Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Corso, Giulia; Heusermann, Wolf; Trojer, Dominic; Görgens, André; Steib, Emmanuelle; Voshol, Johannes; Graff, Alexandra; Genoud, Christel; Lee, Yi; Hean, Justin; Nordin, Joel Z.; Wiklander, Oscar P. B.; Andaloussi, Samir El; Meisner‐Kober, Nicole

doi:10.1080/20013078.2019.1663043

Cited by 117 publications

(162 citation statements)

References 77 publications

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“…However, the enzymes used for EV labelling until now suffer from poor stability and low quantum yield, thus limiting their utility for various in vitro and in vivo applications. In addition, engineering strategies for EV loading used in previous studies often resulted in the labelling of only a minor fraction of the EV population [35]. Therefore, we concentrated on addressing these two critical issues.…”

Section: Screening Luciferase Species For Efficient Ev Labellingmentioning

confidence: 99%

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging

Gupta

Liang

Павлова

et al. 2020

J of Extracellular Vesicle

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143

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Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or realtime imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

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Section: Screening Luciferase Species For Efficient Ev Labellingmentioning

confidence: 99%

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging

Gupta

Liang

Павлова

et al. 2020

J of Extracellular Vesicle

Self Cite

143

139

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“…However, genetic modification may change the property of cells and even exosomes. Uneven loading of probe proteins is another issue that needs to be addressed [68,69].…”

Section: Surface Modificationmentioning

confidence: 99%

Advances in Analysis of Biodistribution of Exosomes by Molecular Imaging

Yi¹,

Lee²,

Kim

et al. 2020

IJMS

141

131

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Exosomes are nano-sized membranous vesicles produced by nearly all types of cells. Since exosome-like vesicles are produced in an evolutionarily conserved manner for information and function transfer from the originating cells to recipient cells, an increasing number of studies have focused on their application as therapeutic agents, drug delivery vehicles, and diagnostic targets. Analysis of the in vivo distribution of exosomes is a prerequisite for the development of exosome-based therapeutics and drug delivery vehicles with accurate prediction of therapeutic dose and potential side effects. Various attempts to evaluate the biodistribution of exosomes obtained from different sources have been reported. In this review, we examined the current trends and the advantages and disadvantages of the methods used to determine the biodistribution of exosomes by molecular imaging. We also reviewed 29 publications to compare the methods employed to isolate, analyze, and label exosomes as well as to determine the biodistribution of labeled exosomes.

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“…As such, this method should not be used to determine the precise number of proteins of interest in a given EV or used to infer that a given protein is completely absent from an EV or population of EVs. This approach is best suited to studies using fluorescent protein fusions to proteins of interest and imaging approaches capable of single molecule detection [22]. However, the ability to define EV populations of interest, according to their incorporation of endogenously expressed, native proteins, provides a platform by which the composition of EVs containing specific cargoes of interest can be interrogated.…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we establish a method to determine the relative levels of exosomal markers and cargoes present in EV populations using quantitative fluorescent microscopy. Prior studies using fluorescent fusion proteins or dyes to label EVs have demonstrated that microscopic methods can be used to effectively visualise and quantify fluorescent signals associated with individual EVs [ 22 – 24 ]. An advantage of the EV-MAC approach described here is the relative ease by which EVs can be spinnoculated onto coverslips and subsequently analysed using conventional immunofluorescent imaging techniques [ 29 , 31 ].…”

Section: Discussionmentioning

confidence: 99%

Cargo and cell‐specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis

Burbidge

Zwikelmaier

Cook

et al. 2020

J of Extracellular Vesicle

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Extracellular vesicles (EVs) have been implicated in a wide variety of biological activities, have been implicated in the pathogenesis of numerous diseases, and have been proposed to serve as potential biomarkers of disease in human patients and animal models. However, characterization of EV populations is often performed using methods that do not account for the heterogeneity of EV populations and require comparatively large sample sizes to facilitate analysis. Here, we describe an imaging-based method that allows for the multiplexed characterization of EV populations at the single EV level following centrifugation of EV populations directly onto cover slips, allowing comprehensive analysis of EV populations with relatively small samples. We observe that canonical EV markers are present on subsets of EVs which differ substantially in a producer cell and cargo specific fashion, including differences in EVs containing different HIV-1 proteins previously reported to be incorporated into pathogenic EVs. We also describe a lectin binding assay to interrogate EVs based on their glycan content, which we observe to change in response to pharmacological modulation of secretory autophagy pathways. These studies collectively reveal that a multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations and/or to interrogate small subsets of EVs of interest within larger EV populations in biological samples.

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Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Abstract: Kober (2019) Systematic characterization of extracellular vesicle sorting domains and quantification at the single moleculesingle vesicle level by fluorescence correlation spectroscopy and single particle imaging,

Cited by 117 publications

References 77 publications

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging

Advances in Analysis of Biodistribution of Exosomes by Molecular Imaging

Cargo and cell‐specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis

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