scholarly journals Characterization of Extracellular Vesicles Labelled with a Lipophilic Dye Using Fluorescence Nanoparticle Tracking Analysis

Membranes ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 779
Author(s):  
Getnet Midekessa ◽  
Kasun Godakumara ◽  
Keerthie Dissanayake ◽  
Mohammad Mehedi Hasan ◽  
Qurat Ul Ain Reshi ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fluorescent Dyes ◽  
Biological Fluid ◽  
Biological Fluids ◽  
Size Exclusion ◽  
Fluorescent Nanoparticles ◽  
Significant Shift ◽  
Reliable Technique ◽  
Exclusion Chromatography ◽  
Human Choriocarcinoma Cells

Research on extracellular vesicles (EVs) has intensified over the past decade, including fluorescent membrane labeling of EVs. An optimal fluorescent method requires the size of EVs to be preserved after labeling. Lipophilic fluorescent dyes, such as CellMask™ Green (CMG), have been widely used for this purpose. Here, we investigated conditions affecting the optimum CMG labeling of EVs derived from human choriocarcinoma cells (JAr) and different biological fluids using fluorescence NTA (fl-NTA). The effect of CMG labeling on the size, concentration and zeta potential (ZP) on JAr EVs purified with different methods were measured along with biological fluid-derived EVs. With the increase of CMG dye concentration, a significant decrease in the mean size of fluorescent nanoparticles (fl-NPs) was observed. The ZP of fl-NPs originating from JAr cells with the lowest and highest dye concentrations showed a significant shift towards more and less negative ZP values, respectively. Differences in the concentration of fl-NPs were observed for JAr EVs purified using size-exclusion chromatography (SEC) alone and SEC in combination with tangential flow filtration. The proportion of CMG labeling of NPs varied across different biological sources. CMG labeling may be a reliable technique for the detection of EVs using fl-NTA.

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

2021 ◽  
Author(s):  
Vasiliy S. Chernyshev ◽  
Roman N. Chuprov-Netochin ◽  
Ekaterina Tsydenzhapova ◽  
Elena Victorovna Svirshchevskaya ◽  
Rimma A. Poltavtseva ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biological Samples ◽  
Biological Fluids ◽  
Scale Up ◽  
Size Exclusion ◽  
Standard Laboratory ◽  
Laboratory Equipment ◽  
Isolation And Purification ◽  
Depth Filtration ◽  
Exclusion Chromatography

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.

scholarly journals Fibrillar pharmacology of functionalized nanocellulose

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sam Wong ◽  
Simone Alidori ◽  
Barbara P. Mello ◽  
Bryan Aristega Almeida ◽  
David Ulmert ◽  
...  
Keyword(s):  
Aspect Ratio ◽  
Fluorescent Dyes ◽  
Pharmacokinetic Profile ◽  
Water Soluble ◽  
Size Exclusion ◽  
Hydroxyl Groups ◽  
Target Tissue ◽  
Exclusion Chromatography ◽  
Specific Accumulation

AbstractCellulose nanocrystals (CNC) are linear organic nanomaterials derived from an abundant naturally occurring biopolymer resource. Strategic modification of the primary and secondary hydroxyl groups on the CNC introduces amine and iodine group substitution, respectively. The amine groups (0.285 mmol of amine per gram of functionalized CNC (fCNC)) are further reacted with radiometal loaded-chelates or fluorescent dyes as tracers to evaluate the pharmacokinetic profile of the fCNC in vivo. In this way, these nanoscale macromolecules can be covalently functionalized and yield water-soluble and biocompatible fibrillar nanoplatforms for gene, drug and radionuclide delivery in vivo. Transmission electron microscopy of fCNC reveals a length of 162.4 ± 16.3 nm, diameter of 11.2 ± 1.52 nm and aspect ratio of 16.4 ± 1.94 per particle (mean ± SEM) and is confirmed using atomic force microscopy. Size exclusion chromatography of macromolecular fCNC describes a fibrillar molecular behavior as evidenced by retention times typical of late eluting small molecules and functionalized carbon nanotubes. In vivo, greater than 50% of intravenously injected radiolabeled fCNC is excreted in the urine within 1 h post administration and is consistent with the pharmacological profile observed for other rigid, high aspect ratio macromolecules. Tissue distribution of fCNC shows accumulation in kidneys, liver, and spleen (14.6 ± 6.0; 6.1 ± 2.6; and 7.7 ± 1.4% of the injected activity per gram of tissue, respectively) at 72 h post-administration. Confocal fluorescence microscopy reveals cell-specific accumulation in these target tissue sinks. In summary, our findings suggest that functionalized nanocellulose can be used as a potential drug delivery platform for the kidneys.

scholarly journals Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 522
Author(s):  
Elmo W. I. Neuberger ◽  
Barlo Hillen ◽  
Katharina Mayr ◽  
Perikles Simon ◽  
Eva-Maria Krämer-Albers ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Affinity Capture ◽  
Healthy Humans ◽  
Dnase Digestion ◽  
And Topology ◽  
Single Bout ◽  
Exclusion Chromatography ◽  
Free Circulating Dna ◽  
Line Elements

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation

Small ◽  
2022 ◽  
pp. 2104470
Author(s):  
Sheng Yuan Leong ◽  
Hong Boon Ong ◽  
Hui Min Tay ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Plasma Protein ◽  
Protein Separation ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ana Gámez-Valero ◽  
Marta Monguió-Tortajada ◽  
Laura Carreras-Planella ◽  
Marcel·la Franquesa ◽  
Katrin Beyer ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Platelet Derived Extracellular Vesicles Promote in Vitro Gingival Wound Healing.

2021 ◽  
Author(s):  
Miquel Antich-Rosselló ◽  
Marta Munar-Bestard ◽  
Maria Antònia Forteza-Genestra ◽  
Javier Calvo ◽  
Antoni Gayà ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Matrix Remodeling ◽  
Platelet Concentrates ◽  
Ldh Activity ◽  
Exclusion Chromatography ◽  
Good Biocompatibility

Abstract Purpose: Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates like platelet lysate (PL). In this article, the use of extracellular vesicles (EVs) derived from PL and their combination with hyaluronic acid biomaterials (HA) in wound healing was investigated.Methods: EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro for biocompatibility (LDH activity and metabolic activity) and by a wound healing assay and gene expression analysis.Results: EVs and EVs-HA treatments were biocompatible and showed an increase in wound healing compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed in gingival keratinocytes and gingival fibroblasts after the treatment with EVs.Conclusion: EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet derived EVs emerge as promising candidates for oral regeneration with the possibility to combine them with biomaterials in order to enhance their application in clinical use.

scholarly journals Analysis of Serum miRNA in Glioblastoma Patients: CD44-Based Enrichment of Extracellular Vesicles Enhances Specificity for the Prognostic Signature

2020 ◽  
Vol 21 (19) ◽  
pp. 7211
Author(s):  
Theophilos Tzaridis ◽  
Katrin S Reiners ◽  
Johannes Weller ◽  
Daniel Bachurski ◽  
Niklas Schäfer ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Healthy Subjects ◽  
Size Exclusion ◽  
Total Serum ◽  
Prognostic Signature ◽  
Quantitative Real Time Pcr ◽  
Serum Mirna ◽  
Exclusion Chromatography ◽  
Prognostic Stratification ◽  
Prediction Of Prognosis

Glioblastoma is a devastating disease, for which biomarkers allowing a prediction of prognosis are urgently needed. microRNAs have been described as potentially valuable biomarkers in cancer. Here, we studied a panel of microRNAs in extracellular vesicles (EVs) from the serum of glioblastoma patients and evaluated their correlation with the prognosis of these patients. The levels of 15 microRNAs in EVs that were separated by size-exclusion chromatography were studied by quantitative real-time PCR, followed by CD44 immunoprecipitation (SEC + CD44), and compared with those from the total serum of glioblastoma patients (n = 55) and healthy volunteers (n = 10). Compared to total serum, we found evidence for the enrichment of miR-21-3p and miR-106a-5p and, conversely, lower levels of miR-15b-3p, in SEC + CD44 EVs. miR-15b-3p and miR-21-3p were upregulated in glioblastoma patients compared to healthy subjects. A significant correlation with survival of the patients was found for levels of miR-15b-3p in total serum and miR-15b-3p, miR-21-3p, miR-106a-5p, and miR-328-3p in SEC + CD44 EVs. Combining miR-15b-3p in serum or miR-106a-5p in SEC + CD44 EVs with any one of the other three microRNAs in SEC + CD44 EVs allowed for a prognostic stratification of glioblastoma patients. We have thus identified four microRNAs in glioblastoma patients whose levels, in combination, can predict the prognosis for these patients.

scholarly journals Size-Exclusion Chromatography as a Technique for the Investigation of Novel Extracellular Vesicles in Cancer

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3156
Author(s):  
Daniel S. K. Liu ◽  
Flora M. Upton ◽  
Eleanor Rees ◽  
Christopher Limb ◽  
Long R. Jiao ◽  
...  
Keyword(s):  
Systematic Review ◽  
Cancer Cells ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Extracellular Vesicle ◽  
Size Exclusion ◽  
Isolation Method ◽  
Isolation And Purification ◽  
Exclusion Chromatography

Cancer cells release extracellular vesicles, which are a rich target for biomarker discovery and provide a promising mechanism for liquid biopsy. Size-exclusion chromatography (SEC) is an increasingly popular technique, which has been rediscovered for the purposes of extracellular vesicle (EV) isolation and purification from diverse biofluids. A systematic review was undertaken to identify all papers that described size exclusion as their primary EV isolation method in cancer research. In all, 37 papers were identified and discussed, which showcases the breadth of applications in which EVs can be utilised, from proteomics, to RNA, and through to functionality. A range of different methods are highlighted, with Sepharose-based techniques predominating. EVs isolated using SEC are able to identify cancer cells, highlight active pathways in tumourigenesis, clinically distinguish cohorts, and remain functionally active for further experiments.

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