Physical characterization and profiling of airway epithelial derived exosomes using light scattering

Abstract

Exosomes and other extracellular vesicles have been gaining interest during the last decade due to their emerging role in biology and, disease pathogenesis and their biomarker potential. Almost all published research related to exosomes and other extracellular vesicles include some form of physical characterization. Therefore, these vesicles should be precisely profiled and characterized physically before studying their biological role as intercellular messengers, biomarkers or therapeutic tools. Using a combination of light scattering techniques, including dynamic light scattering (DLS) and multi-angle laser light scattering combined with size exclusion separation (SEC-MALLS), we physically characterized and compared distinct extracellular vesicles derived from the apical secretions of two different cultured airway epithelial cells. The results indicated that epithelial cells release vesicles with distinct physical properties and sizes. Human primary tracheobronchial cell culture (HTBE) derived vesicles have a hydrodynamic radius (Rh) of approximately 340 nm while their radius of gyration (Rg) is approximately 200 nm. Electron microscopy analysis, however, revealed that their spherical component is 40-100 nm in size, and they carry filamentous, entangled membrane mucins on their surface that increases their overall radius. The mucin decoration on the surface defines their size and charge as measured using light scattering techniques. Their surface properties mirror the properties of the cells from which they are derived. This may provide a unique tool for researchers to elucidate the unanswered questions in normal airway biology and innate and adaptive defense, including the remodeling of airways during inflammation, tumorigenesis and metastasis.

Size-dependent cellular uptake of exosomes

“…Several techniques are available to determine the size of nanoparticles that have been applied to characterise exosomes. These include Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) among the most used [11][12][13][14][15][16][17]. Unfortunately, none of these methods on its own has the ability to estimate the size distribution accurately and at the same time provide high-throughput.…”

Extracellular vesicles in lung microenvironment and pathogenesis

“…Kulshreshtha et al showed that exosome marker proteins were mostly localized to lung epithelial cells and alveolar macrophages in human lung tissue sections [10]. Remarkably, Kesimer et al clarified the physical characterization and profiles of lung epithelial cell-derived exosomes using lightscattering [26]. They showed that exosomes contain differently sized membrane-tethered mucins that can alter the physical structural properties and affect the measured size, conformation, and charges of exosomes [26].…”

“…Remarkably, Kesimer et al clarified the physical characterization and profiles of lung epithelial cell-derived exosomes using lightscattering [26]. They showed that exosomes contain differently sized membrane-tethered mucins that can alter the physical structural properties and affect the measured size, conformation, and charges of exosomes [26]. In general, secretory and membrane-tethered mucins can contribute to mucociliary defense, an innate immune defense system that protects the airway against pathogens and environmental toxins [27].…”

“…Cargoes are located inside the luminal space and can include nucleic acids (i.e., DNA, mRNA, and miRNAs) and proteins. Lung epithelial cell-derived exosomes contain different sizes of membrane-tethered mucins that can alter the physical properties of the structure and affect their measured size[26]. (B) An electron microscopy image of lung epithelial cell (BEAS-2B)-derived exosomes measuring approximately 50-150 nm in diameter.…”

Extracellular Vesicles in Chronic Obstructive Pulmonary Disease

“…Epithelial cell-derived EVs are considered to be key players in the EV-mediated communication in the lungs. It has been reported that epithelial cell-derived exosomes have a size range of 30 to 100 nm and contain different membrane-tethered mucins of different sizes [54,55]. These surface mucins can provide sites of interaction between EVs and inhaled materials or host cells; hence, EVs may have important roles in homeostasis and innate defense.…”