Nanoparticle-Enabled Multiplexed Electrochemical Immunoassay for Detection of Surface Proteins on Extracellular Vesicles

2021 ◽  
Author(s):  
Seonhwa Lee ◽  
Bruno P. Crulhas ◽  
Sonja Suvakov ◽  
Stanislav V. Verkhoturov ◽  
Dmitriy S. Verkhoturov ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Surface Proteins ◽  
Electrochemical Immunoassay

scholarly journals Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

2021 ◽  
Vol 10 (2) ◽  
pp. 319
Author(s):  
Hee Cheol Yang ◽  
Won Jong Rhee
Keyword(s):  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Molecular Beacon ◽  
Disease Diagnosis ◽  
Single Step ◽  
Flow Cytometer ◽  
Surface Proteins ◽  
Biomarker Detection ◽  
In Situ Detection

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals Extracellular Vesicles in Human Preterm Colostrum Inhibit Infection by Human Cytomegalovirus In Vitro

2020 ◽  
Vol 8 (7) ◽  
pp. 1087
Author(s):  
Manuela Donalisio ◽  
Simona Cirrincione ◽  
Massimo Rittà ◽  
Cristina Lamberti ◽  
Andrea Civra ◽  
...  
Keyword(s):  
Breast Milk ◽  
Extracellular Vesicles ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Protective Role ◽  
Surface Proteins ◽  
Human Colostrum ◽  
Clinical Illness

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.

Proteome Analysis of Colorectal Cancer Cell Line SW480 Released Extracellular Vesicles

2018 ◽  
Vol 762 ◽  
pp. 3-7
Author(s):  
Ilva Nakurte ◽  
Kaspars Jekabsons ◽  
Elina Zandberga ◽  
Arturs Abols ◽  
Aija Line ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Extracellular Vesicles ◽  
Cancer Cell Line ◽  
Surface Protein ◽  
Proteome Analysis ◽  
Research Area ◽  
Surface Proteins ◽  
Colorectal Cancer Cell Line ◽  
Protein Fragments ◽  
Early Cancer Diagnosis

The detection and profiling of disease-specific extracellular vesicles (EVs) from body fluids has been challenging research area during recent years. However, the question – can EVs surface proteins be exploited as a credible tool for early cancer diagnosis – is still not answered. Objective of the current study was to find out whether hypoxia induces differences in protein profiles of EVs released from hypoxic human colorectal cancer cells SW480 (EVHyp) and EVs released from these cells grown in normoxic conditions – EVNorm. Obtained results show differences in EVs surface protein samples. Some protein fragments were found only or mostly in EVHyp surface protein samples. Finding of one or two such EVHyp protein fragments allows us to suggest that deciphered EVHyp surface proteins might be indices of hypoxia-induced proteome changes and might serve as a hint to find a cancer specific protein.

scholarly journals Human cytomegalovirus-infected cells release extracellular vesicles that carry viral surface proteins

Virology ◽  
2018 ◽  
Vol 524 ◽  
pp. 97-105 ◽  
Author(s):  
Sonia Zicari ◽  
Anush Arakelyan ◽  
Rogers Alberto Ñahui Palomino ◽  
Wendy Fitzgerald ◽  
Christophe Vanpouille ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Human Cytomegalovirus ◽  
Surface Proteins ◽  
Infected Cells ◽  
Viral Surface

scholarly journals Comprehensive label-free characterization of extracellular vesicles and their surface proteins

2020 ◽  
Author(s):  
E. Priglinger ◽  
J. Strasser ◽  
B. Buchroithner ◽  
F. Weber ◽  
S. Wolbank ◽  
...  
Keyword(s):  
Quality Control ◽  
Extracellular Vesicles ◽  
Impedance Measurement ◽  
Surface Proteins ◽  
Label Free ◽  
Neuroprotective Effects ◽  
Characterization Methods ◽  
Minimal Sample ◽  
Flow Filtration

AbstractInterest in mesenchymal stem cell derived extracellular vesicles (MSC-EVs) as therapeutic agents has dramatically increased over the last decade. Preclinical studies show that MSC-EVs have anti-apoptotic and neuroprotective effects, boost wound healing, and improve the integration of allogeneic grafts through immunomodulation. Current approaches to the characterization and quality control of EV-based therapeutics include particle tracking techniques, Western blotting, and advanced cytometry, but standardized methods are lacking. In this study, we established and verified quartz crystal microbalance (QCM) as highly sensitive label-free immunosensing technique for characterizing clinically approved umbilical cord MSC-EVs enriched by tangential flow filtration and ultracentrifugation. Using QCM in conjunction with common characterization methods, we were able to specifically detect EVs via EV (CD9, CD63, CD81) and MSC (CD44, CD49e, CD73) markers and gauge their prevalence. Additionally, we characterized the topography and elasticity of these EVs by atomic force microscopy (AFM), enabling us to distinguish between EVs and non-vesicular particles (NVPs) in a therapeutic formulation. This measurement modality makes it possible to identify EV sub-fractions, discriminate between EVs and NVPs, and to characterize EV surface proteins, all with minimal sample preparation and using label-free measurement devices with low barriers of entry for labs looking to widen their spectrum of characterization techniques. Our combination of QCM with impedance measurement (QCM-I) and AFM measurements provides a robust multi-marker approach to the characterization of clinically approved EV formulations and opens the door to improved quality control.

scholarly journals Programmable Modulation for Extracellular Vesicles

2019 ◽  
Author(s):  
Yihua Wang ◽  
Richard Melvin ◽  
Lynne T. Bemis ◽  
Gregory A. Worrell ◽  
Hai-Long Wang
Keyword(s):  
Electrical Stimulation ◽  
Electric Fields ◽  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Physical Method ◽  
Surface Proteins ◽  
Stimulation Parameters ◽  
Wide Range ◽  
Novel Method ◽  
Rat Astrocytes

AbstractEvery living cell releases extracellular vesicles (EVs) that are critical for cellular signaling and a wide range of biological functions. The potential diagnostic and therapeutic applications of EVs are well recognized, and rapidly expanding. While a complete understanding of the molecular mechanisms underpinning EVs release remains elusive, here we demonstrate a novel method for programmable control of the release of EVs and their cargo using external electric fields. As a proof of principle, we use cultured rat astrocytes to demonstrate how the frequency of external electrical stimulation selectively modulates EV release, their surface proteins, and microRNA profiles. This method could broadly impact biological science and medical applications. First, it raises an interesting question of how endogenous electrical activity could modulate EV production. Second, it provides a novel mechanism for tuning therapeutic electrical stimulation that may be useful for treating brain disorders. Third, it provides a new way to generate EVs carrying desired cargos by tuning electrical stimulation parameters. Unlike chemical methods for creating EVs, electrical stimulation is a clean physical method with adjustable parameters including stimulation frequency, field strength and waveform morphology.

scholarly journals Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation

2020 ◽  
Vol 21 (12) ◽  
pp. 4407 ◽  
Author(s):  
William A. Banks ◽  
Priyanka Sharma ◽  
Kristin M. Bullock ◽  
Kim M. Hansen ◽  
Nils Ludwig ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Surface Proteins ◽  
Brain Barrier ◽  
Multiple Time ◽  
Blood Brain ◽  
Cancerous Cell ◽  
Wheatgerm Agglutinin ◽  
Mannose 6 Phosphate

Extracellular vesicles can cross the blood–brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVβ6, AVβ3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

scholarly journals Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting

2019 ◽  
Author(s):  
Stefan Vogt ◽  
Gerhard Stadlmayr ◽  
Johannes Grillari ◽  
Florian Rüker ◽  
Gordana Wozniak-Knopp
Keyword(s):  
Extracellular Vesicles ◽  
Surface Proteins ◽  
Tissue Specific ◽  
Specific Targeting

scholarly journals Environmental conditions modulate the protein content and immunomodulatory activity of extracellular vesicles produced by the probiotic Propionibacterium freudenreichii

2020 ◽  
Author(s):  
Vinícius de Rezende Rodovalho ◽  
Brenda Silva Rosa da Luz ◽  
Aurélie Nicolas ◽  
Fillipe Luiz Rosa do Carmo ◽  
Julien Jardin ◽  
...  
Keyword(s):  
Protein Content ◽  
Extracellular Vesicles ◽  
Environmental Conditions ◽  
Culture Medium ◽  
Surface Proteins ◽  
Bioactive Molecules ◽  
Propionibacterium Freudenreichii ◽  
Therapeutic Applications ◽  
Immunomodulatory Properties

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions. Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

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