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WIREs Nanomed Nanobiotechnol
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A systematic approach to exosome‐based translational nanomedicine

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Abstract Exosomes are a type of cell‐derived extracellular nanovesicle. They relay information between cells. Some known exosome functions include immune modulation, promotion of angiogenesis, and tumor metastasis. To date, clinical use of exosomes has focused predominantly on evaluating their efficacy as cancer vaccines or diagnostically as biomarker containers. However, few investigations have explored their potential to serve as a platform for the development of semi‐synthetic nanovesicles. Given their nanoscale size, potential to express targeting ligands in native conformations and deformable structure, exosomes offer a logical biological vesicle platform for adapting and producing semi‐synthetic vesicles with excellent potential for nanomedicine applications. However, there are obstacles associated with realizing this potential that must be addressed. Thus, a systematic approach to isolating, modifying, and testing exosomes is presented to facilitate the introduction of exosome‐based translational nanomedicine. WIREs Nanomed Nanobiotechnol 2012, 4:458–467. doi: 10.1002/wnan.1174 This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Exosome production and contents. Exosomes are formed in multivesicular bodies (MVBs) that subsequently fuse with the inner plasma membrane surface resulting in exosome release into the extracellular microenvironment. Exosomes are approximately 30–200 nm in size and display a variety of surface receptors such as major histocompatibility complexes (MHCs) or contain heat shock proteins (HSPs)5 and exosome shuttle RNA (mRNA and miRNA).6 Exosome messages are transmitted to target cells by either receptor‐mediated uptake or possibly fusion.

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A systematic approach to exosome‐based semi‐synthetic nanovesicle (EBSSN) production and testing. Cellular nanofactories are constructed by selecting cell lines of interest and programing them through traditional molecular biology techniques to produce EBSSNs expressing specific targeting ligands (receptors) or cargo (RNA). Non‐modified exosomes or EBSSNs are collected from cell culture supernatants using differential centrifugation. Immediately prior to the ultracentrifugation step of exosome or EBSSN isolation, dynamic light scattering (DLS) is employed to confirm the presence and size of exosomes or EBSSNs and fluorescent labels (carbocyanine dyes) added. Following ultracentrifugation, exosomes or EBSSNs are purified using sucrose density gradients and fractions collected. Rapid confirmation of exosome or EBSSN isolation, without sample loss, is achieved through the use of in gradient fluorescent detection (IGFD). Fluorescent‐labeled exosomes or EBSSNs are washed and purified by ultracentrifugation. Exosomes, or EBSSNs derived from intracellular modification steps, can be further exogenously modified using universal peptide cargo linkers43 or alternative strategies to add additional targeting ligands and/or therapeutics. EBSSNs are then washed in PBS and re‐isolated by ultracentrifugation. EBSSN properties are assessed using DLS and zeta potential analysis. Quantitative readouts such as ELISAs and/or RT‐RT PCR arrays are used to confirm EBSSN cargo content and induced effects using reducible 3D tissue culture14 or in vivo models such as bilateral lymph node exosome tracking.33

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Special Topics in Nanomedicine and Nanobiotechnology

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Diagnostic Tools > Diagnostic Nanodevices
Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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