Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

Abstract

The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

Overview of extracellular vesicle characterization techniques and introduction to combined reflectance and fluorescence confocal microscopy to distinguish extracellular vesicle subpopulations

“…1(a) ] and then only fluorescently labeled particles are detected and characterized within the solution. 45 However, this approach can be limited by bleaching of the fluorophore, and while labeling EVs with antibodies conjugated to quantum dots can enhance photostability, this restricts the targets against which antibodies can be selected and additional purification steps are required to separate the EVs from unbound fluorophores. 37 …”

Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies

“…Characterized EV spike-in models were produced as previously described [ 15 ]. Briefly, human cell lines HT29, A549, 22RV1 (ATCC) and HEK293-pRTS-CA9 (courtesy of prof. Dr. Reinhard Zeidler, Helmholtz Zentrum München, Germany) were grown in complete medium supplemented with 10% FBS (Euroclone) and 1% pen/strep (Sigma).…”

“…Most popular approaches, such as ultracentrifugation (UC) or size-exclusion chromatography (SEC), rely on physical properties to isolate particles, depending on their density or size [5,12,13]. Despite the major differences between UC and SEC, when purifying low complexity samples such as serum-free cell conditioned medium (CCM), where the majority of particles are indeed EVs, both techniques recover whole EVs equally well and do not enrich for particular surface phenotypes [7,[13][14][15]. Thus, these methodologies are extremely useful to obtain large batches of fairly pure EV samples from cell cultures and enable unbiased characterization of EV subpopulations.…”

Extracellular Vesicles as New Players in Drug Delivery: A Focus on Red Blood Cells-Derived EVs

“…This feature promotes the use of NTA in place of DLS, especially in highly polydisperse samples. Likewise, DLS or even NTA is a technique requiring a relatively short time (less than one hour) and no sample preparation, but, exploiting the same principle as DLS, even in this case, a few large particles might obscure a multitude of smaller particles [ 126 , 127 ].…”