TY - JOUR
T1 - Infrared and Raman spectroscopy for purity assessment of extracellular vesicles
AU - Zini, Jacopo
AU - Saari, Heikki
AU - Ciana, Paolo
AU - Viitala, Tapani
AU - Lõhmus, Andres
AU - Saarinen, Jukka
AU - Yliperttula, Marjo
N1 - Funding Information:
Researchers from the University of Helsinki, Faculty of Natural Sciences at Kumpula campus for allowing the use of the Raman device, Leena Pietilä for the help in cell culturing and BSc Olli-Petteri Nivaro for assisting with EV purifications. We are also thankful to prof Arto Urtti for his valuable help and advice with the revisions of the manuscript. We are thankful for the funding from Academy of Finland projects no 314406 (MY) and no 315406 (MY); Business Finland EVE ecosystem 1842/31/2019 (MY); Aaltonen Foundation (HS).
Funding Information:
Researchers from the University of Helsinki, Faculty of Natural Sciences at Kumpula campus for allowing the use of the Raman device, Leena Pietilä for the help in cell culturing and BSc Olli-Petteri Nivaro for assisting with EV purifications. We are also thankful to prof Arto Urtti for his valuable help and advice with the revisions of the manuscript. We are thankful for the funding from Academy of Finland projects no 314406 (MY) and no 315406 (MY); Business Finland EVE ecosystem 1842/31/2019 (MY); Aaltonen Foundation (HS).
Publisher Copyright:
© 2022
PY - 2022/5/1
Y1 - 2022/5/1
N2 - Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.
AB - Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.
KW - Density gradient
KW - Differential centrifugation
KW - Extracellular vesicles
KW - IR spectroscopy
KW - Nanoparticle tracking analysis
KW - Purification protocols
KW - Raman spectroscopy
KW - Size exclusion chromatography
KW - Ultra-centrifugation
UR - http://www.scopus.com/inward/record.url?scp=85124597824&partnerID=8YFLogxK
U2 - 10.1016/j.ejps.2022.106135
DO - 10.1016/j.ejps.2022.106135
M3 - Article
C2 - 35121019
AN - SCOPUS:85124597824
SN - 0928-0987
VL - 172
JO - European Journal of Pharmaceutical Sciences
JF - European Journal of Pharmaceutical Sciences
M1 - 106135
ER -