Purifying, Profiling, Probing, and Programming: The Chemical Biology of Extracellular Vesicles

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Conspectus Extracellular vesicles (EVs) are membrane-enclosed nanoparticles that mediate intercellular communication and have attracted growing interest as potential diagnostic markers and therapeutic vehicles. At the same time, their nanoscale dimensions, compositional heterogeneity, and context-dependent biogenesis continue to complicate their isolation, characterization, and functional analysis. Many of these challenges are fundamentally rooted in limitations of molecular resolution and specificity, making EV research amenable to chemical biology approaches. In this Account, we summarize work from our laboratory over the past 15 years in which we have developed chemical biology tools to address four recurring methodological needs in EV research: purifying heterogeneous vesicle subpopulations, profiling their molecular composition, probing their cellular fate, and programming their therapeutic function. We began with the challenge of EV isolation. We first discussed membrane curvature-sensing and lipid-affinity probes as modular reagents for the selective enrichment of EV subpopulations based on membrane physical and compositional features. We then turned to molecular profiling of EV contents and presented a structure-resolved lipid MS workflow, including a chiral LC-MS/MS platform, that mapped EV lipidomes at stereoisomeric resolution and revealed PI4P as a low-abundance signaling lipid regulating EV biogenesis under immune stress. To address the long-standing difficulty of tracking EV fate in vivo, we highlighted the bioorthogonal and genetically encoded labeling strategies that overcome the nonspecific transfer artifacts of lipophilic dyes, making EV uptake and cargo delivery quantifiable at the molecular level. Finally, we described stimulus-responsive engineering approaches for EV engineering, including photoactivatable prodrug hybrids and pH-responsive nanocarrier designs, which convert EVs into programmable delivery systems capable of controlled transport across the blood–brain barrier and enhanced drug action in tumor models. Taken together, these studies are not intended to suggest that chemical biology, on its own, can fully resolve the inherent complexity of EV systems. Rather, they underscore the value of introducing molecularly defined tools into EV isolation, compositional analysis, trafficking studies, and functional engineering, thereby rendering longstanding challenges in the field more experimentally tractable and quantitatively interpretable. We hope the design strategies and analytical approaches summarized in this Account will offer a practical reference for researchers working across EV biology and EV-based translational medicine.