Orthogonal analysis reveals inconsistencies in cargo loading of extracellular vesicles

Since extracellular vesicles (EVs) have emerged as a promising drug delivery system, diverse methods have been used to load them with active pharmaceutical ingredients (API) in preclinical and clinical studies. However, there is yet to be an engineered EV formulation approved for human use, a barrier driven in part by the intrinsic heterogeneity of EVs. API loading is rarely assessed in the context of single vesicle measurements of physicochemical properties but is likely administered in a heterogeneous fashion to the detriment of a consistent product. Here, we applied a suite of single-particle resolution methods to determine the loading of rhodamine 6G (R6G) surrogate cargo mimicking hydrophilic small molecule drugs across four common API loading methods: sonication, electroporation, freeze-thaw cycling and passive incubation. Loading efficiencies and alterations in the physical properties of EVs were assessed, as well as co-localization with common EV-associated tetraspanins (i.e., CD63, CD81 and CD9) for insight into EV subpopulations. Sonication had the highest loading efficiency, yet significantly decreased particle yield, while electroporation led to the greatest number of loaded API particles, albeit at a lower efficiency. Moreover, results were often inconsistent between repeated runs within a given method, demonstrating the difficulty in developing a rigorous loading method that consistently loaded EVs across their heterogeneous subpopulations. This work highlights the significance of how chosen quantification metrics can impact apparent conclusions and the importance of single-particle characterization of EV loading

A critical systematic review of extracellular vesicle clinical trials

This systematic review examines the landscape of extracellular vesicle (EV)-related clinical trials to elucidate the field's trends in clinical applications and EV-related methodologies, with an additional focus on the acknowledgement of EV subpopulations. By analysing data from public reporting repositories, we catalogued 471 EV-related clinical trials to date, with indications for over 200 diseases. Diagnostics and companion diagnostics represented the bulk of EV-related clinical trials with cancer being the most frequent application. EV-related therapeutics trials mainly utilized mesenchymal stromal cell (MSC) EVs and were most frequently used for treatment of respiratory illnesses. Ultracentrifugation and RNA-sequencing were the most common isolation and characterization techniques; however, methodology for each was not frequently reported in study records. Most of the reported characterization relied on bulk characterization of EV isolates, with only 11% utilizing EV subpopulations in their experimental design. While this may be connected to a lack of available techniques suitable for clinical implementation, it also highlights the opportunity for use of EV subpopulations to improve translational efforts. As academic research identifies more chemically distinct subpopulations and technologies for their enrichment, we forecast to more refined EV trials in the near future. This review emphasizes the need for meticulous methodological reporting and consideration of EV subpopulations to enhance the translational success of EV-based interventions, pointing towards a paradigm shift in personalized medicine

Harnessing extracellular vesicle heterogeneity for diagnostic and therapeutic applications

Extracellular vesicles (EVs) are diverse nanoparticles with large heterogeneity in size and molecular composition. Although this heterogeneity provides high diagnostic value for liquid biopsy and confers many exploitable functions for therapeutic applications in cancer detection, wound healing and neurodegenerative and cardiovascular diseases, it has also impeded their clinical translation-hence heterogeneity acts as a double-edged sword. Here we review the impact of subpopulation heterogeneity on EV function and identify key cornerstones for addressing heterogeneity in the context of modern analytical platforms with single-particle resolution. We outline concrete steps towards the identification of key active biomolecules that determine EV mechanisms of action across different EV subtypes. We describe how such knowledge could accelerate EV-based therapies and engineering approaches for mimetic artificial nanovesicle formulations. This approach blunts one edge of the sword, leaving only a single razor-sharp edge on which EV heterogeneity can be exploited for therapeutic applications across many diseases

Multiomics Profiling of Extracellular Vesicles Supports Their Involvement in Endothelial Senescence‐Associated Vascular Dysfunction

Dysfunction of vascular endothelium is characteristic of many aging-related diseases, including Alzheimer's disease (AD) and AD-related dementias (ADRD). Although it is widely posited that endothelial cell dysfunction contributes to the pathogenesis and/or progression of AD/ADRD, it is not clear how. A plausible hypothesis is that intercellular trafficking of extracellular vesicles (EVs) from senescent vascular endothelial cells promotes vascular endothelial cell dysfunction. To test this hypothesis, we compared the expression of proteins and miRNAs in EVs isolated from four sets of genetically identical early passage non-senescent (EP) versus late passage senescent (SEN) primary human coronary artery endothelial cells (HCAECs) derived from four donors. Proteomics and miRNA libraries constructed from these EV isolates were evaluated using FunRich gene ontology analysis to compare functional enrichment between EP and SEN endothelial cell EVs (ECEVs). Replicative senescence was associated with altered EV abundance and contents independent of changes in EV size. Unique sets of miRNAs and proteins were differentially expressed in SEN-ECEVs, including molecules related to cell adhesion, barrier integrity, receptor signalling, endothelial-mesenchymal transition and cell senescence. miR-181a-5p was the most upregulated miRNA in SEN-ECEVs, increasing >5-fold. SEN-ECEV proteomes supported involvement in several pro-inflammatory pathways consistent with senescence and the senescence-associated secretory phenotype (SASP). These data indicate that SEN-ECEVs are enriched in bioactive molecules implicated in senescence-associated vascular dysfunction, blood-brain barrier impairment, and AD/ADRD pathology. These observations suggest involvement of SEN-ECEVs in the pathogenesis of vascular dysfunction associated with AD/ADRD

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field

Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its \u27Minimal Information for Studies of Extracellular Vesicles\u27, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal Information for Studies of Extracellular Vesicles (MISEV2023): From Basic to Advanced Approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its \u27Minimal Information for Studies of Extracellular Vesicles\u27, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal information for studies of extracellular vesicles (MISEV2023):From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly