Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution.
EV cargo sorting
ExoView
exosomes
extracellular vesicles
nanoflow cytometry
protein delivery vehicle
single‐molecule localization microscopy
Journal
Journal of extracellular vesicles
ISSN: 2001-3078
Titre abrégé: J Extracell Vesicles
Pays: United States
ID NLM: 101610479
Informations de publication
Date de publication:
08 2021
08 2021
Historique:
received:
14
10
2020
revised:
20
06
2021
accepted:
13
07
2021
entrez:
11
8
2021
pubmed:
12
8
2021
medline:
12
8
2021
Statut:
ppublish
Résumé
Extracellular Vesicles (EVs) have been intensively explored for therapeutic delivery of proteins. However, methods to quantify cargo proteins loaded into engineered EVs are lacking. Here, we describe a workflow for EV analysis at the single-vesicle and single-molecule level to accurately quantify the efficiency of different EV-sorting proteins in promoting cargo loading into EVs. Expi293F cells were engineered to express EV-sorting proteins fused to green fluorescent protein (GFP). High levels of GFP loading into secreted EVs was confirmed by Western blotting for specific EV-sorting domains, but quantitative single-vesicle analysis by Nanoflow cytometry detected GFP in less than half of the particles analysed, reflecting EV heterogeneity. Anti-tetraspanin EV immunostaining in ExoView confirmed a heterogeneous GFP distribution in distinct subpopulations of CD63
Identifiants
pubmed: 34377376
doi: 10.1002/jev2.12130
pii: JEV212130
pmc: PMC8329990
doi:
Substances chimiques
Recombinant Fusion Proteins
0
Tetraspanins
0
Green Fluorescent Proteins
147336-22-9
Types de publication
Journal Article
Langues
eng
Pagination
e12130Informations de copyright
© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.
Références
ACS Nano. 2021 Feb 23;15(2):3212-3227
pubmed: 33470092
Methods. 2020 May 1;177:15-26
pubmed: 31978536
Proteomics. 2009 Nov;9(21):4997-5000
pubmed: 19810033
Org Biomol Chem. 2015 Oct 14;13(38):9775-82
pubmed: 26264754
Science. 2020 Feb 7;367(6478):
pubmed: 32029601
Nat Rev Cardiol. 2020 Nov;17(11):685-697
pubmed: 32483304
J Extracell Vesicles. 2016 May 13;5:31027
pubmed: 27189348
Nanomedicine (Lond). 2019 Nov;14(21):2799-2814
pubmed: 31724479
J Res Natl Inst Stand Technol. 2002 Feb 01;107(1):83-91
pubmed: 27446720
ACS Nano. 2018 Jan 23;12(1):494-503
pubmed: 29286635
Protein Eng Des Sel. 2011 Sep;24(9):671-8
pubmed: 21596686
J Extracell Vesicles. 2013 Mar 19;2:
pubmed: 24009897
J Extracell Vesicles. 2021 Jan;10(3):e12052
pubmed: 33473263
Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2020 Mar;12(2):e1594
pubmed: 31840427
Biochim Biophys Acta Mol Cell Res. 2018 Dec;1865(12):1891-1900
pubmed: 30290236
Nano Lett. 2019 Jan 9;19(1):19-28
pubmed: 30517011
J Extracell Vesicles. 2020 Apr 17;9(1):1751428
pubmed: 32363015
Cancer Res. 2008 Feb 15;68(4):1228-35
pubmed: 18281500
Nanoscale. 2018 Aug 7;10(29):14230-14244
pubmed: 30010165
Nat Biotechnol. 2011 Apr;29(4):341-5
pubmed: 21423189
ACS Nano. 2019 Sep 24;13(9):10499-10511
pubmed: 31469961
J Extracell Vesicles. 2015 Dec 07;4:28533
pubmed: 26649679
Sci Rep. 2016 Nov 17;6:37246
pubmed: 27853258
J Extracell Vesicles. 2021 Apr;10(6):e12079
pubmed: 33850608
Nat Commun. 2019 Sep 12;10(1):4136
pubmed: 31515491
J Thromb Haemost. 2010 Feb;8(2):315-23
pubmed: 19840362
Curr Diab Rep. 2019 Jul 31;19(9):69
pubmed: 31367976
Nat Methods. 2017 Feb 28;14(3):228-232
pubmed: 28245209
PLoS Biol. 2012;10(12):e1001450
pubmed: 23271954
Neural Regen Res. 2020 Apr;15(4):586-596
pubmed: 31638080
J Extracell Vesicles. 2019 Feb 11;8(1):1573051
pubmed: 30788083
Mol Ther. 2018 Feb 7;26(2):634-647
pubmed: 29221804
Sci Rep. 2017 May 12;7(1):1878
pubmed: 28500324
Nat Rev Mol Cell Biol. 2018 Apr;19(4):213-228
pubmed: 29339798
Adv Drug Deliv Rev. 2020;159:332-343
pubmed: 32305351
Sci Rep. 2019 Jul 11;9(1):10041
pubmed: 31296886
Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):E968-77
pubmed: 26858453
Oncotarget. 2015 Mar 10;6(7):4953-67
pubmed: 25669974
Blood Cells Mol Dis. 2005 Sep-Oct;35(2):158-68
pubmed: 16087368
J Extracell Vesicles. 2019 Sep 18;8(1):1663043
pubmed: 31579435
J Extracell Vesicles. 2018 Mar 01;7(1):1440131
pubmed: 29535849
Acta Pharmacol Sin. 2017 Jun;38(6):754-763
pubmed: 28392567
Biol Proced Online. 2017 Sep 15;19:11
pubmed: 28932173
J Extracell Vesicles. 2018 Nov 23;7(1):1535750
pubmed: 30637094
ACS Nano. 2018 Jan 23;12(1):671-680
pubmed: 29300458
Nanoscale. 2019 Apr 4;11(14):6990-7001
pubmed: 30916672
J Extracell Vesicles. 2016 Mar 14;5:31053
pubmed: 26979463
Theranostics. 2019 Oct 17;9(26):8001-8017
pubmed: 31754377
J Extracell Vesicles. 2019 Mar 21;8(1):1587567
pubmed: 30949308
J Extracell Vesicles. 2019 Feb 27;8(1):1579541
pubmed: 30834072
Sci Rep. 2016 Nov 08;6:36338
pubmed: 27821849
Nanomedicine. 2015 May;11(4):879-83
pubmed: 25659648
Biomater Sci. 2020 May 19;8(10):2966-2976
pubmed: 32342086
Mol Ther. 2021 May 5;29(5):1729-1743
pubmed: 33484965
J Extracell Vesicles. 2016 Jul 13;5:31295
pubmed: 27421995
ACS Nano. 2017 Jan 24;11(1):69-83
pubmed: 28068069