Before Translating Extracellular Vesicles into Personalized Diagnostics and Therapeutics: What We Could Do

Abstract

Extracellular vesicle (EV) research is rapidly advancing from fundamental science to translational applications in EV-based personalized therapeutics and diagnostics. Yet, fundamental questions persist regarding EV biology and mechanisms, particularly concerning the heterogeneous interactions between EVs and cells. While we have made strides in understanding virus delivery and intracellular vesicle transport, our comprehension of EV trafficking remains limited. EVs are believed to mediate intercellular communication through cargo transfer, but uncertainties persist regarding the occurrence and quantification of EV-cargo delivery within acceptor cells. This ambiguity is crucial to address, given the significant translational impact of EVs on therapeutics and diagnostics. This perspective article does not seek to provide exhaustive recommendations and guidance on EV-related studies, as these are well-articulated in position papers and statements by the International Society for Extracellular Vesicles (ISEV), including the 'Minimum Information for Studies of Extracellular Vesicles' (MISEV) 2014, MISEV2018, and the recent MISEV2023. Instead, recognizing the multilayered heterogeneity of EVs as both a challenge and an opportunity, this perspective emphasizes novel approaches to facilitate our understanding of diverse EV biology, address uncertainties, and leverage this knowledge to advance EV-based personalized diagnostics and therapeutics. Specifically, this perspective synthesizes current insights, identifies opportunities, and highlights exciting technological advancements in ultrasensitive single EV or "digital" profiling developed within the author's multidisciplinary group. These newly developed technologies address technical gaps in dissecting the molecular contents of EV subsets, contributing to the evolution of EVs as next-generation liquid biopsies for diagnostics and providing better quality control for EV-based therapeutics.

Keywords: diagnostics; drug delivery; exosome; extracellular vesicle; personalized medicine; protein biomarker; single molecule array.

Figure 1

Typical structure and biomolecular cargo of an extracellular vesicle (EV). This vesicle carries a diverse payload, which can consist of various nucleic acids (such as miRNAs, mRNAs, and DNAs), proteins, lipids, receptors, adhesion molecules, and other biomolecules.

Figure 2

Challenges encountered in the EV field from both biological and technological perspectives. (a) The general understanding of the EV-cell interplay, including processes such as EV biogenesis, release, uptake, fusion, and functional transfer between cells. However, detailed mechanisms remain understudied despite this general understanding, which may be attributed to technological limitations highlighted in (b). The collective challenges presented here underscore the complexity of standardizing and specifying EVs for clinical translation.

Figure 3

Potential engineering workflow of EV-nanoparticle drug delivery system for personalized therapeutics. Step 1 (top): Heterogeneous EVs released from donor cells undergo EV standardization to purify and recover the EV subpopulation of interest. Step 2: The recovered EV subpopulation with improved uniformity will be hybridized with nanoparticles to form an EV-nanoparticle hybrid. Step 3: To enhance the targeting ability of the hybrid particle, ligands will be modified to the particle’s surface, and the drug will be loaded into the EV-nanoparticle hybrid. This resulting EV-nanoparticle hybrid exhibits improved uniformity and thus better quality control compared to the hybrid lacking EV standardization (bottom).

Figure 4

Workflow of the EV single-molecule array (eSimoa) framework for spatial decoding of EV-associated proteins. The eSimoa framework combines EV isolation with ultrasensitive protein detection to profile EV proteins with exceptional sensitivity and specificity. The eSimoa framework comprises three complementary pipelines. Pipeline (i): surface eSimoa, capturing and detecting two EV surface proteins. Pipeline (ii): luminal eSimoa, analyzing EV luminal proteins. Pipeline (iii): surface-luminal eSimoa or pulldown eSimoa, integrating the surface and luminal eSimoa approaches by selectively targeting a subpopulation of EVs with a specific surface protein using pulldown beads, followed by the analysis of luminal proteins within this subpopulation. Reproduced from ref (77). Copyright 2023 The Authors, published by Wiley-VCH GmbH under a CC-BY 4.0 license.

Figure 5

Proposed strategies to advance EV-based personalized medicine. Because of the multilayered heterogeneity of EVs, thorough EV standardization and specification are essential prerequisites for translating EVs into therapeutics. This can be achieved from three perspectives: leveraging advanced technologies such as single EV or digital methods (e.g., eSimoa), deepening our knowledge of diverse EV biology, and embracing multidisciplinary approaches. Once fulfilled, such EV therapeutics has the potential to become next-generation personalized medicine.