Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA

Abstract

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Longitudinal characterization of circulating extracellular vesicles and small RNA during simian immunodeficiency virus infection and antiretroviral therapy

“…To be sure, overlapping contents and properties make EVs and virions (or ''host EVs'' and ''viral EVs'') nontrivial to separate [37][38][39][40]. Although density gradients are a standard method for virion/extracellular vesicle separation [41][42][43][44][45], most methods have been optimized for cell culture medium [42][43][44] or large quantities of virions spiked into plasma [45]. However, our extracellular vesicle and extracellular vesicle exRNA results are likely not skewed by the presence of virions.…”

Emerging Drug Delivery Vectors: Engineering of Plant-Derived Nanovesicles and Their Applications in Biomedicine

“…Therefore, differential centrifugation is often combined with density gradient centrifugation (Figure 2), and commonly used gradient media include sucrose, iodophor, and cesium chloride, many of which have been commercialized. 13,[27][28][29] Ultrafiltration is another commonly used separation method that relies on fluid pressure to drive small molecules through polymeric membranes, migrating while retaining large molecules. 30 There are typically two membrane separation modes, direct flow filtration (DFF) and tangential flow filtration (TFF), where the flow channel is perpendicular or parallel to the membrane surface.…”