Formation and Evaluation of a Two-Phase Polymer System in Human Plasma as a Method for Extracellular Nanovesicle Isolation

Slyusarenko, Maria; Nikiforova, Nadezhda; Sidina, Elena; Nazarova, Inga; Егоров, Владимир В.; Garmay, Yuri; Merdalimova, Anastasiia; Yevlampieva, N. P.; Gorin, Dmitry A.; Malek, Anastasia

doi:10.3390/polym13030458

Cited by 19 publications

(24 citation statements)

References 41 publications

(65 reference statements)

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“…Raman spectrum of EVs isolated by DF (Figure S5) agrees with previous reports by Krafft et al 50 and Slyusarenko et al 51 and contains the expected peaks assigned in Table S3. Specifically, the peak near 704 cm -1 corresponds to cholesterol and cholesterol esters.…”

Section: Ev Isolation By Depth-filtrationsupporting

confidence: 89%

Asymmetric depth-filtration – a versatile and scalable approach for isolation and purification of extracellular vesicles

Chernyshev

Chuprov‐Netochin

Tsydenzhapova

et al. 2021

Preprint

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A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

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Section: Ev Isolation By Depth-filtrationsupporting

confidence: 89%

Asymmetric depth-filtration – a versatile and scalable approach for isolation and purification of extracellular vesicles

Chernyshev

Chuprov‐Netochin

Tsydenzhapova

et al. 2021

Preprint

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“…The small extracellular nanovesicles (SEVs) were isolated from the PPP using a two-polymer system, previously described in [ 19 ]. The polymers, polyethylene glycol (PEG, 20 kDa), and dextran (DEX, 450–650 kDa) (both from Sigma-Aldrich, St. Louis, MO, USA), were dissolved in PBS at concentrations of 30% and 35%, respectively.…”

Section: Methodsmentioning

confidence: 99%

AuNP Aptasensor for Hodgkin Lymphoma Monitoring

Slyusarenko

Shalaev²,

Valitova³

et al. 2022

Biosensors

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A liquid biopsy based on circulating small extracellular vesicles (SEVs) has not yet been used in routine clinical practice due to the lack of reliable analytic technologies. Recent studies have demonstrated the great diagnostic potential of nanozyme-based systems for the detection of SEV markers. Here, we hypothesize that CD30-positive Hodgkin and Reed–Sternberg (HRS) cells secrete CD30 + SEVs; therefore, the relative amount of circulating CD30 + SEVs might reflect classical forms of Hodgkin lymphoma (cHL) activity and can be measured by using a nanozyme-based technique. A AuNP aptasensor analytics system was created using aurum nanoparticles (AuNPs) with peroxidase activity. Sensing was mediated by competing properties of DNA aptamers to attach onto surface of AuNPs inhibiting their enzymatic activity and to bind specific markers on SEVs surface. An enzymatic activity of AuNPs was evaluated through the color reaction. The study included characterization of the components of the analytic system and its functionality using transmission and scanning electron microscopy, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and spectrophotometry. AuNP aptasensor analytics were optimized to quantify plasma CD30 + SEVs. The developed method allowed us to differentiate healthy donors and cHL patients. The results of the CD30 + SEV quantification in the plasma of cHL patients were compared with the results of disease activity assessment by positron emission tomography/computed tomography (PET-CT) scanning, revealing a strong positive correlation. Moreover, two cycles of chemotherapy resulted in a statistically significant decrease in CD30 + SEVs in the plasma of cHL patients. The proposed AuNP aptasensor system presents a promising new approach for monitoring cHL patients and can be modified for the diagnostic testing of other diseases.

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“…The total population of nanovesicles was isolated from pellet pure plasma (PPP) samples using a two-phase polymer system [ 13 ]. Unless otherwise specified, SEVs were isolated from 2 mL of PPP and resuspended in 100 μL of PBS.…”

Section: Resultsmentioning

confidence: 99%

“…The total EV population was isolated from PPP using a two-phase polymer system as previously described [ 13 ], with slight modifications. Briefly, a solution of dextran (450–650 kDa, 1.5%) and polyethylene glycol (35 kDa, 3.5%) (both from Sigma-Aldrich, St. Louis, MO, USA) was prepared in the required volume of PPP.…”

Section: Methodsmentioning

confidence: 99%

“…To explore this hypothesis, we developed a method to isolate PSMA(+)SEVs using super-paramagnetic beads functionalized by PSMA-specific DNA aptamer [ 12 ] via the click reaction. In combination with the previously described two-phase polymer system for plasma SEV isolation [ 13 ], the proposed technique provides a scaffold for an effective and economic approach to vesicle-based liquid biopsy. We analyzed four PC-associated miRNAs in PSMA(+)SEVs and the total population of plasma vesicles, and compared the diagnostic potency of these tests.…”

Section: Introductionmentioning

confidence: 99%

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A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles

Zabegina¹,

Nazarova²,

Nikiforova³

et al. 2021

Cells

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Vesicular miRNA has emerged as a promising marker for various types of cancer, including prostate cancer (PC). In the advanced stage of PC, the cancer-cell-derived small extracellular vesicles (SEVs) may constitute a significant portion of circulating vesicles and may mediate a detectable change in the plasma vesicular miRNA profile. However, SEVs secreted by small tumor in the prostate gland constitute a tiny fraction of circulating vesicles and cause undetectable miRNA pattern changes. Thus, the isolation and miRNA profiling of a specific prostate-derived fraction of SEVs can improve the diagnostic potency of the methods based on vesicular miRNA analysis. Prostate-specific membrane antigen (PSMA) was selected as a marker of prostate-derived SEVs. Super-paramagnetic beads (SPMBs) were functionalized by PSMA-binding DNA aptamer (PSMA–Apt) via a click reaction. The efficacy of SPMB–PSMA–Apt complex formation and PSMA(+)SEVs capture were assayed by flow cytometry. miRNA was isolated from the total population of SEVs and PSMA(+)SEVs of PC patients (n = 55) and healthy donors (n = 30). Four PC-related miRNAs (miR-145, miR-451a, miR-143, and miR-221) were assayed by RT-PCR. The click chemistry allowed fixing DNA aptamers onto the surface of SPMB with an efficacy of up to 89.9%. The developed method more effectively isolates PSMA(+)SEVs than relevant antibody-based technology. The analysis of PC-related miRNA in the fraction of PSMA(+)SEVs was more sensitive and revealed distinct diagnostic potency (AUC: miR-145, 0.76; miR-221, 0.7; miR-451a, 0.65; and miR-141, 0.64) than analysis of the total SEV population. Thus, isolation of prostate-specific SEVs followed by analysis of vesicular miRNA might be a promising PC diagnosis method.

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Formation and Evaluation of a Two-Phase Polymer System in Human Plasma as a Method for Extracellular Nanovesicle Isolation

Cited by 19 publications

References 41 publications

Asymmetric depth-filtration – a versatile and scalable approach for isolation and purification of extracellular vesicles

Asymmetric depth-filtration – a versatile and scalable approach for isolation and purification of extracellular vesicles

AuNP Aptasensor for Hodgkin Lymphoma Monitoring

A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles

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