Aiming to Compare Apples to Apples: Analysis of Extracellular Vesicles and Other Nanosized Particles by Flow Cytometry

Görgens, André; Nolan, John P.

doi:10.1002/cyto.a.24173

Cited by 5 publications

(2 citation statements)

References 19 publications

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“…A multi-institutional team led by Louise Laurent (University of California San Diego, contact PI) will focus on using immunoaffinity separation (IMS) for isolation of exRNA carrier subclasses (CSs). This transdisciplinary team, with expertise in exRNA and lipoprotein biology ( Li et al., 2018b ), exRNA biomarker discovery ( Chen-Plotkin et al., 2018 ; Srinivasan et al., 2020 ; Xiao et al., 2017 ), exRNA sequencing ( Rodosthenous et al., 2020 ; Srinivasan et al., 2019 ), low-input proteomics ( Tsai et al., 2021 ), lipidomics ( Wang et al., 2021b ), and flow cytometry ( Görgens and Nolan, 2020 ; Nolan and Duggan, 2018 ; Welsh et al., 2020 ), will work together to develop and apply a rigorous, reproducible, efficient, scalable, and cost-effective workflow for preparative isolation of subtypes of EVs, RNPs, and LPPs for downstream-omics analysis. This team’s initial approach will be to use antibodies conjugated to magnetic nanoparticles, but they will also explore the use of multiplex bead-based flow sorting for even more efficient separation of CSs.…”

Section: Introductionmentioning

confidence: 99%

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research

Mateescu

Jones

Alexander³

et al. 2022

iScience

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Section: Introductionmentioning

confidence: 99%

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research

Mateescu

Jones

Alexander³

et al. 2022

iScience

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“…For example, a commonly used parameter, encapsulation efficiency, was defined as the ratio of the overall nanocarrier‐incorporated cargo quantity to the initial input (Shen et al., 2017 ; J. Tang, Zhang, et al., 2018 ). However, EVs are naturally heterogeneous in particle size and molecular content, and often contaminated with co‐precipitated proteins and other impurities, which can lead to the unequal drug encapsulation (Gorgens & Nolan, 2020 ; Ramirez et al., 2018 ; Roy et al., 2019 ). Hence, the unknown heterogeneity necessitates quantitative characterization at the single‐particle level for evaluation and even purification of cargo‐loaded EVs (Ferguson & Nguyen, 2016 ; Shao et al., 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour

Chen

Sun

Wang

et al. 2021

J of Extracellular Vesicle

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Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named “Sonication and Extrusion‐assisted Active Loading” (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin‐loaded milk‐derived EVs (Dox‐mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10‐fold enhancement of drug encapsulation efficiency compared with passive loading. Single‐particle analysis by nano‐flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox‐mEVs which would otherwise be overlooked by bulk‐based approaches. Correlation analysis between doxorubicin auto‐fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid‐enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid‐probe‐ and immuno‐mediated magnetic isolation techniques to selectively remove the contaminants of non‐lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL‐prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as‐developed cargo‐loading approach and nano‐flow cytometry‐based characterization method will provide an instructive insight in the development of EV‐based delivery systems.

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Biogenesis and Identification of Extracellular Vesicles

Ding,

Zhang,

Zhao

et al. 2023

Biomedical Applications of Extracellular Vesicles

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Aiming to Compare Apples to Apples: Analysis of Extracellular Vesicles and Other Nanosized Particles by Flow Cytometry

Cited by 5 publications

References 19 publications

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour

Biogenesis and Identification of Extracellular Vesicles

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