Analysis of Neat Biofluids Obtained During Cardiac Surgery Using Nanoparticle Tracking Analysis: Methodological Considerations

Shearn, Andrew I U; Aday, Sezin; Ben‐Aicha, Soumaya; Carnell-Morris, Pauline; Siupa, Agnieszka; Angelini, Gianni D; Clayton, Aled; Boulanger, Chantal M.; Punjabi, Prakash P; Emanueli, Costanza; Biglino, Giovanni

doi:10.3389/fcell.2020.00367

Cited by 7 publications

(10 citation statements)

References 31 publications

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“…However, similar sizes of lipoproteins could confound the results of EVs quantification. It is suggested that the user strictly follow the protocols and report data accurately [ 96 ].…”

Section: Methods Of Quantificationmentioning

confidence: 99%

Elucidating Methods for Isolation and Quantification of Exosomes: A Review

et al. 2021

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Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.

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“…However, similar sizes of lipoproteins could confound the results of EVs quantification. It is suggested that the user strictly follow the protocols and report data accurately [ 96 ].…”

Section: Methods Of Quantificationmentioning

confidence: 99%

Elucidating Methods for Isolation and Quantification of Exosomes: A Review

et al. 2021

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“…The DMEM cell culture medium is a highly complex matrix that strongly scatters laser light, which can interfere with the scattered light from the particles [ 22 , 26 ]. A full separation of the PS100 beads from proteins in the sample by a precedent EAF4 purification step is a viable way to reduce the interfering protein background leading to individual PS100 particle fractions that are much better amenable to NTA analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Over the past years, both FFF and NTA have significantly grown in popularity for the analysis of complex biological samples including extracellular vesicles such as exosomes [ 23 , 24 , 25 , 26 , 27 , 28 , 29 ], and liposomes [ 4 , 30 , 31 , 32 , 33 ]. It was therefore just a matter of time until both technologies were coupled in order to combine the advantages of both worlds.…”

Section: Introductionmentioning

confidence: 99%

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection

Drexel

Siupa²,

Carnell-Morris³

et al. 2020

Molecules

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Accurate physico-chemical characterization of exosomes and liposomes in biological media is challenging due to the inherent complexity of the sample matrix. An appropriate purification step can significantly reduce matrix interferences, and thus facilitate analysis of such demanding samples. Electrical Asymmetrical Flow Field-Flow Fractionation (EAF4) provides online sample purification while simultaneously enabling access to size and Zeta potential of sample constituents in the size range of approx. 1–1000 nm. Hyphenation of EAF4 with Multi-Angle Light Scattering (MALS) and Nanoparticle Tracking Analysis (NTA) detection adds high resolution size and number concentration information turning this setup into a powerful analytical platform for the comprehensive physico-chemical characterization of such challenging samples. We here present EAF4-MALS hyphenated with NTA for the analysis of liposomes and exosomes in complex, biological media. Coupling of the two systems was realized using a flow splitter to deliver the sample at an appropriate flow speed for the NTA measurement. After a proof-of-concept study using polystyrene nanoparticles, the combined setup was successfully applied to analyze liposomes and exosomes spiked into cell culture medium and rabbit serum, respectively. Obtained results highlight the benefits of the EAF4-MALS-NTA platform to study the behavior of these promising drug delivery vesicles under in vivo like conditions.

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“…The NTA apparatus NanoSight NS300 (Malvern Panalytical, Malvern, UK), with NTA software version 3.2 (Malvern Instruments), was employed to characterize the size and concentration of the sEVs isolated from the pericyte CM. 53 For the analyses, sEVs were diluted 5 times in Dulbecco’s phosphate-buffered saline (DPBS). Then, each sample was filtered using 0.22 μm Millex syringe filter unit (Millipore, Burlington, MA, USA) and injected into the flow cell of the NanoSight NS300.…”

Section: Methodsmentioning

confidence: 99%

Bioinspired artificial exosomes based on lipid nanoparticles carrying let-7b-5p promote angiogenesis in vitro and in vivo

et al. 2021

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MicroRNAs (miRNAs) regulate gene expression by post-transcriptional inhibition of target genes. Proangiogenic small extracellular vesicles (sEVs; popularly identified with the name "exosomes") with a composite cargo of miRNAs are secreted by cultured stem cells and present in human biological fluids. Lipid nanoparticles (LNPs) represent an advanced platform for clinically approved delivery of RNA therapeutics. In this study, we aimed to (1) identify the miRNAs responsible for sEV-induced angiogenesis; (2) develop the prototype of bioinspired "artificial exosomes" (AEs) combining LNPs with a proangiogenic miRNA, and (3) validate the angiogenic potential of the bioinspired AEs. We previously reported that human sEVs from bone marrow (BM)-CD34 + cells and pericardial fluid (PF) are proangiogenic. Here, we have shown that sEVs secreted from saphenous vein pericytes and BM mesenchymal stem cells also promote angiogenesis. Analysis of miRNA datasets available in-house or datamined from GEO identified the let-7 family as common miRNA signature of the proangiogenic sEVs. LNPs with either hsa-let-7b-5p or cyanine 5 (Cy5)-conjugated Caenorhabditis elegans miR-39 (Cy5-cel-miR-39; control miRNA) were prepared using microfluidic micromixing. let-7b-5p-AEs did not cause toxicity and transferred functionally active let-7b-5p to recipient endothelial cells (ECs). let-7b-AEs also improved EC survival under hypoxia and angiogenesis in vitro and in vivo. Bioinspired proangiogenic AEs could be further developed into innovative nanomedicine products targeting ischemic diseases.

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Analysis of Neat Biofluids Obtained During Cardiac Surgery Using Nanoparticle Tracking Analysis: Methodological Considerations

Cited by 7 publications

References 31 publications

Elucidating Methods for Isolation and Quantification of Exosomes: A Review

Elucidating Methods for Isolation and Quantification of Exosomes: A Review

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection

Bioinspired artificial exosomes based on lipid nanoparticles carrying let-7b-5p promote angiogenesis in vitro and in vivo

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