Extracellular vesicles (EVs) hold great promise as therapeutic modalities due to their endogenous characteristics, however, further bioengineering refinement is required to address clinical and commercial limitations. Clinical applications of EV-based therapeutics are being trialed in immunomodulation, tissue regeneration and recovery, and as delivery vectors for combination therapies. Native/biological EVs possess diverse endogenous properties that offer stability and facilitate crossing of biological barriers for delivery of molecular cargo to cells, acting as a form of intercellular communication to regulate function and phenotype. Moreover, EVs are important components of paracrine signaling in stem/progenitor cell-based therapies, are employed as standalone therapies, and can be used as a drug delivery system. Despite remarkable utility of native/biological EVs, they can be improved using bio/engineering approaches to further therapeutic potential. EVs can be engineered to harbor specific pharmaceutical content, enhance their stability, and modify surface epitopes for improved tropism and targeting to cells and tissues in vivo. Limitations currently challenging the full realization of their therapeutic utility include scalability and standardization of generation, molecular characterization for design and regulation, therapeutic potency assessment, and targeted delivery. The fields’ utilization of advanced technologies (imaging, quantitative analyses, multi-omics, labeling/live-cell reporters), and utility of biocompatible natural sources for producing EVs (plants, bacteria, milk) will play an important role in overcoming these limitations. Advancements in EV engineering methodologies and design will facilitate the development of EV-based therapeutics, revolutionizing the current pharmaceutical landscape.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function.

Extracellular vesicles (EVs) are membrane-enclosed vesicles released by cells. They carry proteins, nucleic acids and metabolites which can be transferred to a recipient cell, locally or at a distance, to elicit a functional response. Since their discovery over 30 years ago, the functional repertoire of EVs in both physiological (e.g., organ morphogenesis, embryo implantation) and pathological (e.g., cancer, neurodegeneration) conditions has cemented their crucial role in intercellular communication. Moreover, because the cargo encapsulated within circulating EVs remains protected from degradation, their diagnostic as well as therapeutic (such as drug delivery tool) applications have garnered vested interest. Global efforts have been made to purify EV subtypes from biological fluids and in vitro cell culture media using a variety of strategies and techniques, with a major focus on EVs of endocytic origin called exosomes (30-150 nm in size). Given that the secretome comprises of soluble secreted proteins, protein aggregates, RNA granules and EV subtypes (such as exosomes, shed microvesicles, apoptotic bodies), it is imperative to purify exosomes to homogeneity if we are to perform biochemical and biophysical characterization and importantly, functional dissection. Besides understanding the composition of EV-subtypes, defining molecular bias of how they reprogram target cells also remains of paramount importance in this area of active research. Here, we outline a systematic "how to" protocol (along with useful insights/tips) to obtain highly purified exosomes and perform their biophysical and biochemical characterization. This protocol employs a mass spectrometry-based proteomics approach to characterize the protein composition of exosomes. We also provide insights on different isolation strategies and their usefulness in various downstream applications. We outline protocols for lipophilic-labelling of exosomes to study uptake by a recipient cell, investigating cellular reprogramming using proteomics and studying functional response to exosomes in the Transwell-Matrigel TM Invasion assay.