher. Sci. · 2015 · 53 · 15310.1016/j.transci.2015.10.012 ↗
20. Salzer · Transfusion · 2008 · 48 · 45110.1111/j.1537-2995.2007.01549.x ↗
21. de Oliveira · J. Extracell. Biol. · 2023 · 2 · e10710.1002/jex2.107 ↗
22. Kusuma · Front. Pharmacol. · 2018 · 9 · 119910.3389/fphar.2018.01199 ↗
23. Almizraq · Transfus. Apher. Sci. · 2017 · 56 · 25410.1016/j.transci.2017.03.007 ↗
24. Bebesi · Sci. Rep. · 2022 · 12 · 97710.1038/s41598-022-04915-7 ↗
25. Straat · Transfus. Med. Hemother. · 2016 · 43 · 30210.1159/000442681 ↗
26. Paolini · J. Extracell. Biol. · 2022 · 1 · e6310.1002/jex2.63 ↗
27. Nelson · Nanomedicine · 2020 · 15 · 214910.2217/nnm-2020-0206 ↗
28. ICH Q1A(R2) Stability Testing of New Drug Substances and Products , Food Drug Adm. , 2003 , https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q1ar2-stability-testing-new-drug-substances-and-products
29. ICH Q1A (R2) Stability testing of new drug substances and drug products – Scientific guideline . European Medicines Agency (EMA) , 2003 , https://www.ema.Europa.eu/en/ich-q1a-r2-stability-testing-new-drug-substances-drug-products-scientific-guideline#current-effective-version-8899
30. Saugstad · J. Extracell. Vesicles · 2017 · 6 · 131757710.1080/20013078.2017.1317577 ↗
31. Tian · J. Extracell. Vesicles · 2020 · 9 · 169702810.1080/20013078.2019.1697028 ↗
32. Gori · J. Extracell. Vesicles · 2020 · 9 · 175142810.1080/20013078.2020.1751428 ↗
33. Ridolfi · Anal. Chem. · 2020 · 92 · 1027410.1021/acs.analchem.9b05716 ↗
34. Raposo · J. Cell Biol. · 2013 · 200 · 37310.1083/jcb.201211138 ↗
35. Carlomagno · Front. Bioeng. Biotechnol. · 2021 · 9 · 64061710.3389/fbioe.2021.640617 ↗
36. Paolini · J. Extracell. Vesicles · 2020 · 9 · 174117410.1080/20013078.2020.1741174 ↗
37. Koponen · Biosens. Bioelectron. · 2020 · 168 · 11251010.1016/j.bios.2020.112510 ↗
38. Kashkanova · Nat. Methods · 2022 · 19 · 58610.1038/s41592-022-01460-z ↗
39. Kashkanova · J. Extracell. Vesicles · 2023 · 12 · e1234810.1002/jev2.12348 ↗
40. Van Deun · Nat. Methods · 2017 · 14 · 22810.1038/nmeth.4185 ↗
41. Grossi · Int. J. Mol. Med. · 2020 · 47 · 53310.3892/ijmm.2020.4806 ↗
42. Radeghieri · J. Extracell. Biol. · 2022 · 1 · e5710.1002/jex2.57 ↗
43. Paolini · Traffic · 2011 · 12 · 160410.1111/j.1600-0854.2011.01262.x ↗
44. Di Noto · Ann. Clin. Biochem. Int. J. Lab. Med. · 2015 · 52 · 32710.1177/0004563214553808 ↗
45. Alvisi · Biochem. J. · 2018 · 475 · 145510.1042/BCJ20170897 ↗
46. Salvi · Int. J. Mol. Med. · 2019 · 2 · 768
47. Shah · PLoS One · 2016 · 11 · e015320010.1371/journal.pone.0153200 ↗
48. Maiolo · Anal. Chem. · 2015 · 87 · 416810.1021/ac504861d ↗
49. Zendrini · Front. Bioeng. Biotechnol. · 2020 · 7 · 45210.3389/fbioe.2019.00452 ↗
50. Borup · J. Extracell. Biol. · 2022 · 1 · e4110.1002/jex2.41 ↗
51. Kashkanova · ACS Nano · 2024 · 18 · 1916110.1021/acsnano.4c04396 ↗
52. Ho · J. Neurochem. · 1969 · 16 · 150510.1111/j.1471-4159.1969.tb09905.x ↗
53. Savina · J. Cell Sci. · 2002 · 115 · 250510.1242/jcs.115.12.2505 ↗
54. Welsh · J. Extracell. Vesicles · 2024 · 13 · e1240410.1002/jev2.12404 ↗
55. Lucien · J. Extracell. Vesicles · 2023 · 12 · e1238510.1002/jev2.12385 ↗
56. Gori · Adv. Sci. · 2024 · 11 · 240053310.1002/advs.202400533 ↗
57. Musicò · Nanoscale Horiz. · 2025 · 10 · 10410.1039/D4NH00320A ↗
58. Caponnetto · Nanomedicine · 2017 · 13 · 101110.1016/j.nano.2016.12.009 ↗
59. V.Mangolini , A.Radeghieri , S.Piva , S.Cattaneo , M.Brucale , F.Valle , et al. , Universal protocol to separate and compare Extracellular Vesicles from human plasma and skeletal muscle biopsy, bioRxiv , 2025, preprint, 10.1101/2024.02.19.580950v210.1101/2024.02.19.580950v2 ↗
60. Huang · PLoS One · 2013 · 8 · e8170810.1371/journal.pone.0081708 ↗
61. Cheng · Ecotoxicol. Environ. Saf. · 2024 · 284 · 11686410.1016/j.ecoenv.2024.116864 ↗
62. Shuklov · Mater. Today Chem. · 2020 · 18 · 10035710.1016/j.mtchem.2020.100357 ↗
63. Calvini · Restaurator · 2006 · 27 · 275
64. L.Fambri , R.Caria , F.Atzori , R.Ceccato and D.Lorenzi , et al. , Controlled Ageing and Degradation of Selected Plastics in Marine Environment: 12 Months of Follow-up , in Proceedings of the 2nd International Conference on Microplastic Pollution in the Mediterranean Sea , ed. M. Cocca , E. Di Pace , M.E Errico , G. Gentile , A. Montarsolo and R. Mossotti , Springer International Publishing , Cham , 2020 , pp. 89–100
65. Kockott · Polym. Test. · 2022 · 110 · 10755010.1016/j.polymertesting.2022.107550 ↗
66. Fenollosa · Plant Methods · 2020 · 16 · 6410.1186/s13007-020-00607-3 ↗
67. Waterman · Int. J. Pharm. · 2005 · 293 · 10110.1016/j.ijpharm.2004.12.013 ↗
68. Zou · Cellulose · 1996 · 3 · 24310.1007/BF02228805 ↗
69. Gelibter · J. Extracell. Vesicles · 2022 · 11 · e1216210.1002/jev2.12162 ↗
70. Gubicza · Mater. Chem. Phys. · 2013 · 138 · 44910.1016/j.matchemphys.2013.01.012 ↗
71. Huang · Anticancer Res. · 2010 · 30 · 46310.1158/0008-5472.CAN-09-2501 ↗
72. Yun · Nanomaterials · 2023 · 13 · 261810.3390/nano13192618 ↗
73. Sivanantham · Life · 2022 · 12 · 69710.3390/life12050697 ↗
74. Grosfils · J. Colloid Interface Sci. · 2023 · 652 · 138110.1016/j.jcis.2023.08.059 ↗
75. Svetina · Anat. Rec. · 2002 · 268 · 21510.1002/ar.10156 ↗
76. Maas · Trends Cell Biol. · 2017 · 27 · 17210.1016/j.tcb.2016.11.003 ↗
77. Altamura · Synth. Biol. · 2018 · 3 · ysy01110.1093/synbio/ysy011 ↗
78. Wang · Anim. Reprod. Sci. · 2024 · 270 · 10757610.1016/j.anireprosci.2024.107576 ↗
79. Andar · Pharm. Res. · 2014 · 31 · 40110.1007/s11095-013-1171-8 ↗
80. Jurney · J. Control Release · 2017 · 245 · 17010.1016/j.jconrel.2016.11.033 ↗
81. Daniels · Vox Sang. · 2007 · 93 · 33110.1111/j.1423-0410.2007.00970.x ↗
82. Liao · J. Extracell. Vesicles · 2019 · 8 · 162859210.1080/20013078.2019.1628592 ↗
83. Tóth · J. Extracell. Vesicles · 2021 · 10 · e1214010.1002/jev2.12140 ↗
84. Tassoni · Nanomedicine · 2025 · 20 · 201310.1080/17435889.2025.2538426 ↗
85. Carney · Nat. Nanotechnol. · 2025 · 20 · 1410.1038/s41565-024-01774-3 ↗
86. Manno · Nat. Rev. Bioeng. · 2024 · 3 · 6810.1038/s44222-024-00255-5 ↗
87. Voigt · Nat. Commun. · 2020 · 11 · 637910.1038/s41467-020-20122-2 ↗
88. Garner · Essays Biochem. · 2021 · 65 · 79110.1042/EBC20200059 ↗
89. Ciferri · Nanotheranostics · 2025 · 9 · 29910.7150/ntno.116627 ↗