Abstract
Extracellular vesicles (EVs) are studied extensively as natural biomolecular shuttles and for their diagnostic and therapeutic potential. This exponential rise in interest has highlighted the need for highly robust and reproducible approaches for EV characterisation. Here we optimise quantitative nanomechanical tools and demonstrate the advantages of EV population screening by atomic force microscopy (AFM). Our high-content informatics analytical tools are made available for use by the EV community for widespread, standardised determination of structural stability. Ultracentrifugation (UC) and sonication, the common mechanical techniques used for EV isolation and loading respectively, are used to demonstrate the utility of optimised PeakForce-Quantitative Nano Mechanics (PF-QNM) analysis. EVs produced at an industrial scale exhibited biochemical and biomechanical alterations after exposure to these common techniques. UC resulted in slight increases in physical dimensions, and decreased EV adhesion concurrent with a decrease in CD63 content. Sonicated EVs exhibited significantly reduced levels of CD81, a decrease in size, increased Young's modulus and decreased adhesive force. These biomechanical and biochemical changes highlight the effect of EV sample preparation techniques on critical properties linked to EV cellular uptake and biological function. PF-QNM offers significant additional information about the structural information of EVs following their purification and downstream processing, and the analytical tools will ensure consistency of analysis of AFM data by the EV community, as this technique continues to become more widely implemented.
Highlights
Extracellular vesicles (EVs) are a superfamily of small lipid bilayer enclosed vesicles, present in the majority of human bodily fluids, including blood, urine, saliva, tears and milk.[1,2,3] The general term EVs encompasses many small naturally occurring vesicles, including microvesicles (MVs), exosomes or small EVs, oncosomes and other vesicles such as apoptotic bodies
The protein and RNA composition of EVs generally reflects that of the progenitor cells. small EVs (sEVs) can be identified by specific markers indicating their endocytic origin, such as ALG-2 interacting protein X (Alix), tumour susceptibility gene 101 (TSG101) and the common tetraspanin markers such as CD9, CD63 and CD81, whereas additional markers appear to define specific sub-types.[5]
EVs isolated for this study expressed typical sEV markers, namely CD9, CD63 and CD81, but lacked the expression of internal sEV markers, Alix and TSG101, Fig. 1, demonstrating that intact EVs were isolated
Summary
Extracellular vesicles (EVs) are a superfamily of small lipid bilayer enclosed vesicles, present in the majority of human bodily fluids, including blood, urine, saliva, tears and milk.[1,2,3] The general term EVs encompasses many small naturally occurring vesicles, including microvesicles (MVs), exosomes or small EVs (sEVs), oncosomes and other vesicles such as apoptotic bodies. These groups of EVs can be defined by size, origin and marker expression profiles.[2] The two most prominent classes of EVs are sEVs ( known as exosomes), with dimensions between 30 and 150 nm and originate from the endosomal system,[4,5] and larger MVs with dimensions between 100 and 1000 nm and are created by the budding of. These membrane proteins may target EVs to specific cells and EVs may contain cellular markers crucial in allowing the transport of molecules to target specific cell populations, making isolated EVs a strong candidate to develop advanced therapeutics
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