TY - JOUR
T1 - Importance of isolation method on characteristics and bioactivity of extracellular vesicles from tomatoes
AU - Huang, Ziyu
AU - Nielsen, Søren Drud Heydary
AU - Whitehead, Bradley
AU - Nejsum, Peter
AU - Corredig, Milena
AU - Rasmussen, Martin Krøyer
N1 - Publisher Copyright:
© 2024 The Authors
PY - 2024/5
Y1 - 2024/5
N2 - Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.
AB - Plant-derived extracellular vesicles (EVs), including exosomes, are usually isolated by ultracentrifugation, followed by density gradient purification, without critically assessing the obtained material. The lack of characterization makes it difficult to compare findings between studies or ascribe effects to specific compounds. Thus, the study aimed to make a comprehensive characterization of the different subpopulations of EVs isolated using a range of ultracentrifugation forces and subsequent glucose gradient purification. Results showed that EVs obtained using 100,000 × g had a bilayer membrane structure, the highest abundance of proteins and microRNAs, and characteristics resembling exosomes. Additional gradient purification resulted in three distinct bands of EVs. EVs from the middle band had most characteristics resembling exosomes, such as the bilayer structure and high abundance of proteins and microRNAs. Furthermore, proteomics identified most proteins to be common between the different fractions, while band-specific proteins were also observed. In addition, the bioactive properties of the EVs also differed between bands, as assessed by evaluating the viability and proliferation of intestinal cells (Caco-2). In conclusion, the present findings clearly demonstrate that the characteristics and bioactive properties of plant EVs are highly dependable on isolation methods, which advocate for the development of standardized methods.
KW - Exosome-like nanovesicle
KW - Non-coding RNA
KW - Proliferation
KW - Sucrose gradient
KW - Tomato fruits
KW - Ultra-centrifugal force
KW - Viability
UR - http://www.scopus.com/inward/record.url?scp=85186074752&partnerID=8YFLogxK
U2 - 10.1016/j.jfca.2024.106064
DO - 10.1016/j.jfca.2024.106064
M3 - Journal article
AN - SCOPUS:85186074752
SN - 0889-1575
VL - 129
JO - Journal of Food Composition and Analysis
JF - Journal of Food Composition and Analysis
M1 - 106064
ER -