Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma

Woo, Hyun‐Kyung; Cho, Young-Kwon; Lee, Chang Yeol; Lee, Haeun; Castro, Cesar M.; Lee, Hakho

doi:10.7150/thno.69094

Cited by 34 publications

(23 citation statements)

References 34 publications

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“…We also conducted several pre-analytical experiments to determine which EV preparations were most helpful. Specifically, we compared ultracentrifugation, traditional 17 , and dual-mode size exclusion chromatography (eDMC) 30 to identify practical methods that yielded high amounts of EV. Our results show that the enhanced dual size-exclusion mode yielded high quantities of EV, was efficient, cost-effective, and allowed processing small sample sizes ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We first compared different EV isolation methods to determine which ones would be practical for a clinical workflow (small sample volume, high throughput, fast separation, and low cost). Specifically, EV obtained from i) cell line supernatant or pooled plasma and) were isolated using the following methods: ultracentrifugation, qEV IZON size exclusion chromatography, dual-mode chromatography (DMC) 17 and enhanced dual-mode chromatography (eDMC) 30 .…”

Section: Methodsmentioning

confidence: 99%

“…DMC 17 and eDMC 30 EV purification were done according to the methods of each respective publication. Briefly, size exclusion columns were prepared with an 11 µm nylon net filter (NY1102500, Millipore) placed in the bottom of a 10 mL syringe (BD 302995).…”

Section: Ev Isolationmentioning

confidence: 99%

“…Participation of the side chain in the subsequent cascade elimination achieves quantitative release with a cleavage half-life

Fig. S3: Pre-analytical purification. EV were purified by ultracentrifugation, size exclusion chromatography (IZON qEV single), dual-mode chromatography 17 , and enhanced dual-mode chromatography 30 . EV were compared for total protein concentration (Qubit), nanoparticle tracking analysis (Nanosight), Western blot (ApoB100, TSG101, CD63), and MASEV analysis.…”

Section: Supplemental Informationmentioning

confidence: 99%

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Multiplexed analysis of a single EV (MASEV) reveals unique biomarker composition with diagnostic impact

Spitzberg

Ferguson

Yang

et al. 2022

Preprint

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Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of single EV technologies. Highly multiplexed analyses of single EVs have been challenging to implement beyond a few colors during spectral sensing. We use a multiplexed analysis of the single EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that i) several “ubiquitous” markers are much less common than believed; ii) that multiple biomarkers concur in single vesicles but only in small fractions, iii) that affinity purification can lead to loss of rare EV subtypes, and iv) that deep profiling allows detailed analysis of EV potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity. Abstract Figure Graphical abstract Multiplexed analysis of single EV (MASEV) allows robust protein profiling at the single EV level, a prerequisite for early cancer detection or organ of origin determination.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ev Isolationmentioning

confidence: 99%

“…Participation of the side chain in the subsequent cascade elimination achieves quantitative release with a cleavage half-life

Section: Supplemental Informationmentioning

confidence: 99%

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Multiplexed analysis of a single EV (MASEV) reveals unique biomarker composition with diagnostic impact

Spitzberg

Ferguson

Yang

et al. 2022

Preprint

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“…The above targeting effect is mainly determined by the molecules on the surface of EVs, and EVs inherit some proteins from parental cells when they are secreted [51], so the cell source is an important factor influencing targeting (Fig. 2).…”

Section: Cell Sources Point the Way For Evs To Gomentioning

confidence: 99%

Nanoengineering facilitating the target mission: targeted extracellular vesicles delivery systems design

et al. 2022

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Precision medicine has put forward the proposition of "precision targeting" for modern drug delivery systems. Inspired by techniques from biology, pharmaceutical sciences, and nanoengineering, numerous targeted drug delivery systems have been developed in recent decades. But the large-scale applications of these systems are limited due to unsatisfactory targeting efficiency, cytotoxicity, easy removability, and instability. As such, the natural endogenous cargo delivery vehicle—extracellular vesicles (EVs)—have sparked significant interest for its unique inherent targeting properties, biocompatibility, transmembrane ability, and circulatory stability. The membranes of EVs are enriched for receptors or ligands that interact with target cells, which endows them with inherent targeting mission. However, most of the natural therapeutic EVs face the fate of being cleared by macrophages, resulting in off-target. Therefore, the specificity of natural EVs delivery systems urgently needs to be further improved. In this review, we comprehensively summarize the inherent homing mechanisms of EVs and the effects of the donor cell source and administration route on targeting specificity. We then go over nanoengineering techniques that modify EVs for improving specific targeting, such as source cell alteration and modification of EVs surface. We also highlight the auxiliary strategies to enhance specificity by changing the external environment, such as magnetic and photothermal. Furthermore, contemporary issues such as the lack of a gold standard for assessing targeting efficiency are discussed. This review will provide new insights into the development of precision medicine delivery systems. Graphical Abstract

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Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization

Hsia,

You,

Politis

et al. 2023

Advanced Biology

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Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike‐in into healthy donor plasma samples is performed to further validate biofluid‐derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC‐derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA‐isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

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Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma

Cited by 34 publications

References 34 publications

Multiplexed analysis of a single EV (MASEV) reveals unique biomarker composition with diagnostic impact

Multiplexed analysis of a single EV (MASEV) reveals unique biomarker composition with diagnostic impact

Nanoengineering facilitating the target mission: targeted extracellular vesicles delivery systems design

Rigorous Comparison of Extracellular Vesicle Processing to Enhance Downstream Analysis for Glioblastoma Characterization

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