Acidification of blood plasma facilitates the separation and analysis of extracellular vesicles

Mladenović, Danilo; Khamari, Delaram; Kittel, Ágnes; Koort, Kairi; Buzás, Edit I.; Zarovni, Nataša

doi:10.1016/j.jtha.2023.01.007

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…By restoring to pH 8, captured EVs can be released into a free solution. Similar acidification treatment on EV samples for improving isolation has been previously reported 59–62 , evidencing a rentention of the EV membrane fidelity at the pH used here. ExCy’s capture-release process is a simple, fast, low-cost EV isolation workflow requiring only a magnet, acidic buffer and dye, centrifuge, and pipette.…”

Section: Introductionsupporting

confidence: 85%

Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment

Greenberg,

Ali,

Schmittgen

et al. 2024

Preprint

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Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.

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Section: Introductionsupporting

confidence: 85%

Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment

Greenberg,

Ali,

Schmittgen

et al. 2024

Preprint

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“…Another recently described pre‐treatment method is the acidification of plasma to facilitate the separation of EVs from lipoproteins. [ 77 ] The acidification method combined with SEC results in ≈20% reduction in ApoB levels per particle, [ 77 ] while SMA pre‐treatment according to the accelerated protocol (one‐hour) followed by SEC resulted in a 91% reduction in ApoB/particle (Figure 5C), demonstrating the superiority of the method described in this study. Additionally, with the acidification method, protein‐based contaminants are likely to increase due to pH‐induced denaturation and aggregation.…”

Section: Discussionmentioning

confidence: 67%

Chemically‐Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification

Iannotta,

A.,

Lai

et al. 2023

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Extracellular vesicles (EVs) are nanosized biomolecular packages involved in intercellular communication. EVs are released by all cells, making them broadly applicable as therapeutic, diagnostic, and mechanistic components in (patho)physiology. Sample purity is critical for correctly attributing observed effects to EVs and for maximizing therapeutic and diagnostic performance. Lipoprotein contaminants represent a major challenge for sample purity. Lipoproteins are approximately six orders of magnitude more abundant in the blood circulation and overlap in size, shape, and density with EVs. This study represents the first example of an EV purification method based on the chemically‐induced breakdown of lipoproteins. Specifically, a styrene‐maleic acid (SMA) copolymer is used to selectively breakdown lipoproteins, enabling subsequent size‐based separation of the breakdown products from plasma EVs. The use of the polymer followed by tangential flow filtration or size‐exclusion chromatography results in improved EV yield, preservation of EV morphology, increased EV markers, and reduced contaminant markers. SMA‐based EV purification enables improved fluorescent labeling, reduces interactions with macrophages, and enhances accuracy, sensitivity, and specificity to detect EV biomarkers, indicating benefits for various downstream applications. In conclusion, SMA is a simple and effective method to improve the purity and yield of plasma‐derived EVs, which favorably impacts downstream applications.

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“…Given the number of bacteria in the gut (in 10 13 range) and estimated productivity of bacteria in exponential phase of ~10 EVs per bacteria [22], over 10 14 bacterial EVs can be produced daily, some of which have been shown to cross intestinal barrier and diffuse into blood circulation in mice and humans. Although absolute measurement of outer membrane protein A (OmpA) positive vesicles in human blood has not been done yet, some relative quantifications of OmpA signals in blood EV extracts, and considerations on average size of OMVs (<200 nm) over average size of a blood EV (~75 nm) [16,21], indicate that the number of OMVs could reach up to 10% of all circulating vesicles, or 2x10 9 particles per mL of plasma (Table 1).…”

Section: Breaking Down the Milieu Of Bloodmentioning

confidence: 99%

“…Total protein content in EVs has been differently estimated across years, using diverse experimental and computational approaches. For instance, calculations based on size exclusion chromatography (SEC)-purified plasma, suggest that EVs could constitute approximately ~0.1% of the plasma proteome [8,16,36,37]. However, this figure is likely an overestimation due to a co-isolation of different particles such as LPs or protein aggregates.…”

Section: Breaking Down the Milieu Of Bloodmentioning

confidence: 99%

“…Notably, similar estimates, in the same order of magnitude, of the EV concentration in blood with respect to this number obtained by single vesicle detection, was made based on the mass of isolated EVs. Reported protein content of SEC-purified EVs obtained from 1 mL of plasma is in the range of 30-90 μg [8,16,36,37]. We can estimate the mass of a single EV by considering the proportion with respect to a cell size and mass, as previously reviewed [47].…”

Section: Breaking Down the Milieu Of Bloodmentioning

confidence: 99%

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Stoichiometric constraints for detection of EV-borne biomarkers in blood

Zarovni,

Mladenović,

Brambilla

et al. 2024

Preprint

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Stochiometric issues, encompassing both the quantity and heterogeneity of extracellular vesicles (EVs) derived from tumor or other tissues in blood, pose important challenges across various stages of biomarker discovery and detection, affecting the integrity of data, introducing losses and artifacts during blood processing, EV purification, and analysis. These challenges shape the diagnostic utility of EVs especially within the framework of established and emerging methodologies. By addressing these challenges, we aim to delineate crucial parameters and requirements for tumor-specific EV detection, or more precisely, for tumor identification via EV based assays. Our endeavor involves a comprehensive examination of the layers that mask or confound the traceability of EV markers such as nucleic acids and proteins, and focus on "Low abundance - low occupancy" scenario. Finally, we evaluate the advantages vs. limitations of digital technologies over more conventional bulk assays, suggesting that the combined use of both to capture and interpret the EV signals, in particular the EV surface displayed proteins, may ultimately provide quantitative information on their absolute abundance and distribution.

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Acidification of blood plasma facilitates the separation and analysis of extracellular vesicles

Cited by 5 publications

References 28 publications

Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment

Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment

Chemically‐Induced Lipoprotein Breakdown for Improved Extracellular Vesicle Purification

Stoichiometric constraints for detection of EV-borne biomarkers in blood

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