Extracellular Vesicles: A Comprehensive Review of Their Origins, Functions, and Therapeutic Potential.

Extracellular vesicles (EVs) are membrane-bound particles secreted by most cell types that play a pivotal role in intercellular communication via transporting protein, nucleic acid, lipid, and metabolite cargos. Among EVs, exosomes are a well-characterized subtype, typically ranging from 10-150 nm in diameter and originating from the endosomal pathway via the formation of multivesicular bodies that fuse with the plasma membrane. EVs/exosomes can be isolated from various biological fluids and cultured cells, with production and yield influenced by the cell type and culture conditions. Isolation methods, including ultracentrifugation or density-based ultracentrifugation, tangential flow filtration, size-exclusion chromatography, immunoaffinity and membrane-affinity capture, and recently developed commercial equipment, offer distinct advantages and limitations in terms of purity, scalability, and exosome integrity. Characterization techniques, such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), cryogenic electron microscopy (cryo-EM), atomic force microscopy (AFM), Western blotting, flow cytometry, and dynamic light scattering (DLS), assess exosome size, morphology, and biomarker expression. Given their biocompatibility and inherent targeting capabilities across a diverse range of diseases, EVs/exosomes hold clinical promise as diagnostic biomarkers, cell-free therapeutics, drug delivery vehicles, immune modulators, and in regenerative medicine. However, these emerging fields in exosome medicine continue to face challenges in standardizing EV sourcing, production, purification, yield, bio-targeting, drug loading, and drug delivery. While EVs/exosomes represent a rapidly advancing frontier in biomedical science, robust protocols for standardization and scalable production will be essential for their successful translation into clinical applications. This article provides a comprehensive overview of EV/exosome origins, their biological functions, the approaches for their isolation and characterization, and their therapeutic potential.