Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

AbstractAssessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Download Full-text

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery

Scientific Reports ◽

10.1038/s41598-020-78422-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Marija Holcar ◽

Jana Ferdin ◽

Simona Sitar ◽

Magda Tušek-Žnidarič ◽

Vita Dolžan ◽

...

Keyword(s):

Biomarker Discovery ◽

Plasma Source ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Enzyme Linked Immunosorbent Assay ◽

Enrichment Method ◽

Transmission Electron ◽

Relative Standard ◽

Exclusion Chromatography ◽

Reliable Quantification

AbstractHuman plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation—sUC) and size- (size exclusion chromatography—SEC) based methods. According to nanoparticle tracking analysis (NTA), asymmetrical flow field-flow fractionation coupled to detectors (AF4-UV-MALS), miRNA quantification, transmission electron microscopy and enzyme-linked immunosorbent assay, enrichment of EVs from plasma with sUC method lead to high purity of EVs in the samples. High nanoparticle concentrations after SEC resulted from substantial contamination with lipoproteins and other aggregates of EV-like sizes that importantly affect downstream EV quantification. Additionally, sUC EV-enrichment method linked to quantification with NTA or AF4-UV-MALS is repeatable, as the relative standard deviation of EV size measured in independently processed samples from the same plasma source was 5.4% and 2.1% when analyzed by NTA or AF4-UV-MALS, respectively. In conclusion, the sUC EV-enrichment method is compatible with reliable measurement of concentration and size of EVs from plasma and should in the future be tested on larger cohorts in relation to different diseases. This is one of the first studies using AF4-UV-MALS to quantify EVs in blood plasma, which opens new possible clinical utility for the technique.

Download Full-text

Mass-Spectrometry Based Proteome Comparison of Extracellular Vesicle Isolation Methods: Comparison of ME-kit, Size-Exclusion Chromatography, and High-Speed Centrifugation

Biomedicines ◽

10.3390/biomedicines8080246 ◽

2020 ◽

Vol 8 (8) ◽

pp. 246 ◽

Cited By ~ 2

Author(s):

Anders Askeland ◽

Anne Borup ◽

Ole Østergaard ◽

Jesper V. Olsen ◽

Sigrid M. Lund ◽

...

Keyword(s):

Mass Spectrometry ◽

Size Exclusion Chromatography ◽

High Speed ◽

Proteome Analysis ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Label Free ◽

Protein Biomarkers ◽

Transmission Electron ◽

Exclusion Chromatography

Extracellular vesicles (EVs) are small membrane-enclosed particles released by cells under various conditions specific to cells’ biological states. Hence, mass-spectrometry (MS) based proteome analysis of EVs in plasma has gained much attention as a method to discover novel protein biomarkers. MS analysis of EVs in plasma is challenging and EV isolation is usually necessary. Therefore, we compared differences in abundance, subtypes, and contamination for EVs isolated by high-speed centrifugation, size exclusion chromatography (SEC), and peptide-affinity precipitation (PAP/ME kit) for subsequent MS-based proteome analysis. Successful EV isolation was evaluated by nanoparticle-tracking analysis, immunoblotting, and transmission electron microscopy, while EV abundance, EV subtypes, and contamination was evaluated by label-free tandem MS. High-speed centrifugation and SEC isolates showed high EV abundance at the expense of contamination by non-EV proteins and lipoproteins, respectively. These two methods also resulted in EVs of a similar type, however, with smaller EVs in SEC isolates. PAP isolates had a relatively low EV abundance and high contamination. We consider high-speed centrifugation and SEC suitable as EV isolation for MS biomarker studies, where the choice between the two should depend on the scientific questions and whether the focus is on larger or smaller EVs or a combination of both.

Download Full-text

A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽

10.3390/vaccines9020157 ◽

2021 ◽

Vol 9 (2) ◽

pp. 157

Author(s):

Kinga Böszörményi ◽

Janet Hirsch ◽

Gwendoline Kiemenyi Kayere ◽

Zahra Fagrouch ◽

Nicole Heijmans ◽

...

Keyword(s):

Envelope Protein ◽

Neutralizing Antibodies ◽

Size Exclusion ◽

Vitro Assay ◽

Usutu Virus ◽

Transmission Electron ◽

Exclusion Chromatography ◽

Efficacious Vaccine ◽

Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

Download Full-text

Serum Extracellular Vesicle-Derived circHIPK3 and circSMARCA5 Are Two Novel Diagnostic Biomarkers for Glioblastoma Multiforme

Pharmaceuticals ◽

10.3390/ph14070618 ◽

2021 ◽

Vol 14 (7) ◽

pp. 618

Author(s):

Michele Stella ◽

Luca Falzone ◽

Angela Caponnetto ◽

Giuseppe Gattuso ◽

Cristina Barbagallo ◽

...

Keyword(s):

Glioblastoma Multiforme ◽

Roc Analysis ◽

Characteristic Curve ◽

Area Under The Curve ◽

Digital Pcr ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Circular Rnas ◽

Diagnostic Biomarkers ◽

Exclusion Chromatography

Glioblastoma multiforme (GBM) is the most frequent and deadly human brain cancer. Early diagnosis through non-invasive biomarkers may render GBM more easily treatable, improving the prognosis of this currently incurable disease. We suggest the use of serum extracellular vesicle (sEV)-derived circular RNAs (circRNAs) as highly stable minimally invasive diagnostic biomarkers for GBM diagnosis. EVs were isolated by size exclusion chromatography from sera of 23 GBM and 5 grade 3 glioma (GIII) patients, and 10 unaffected controls (UC). The expression of two candidate circRNAs (circSMARCA5 and circHIPK3) was assayed by droplet digital PCR. CircSMARCA5 and circHIPK3 were significantly less abundant in sEVs from GBM patients with respect to UC (fold-change (FC) of −2.15 and −1.92, respectively) and GIII (FC of −1.75 and −1.4, respectively). Receiver operating characteristic curve (ROC) analysis, based on the expression of sEV-derived circSMARCA5 and circHIPK3, allowed us to distinguish GBM from UC (area under the curve (AUC) 0.823 (0.667–0.979) and 0.855 (0.704 to 1.000), with a 95% confidence interval (CI), respectively). Multivariable ROC analysis, performed by combining the expression of sEV-derived circSMARCA5 and circHIPK3 with preoperative neutrophil to lymphocyte (NLR), platelet to lymphocyte (PLR) and lymphocyte to monocyte (LMR) ratios, three known diagnostic and prognostic GBM markers, allowed an improvement in the GBM diagnostic accuracy (AUC 0.901 (0.7912 to 1.000), 95% CI). Our data suggest sEV-derived circSMARCA5 and circHIPK3 as good diagnostic biomarkers for GBM, especially when associated with preoperative NLR, PLR and LMR.

Download Full-text

Fibrillar pharmacology of functionalized nanocellulose

Scientific Reports ◽

10.1038/s41598-020-79592-5 ◽

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.

Download Full-text

Electromagnetic Piezoelectric Acoustic Sensor Detection of Extracellular Vesicles through Interaction with Detached Vesicle Proteins

Biosensors ◽

10.3390/bios10110173 ◽

2020 ◽

Vol 10 (11) ◽

pp. 173

Author(s):

Loránd Románszki ◽

Zoltán Varga ◽

Judith Mihály ◽

Zsófia Keresztes ◽

Michael Thompson

Keyword(s):

Storage Period ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Frequency Change ◽

Acoustic Sensor ◽

C Storage ◽

Thaw Cycle ◽

Signal Change ◽

Freeze Thaw Cycle ◽

Exclusion Chromatography

An electromagnetic piezoelectric acoustic sensor (EMPAS) was used to study the non-specific adsorption of human red blood cell-derived extracellular vesicle preparations. Vesicle storage history (temperature and duration) highly affected the obtained results: The signal change, namely the frequency decrease of the crystal measured at 20 °C, was negligibly small (<1 s−2) when the vesicle solutions had previously been stored at 4 °C, and was in the order of 10 s−2 when the vesicle solutions had been stored at −30 °C. Moreover, the rate of frequency decrease increased exponentially with the storage time at −30 °C. Upon a 4 °C storage period following the −30 °C storage period of the same sample, the measured frequency decrease dropped, suggesting a partial relaxation of the system. The results are explained by the disintegration of the vesicles triggered by the freeze–thaw cycle, likely due to the detachment of proteins from the vesicle surface as was proved by size-exclusion chromatography. Surface modification of the sensor crystal provided the possibility of signal enhancement, as the maximum rate of the frequency change for the same vesicle concentrations was higher on hydrophobic, octadecyl trichlorosilane–modified quartz than on hydrophilic, bare quartz. The EMPAS signal has been associated with the amount of detached proteins, which in turn is proportional to the originating vesicle concentration.

Download Full-text

A Method for the Isolation of Exosomes from Human and Bovine Milk

Journal of Nutrition and Metabolism ◽

10.1155/2019/5764740 ◽

2019 ◽

Vol 2019 ◽

pp. 1-6 ◽

Cited By ~ 10

Author(s):

Kanchan Vaswani ◽

Murray D. Mitchell ◽

Olivia J. Holland ◽

Yong Qin Koh ◽

Rebecca J. Hill ◽

...

Keyword(s):

Human Milk ◽

Particle Concentration ◽

Infant Nutrition ◽

Bovine Milk ◽

Size Exclusion ◽

Enrichment Method ◽

Transmission Electron ◽

Exclusion Chromatography ◽

Technical Study ◽

Natural Means

Scope. Milk provides a natural means of nutrient supply to infants. Exosomes are an important component of milk that are not only being studied for their promise in translational medicine but also in infant nutrition. They also play important roles in intercellular communication and immune function in mammary glands and are able to transfer their materials to the recipient. Therefore, the isolation of high-quality exosomes is an important aspect of exosome research. Methods and Results. This study is a technical study, which provides a detailed methodology for the isolation and enrichment of exosomes from milk. In this study, we evaluate the suitability of using the exosome enrichment method that we have recently published for bovine milk, on human milk. We initially isolated extracellular vesicles from human and bovine milk on a fresh set of samples, using ultracentrifugation, and then exosomes were subsequently enriched via size exclusion chromatography (SEC). Following isolation and enrichment, exosomes from both species were characterized by particle concentration (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), and the presence of exosomal markers (immunoblotting and mass spectrometry using information dependant acquisition (IDA)). The key exosomal characteristics of spherical/donut-shaped morphology, the presence of exosomal markers, e.g., FLOT-1 and the tetraspanins, CD9 and CD81), and particle concentration were confirmed in both human and bovine milk exosomes. Conclusion. We conclude that our robust exosome enrichment method, previously published for bovine milk, is suitable for use on human milk.

Download Full-text

Low-resolution structures of modular nanotransporters shed light on their functional activity

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320013765 ◽

2020 ◽

Vol 76 (12) ◽

pp. 1270-1279

Author(s):

Yuri V. Khramtsov ◽

Anastasiia D. Vlasova ◽

Alexey V. Vlasov ◽

Andrey A. Rosenkranz ◽

Alexey V. Ulasov ◽

...

Keyword(s):

Structural Information ◽

3D Structure ◽

Cytotoxic Agents ◽

Endosomal Escape ◽

Size Exclusion ◽

Target Cells ◽

Low Resolution ◽

Transmission Electron ◽

Exclusion Chromatography ◽

A Cell

Modular nanotransporters (MNTs) are multifunctional chimeric polypeptides for the multistep transport of locally acting cytotoxic agents into the nuclei of cancer target cells. MNTs consist of several polypeptide domains (functional modules) for the recognition of a cell-surface internalizable receptor, pH-dependent endosomal escape and subsequent transport into the nucleus through the nuclear pores. MNTs are a promising means for cancer treatment. As has been shown previously, all of the modules of MNTs retain their functionalities. Despite their importance, there is no structural information available about these chimeric polypeptides, which hampers the creation of new MNT variants. Here, a low-resolution 3D structure of an MNT is presented which was obtained by atomic force microscopy, transmission electron microscopy and small-angle X-ray scattering coupled to size-exclusion chromatography. The data suggest that the MNT can adopt two main conformations, but in both conformations the protein N- and C-termini are distanced and do not influence each other. The change in the MNT conformation during acidification of the medium was also studied. It was shown that the fraction of the elongated conformation increases upon acidification. The results of this work will be useful for the development of MNTs that are suitable for clinical trials and possible therapeutic applications.

Download Full-text

Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

10.1101/2021.04.01.437817 ◽

2021 ◽

Author(s):

Marvin Droste ◽

Tobias Tertel ◽

Stefanie Jeruschke ◽

Robin Dittrich ◽

Evangelia Kontopoulou ◽

...

Keyword(s):

Flow Cytometry ◽

Method Comparison ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Western Blots ◽

Preparation Methods ◽

Imaging Flow Cytometry ◽

Particle Yield ◽

Exclusion Chromatography ◽

Ultra Filtration

Extracellular vesicles (EVs) from several body fluids, including urine, appear as promising biomarkers. Within the last decade, numerous groups have compared the efficacy of EV preparation protocols. Frequently, the efficacy of EV preparation methods is judged by the recovery of particles as estimated by conventional nanoparticle tracking analysis (NTA) or other particle quantification devices. Here, at the example of different urinary EV (uEV) preparation methods, we determined the particle yield in obtained samples with conventional NTA, analyzed their EV content by imaging flow cytometry (IFCM) and quantified the intensity of TSG101 and the contaminant protein uromodulin (UMOD) in Western blots. Our results demonstrate a correlation among CD9-positive objects detected by IFCM and TSG101 Western blot intensities, while particle numbers as determined by NTA correlated with the amount of UMOD. Consequently, our results question the reliability of conventional NTA analyses for identifying the optimal EV preparation method. Here, in our method comparison, a combination of size exclusion chromatography followed by ultra-filtration showed the highest CD9-positive object and TSG101 protein recovery, and in relation to the number of CD9-positive objects, the lowest amount of UMOD contamination.

Download Full-text