Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)
Abstract Small extracellular vesicles and nanoparticles (sEVPs) are cell‐secreted entities with potential as diagnostic biomarkers and therapeutic vehicles. However, significant intrinsic sEVP heterogeneity impedes analysis and understanding of their composition and functions. We employ multidimensi...
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| Format: | Article |
| Language: | English |
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Wiley
2024-12-01
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| Series: | Journal of Extracellular Vesicles |
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| Online Access: | https://doi.org/10.1002/jev2.12520 |
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| author | Simou Sun Sarah J. Cox‐Vázquez Nam‐Joon Cho Guillermo C. Bazan Jay T. Groves |
| author_facet | Simou Sun Sarah J. Cox‐Vázquez Nam‐Joon Cho Guillermo C. Bazan Jay T. Groves |
| author_sort | Simou Sun |
| collection | DOAJ |
| description | Abstract Small extracellular vesicles and nanoparticles (sEVPs) are cell‐secreted entities with potential as diagnostic biomarkers and therapeutic vehicles. However, significant intrinsic sEVP heterogeneity impedes analysis and understanding of their composition and functions. We employ multidimensional fluorescent labelling on sEVPs, leveraging the robustness of a newly developed membrane probe—conjugated oligoelectrolytes (COEs), and conduct total internal reflection fluorescence (TIRF) microscopy on sEVP arrays. These arrays comprise single sEVPs anchored to a soft material functionalized surface with little bias. We then develop an enhanced algorithm for colocalization analysis of the multiple labels on individual sEVPs and perform deep profiling of particle content. We categorize sEVPs derived from the same cell type into seven distinct subpopulations—some vesicular whereas others non‐vesicular, and we demonstrate that sEVPs from four cell types exhibit quantitatively distinguishable subpopulation distributions. Furthermore, we gain insights into specific particle features within each subpopulation, including CD63 counts, relative particle size, relative concentration of cargoes, and correlations among different cargoes. This high‐content analysis reveals common cargo sorting features in sEVP subpopulations across different cell types and suggests new statistics within the sEVP inherent heterogeneity that could differentiate sEVPs from two types of cancer cells and two types of normal cells. Collectively, our study presents a robust single‐sEVP characterization platform, combining high‐content imaging with comprehensive analysis. This platform is poised to advance sEVP‐based theranostic assays and facilitate exploration into disease‐associated sEVP biogenesis and sEVP‐mediated intercellular communication. |
| format | Article |
| id | doaj-art-3e40ca6265224139862ceddf031b4a62 |
| institution | Kabale University |
| issn | 2001-3078 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Extracellular Vesicles |
| spelling | doaj-art-3e40ca6265224139862ceddf031b4a622025-01-17T11:11:12ZengWileyJournal of Extracellular Vesicles2001-30782024-12-011312n/an/a10.1002/jev2.12520Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs)Simou Sun0Sarah J. Cox‐Vázquez1Nam‐Joon Cho2Guillermo C. Bazan3Jay T. Groves4Institute for Digital Molecular Analytics and Science Nanyang Technological University Singapore SingaporeDepartment of Chemistry National University of Singapore Singapore SingaporeSchool of Materials Science and Engineering Nanyang Technological University Singapore SingaporeInstitute for Digital Molecular Analytics and Science Nanyang Technological University Singapore SingaporeInstitute for Digital Molecular Analytics and Science Nanyang Technological University Singapore SingaporeAbstract Small extracellular vesicles and nanoparticles (sEVPs) are cell‐secreted entities with potential as diagnostic biomarkers and therapeutic vehicles. However, significant intrinsic sEVP heterogeneity impedes analysis and understanding of their composition and functions. We employ multidimensional fluorescent labelling on sEVPs, leveraging the robustness of a newly developed membrane probe—conjugated oligoelectrolytes (COEs), and conduct total internal reflection fluorescence (TIRF) microscopy on sEVP arrays. These arrays comprise single sEVPs anchored to a soft material functionalized surface with little bias. We then develop an enhanced algorithm for colocalization analysis of the multiple labels on individual sEVPs and perform deep profiling of particle content. We categorize sEVPs derived from the same cell type into seven distinct subpopulations—some vesicular whereas others non‐vesicular, and we demonstrate that sEVPs from four cell types exhibit quantitatively distinguishable subpopulation distributions. Furthermore, we gain insights into specific particle features within each subpopulation, including CD63 counts, relative particle size, relative concentration of cargoes, and correlations among different cargoes. This high‐content analysis reveals common cargo sorting features in sEVP subpopulations across different cell types and suggests new statistics within the sEVP inherent heterogeneity that could differentiate sEVPs from two types of cancer cells and two types of normal cells. Collectively, our study presents a robust single‐sEVP characterization platform, combining high‐content imaging with comprehensive analysis. This platform is poised to advance sEVP‐based theranostic assays and facilitate exploration into disease‐associated sEVP biogenesis and sEVP‐mediated intercellular communication.https://doi.org/10.1002/jev2.12520Single‐EV CharacterizationsHigh‐Throughput ArraysQuantitative ImagingHigh‐Content Analysis |
| spellingShingle | Simou Sun Sarah J. Cox‐Vázquez Nam‐Joon Cho Guillermo C. Bazan Jay T. Groves Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) Journal of Extracellular Vesicles Single‐EV Characterizations High‐Throughput Arrays Quantitative Imaging High‐Content Analysis |
| title | Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) |
| title_full | Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) |
| title_fullStr | Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) |
| title_full_unstemmed | Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) |
| title_short | Direct imaging with multidimensional labelling and high‐content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles (sEVPs) |
| title_sort | direct imaging with multidimensional labelling and high content analysis allows quantitative categorization and characterizations of individual small extracellular vesicles and nanoparticles sevps |
| topic | Single‐EV Characterizations High‐Throughput Arrays Quantitative Imaging High‐Content Analysis |
| url | https://doi.org/10.1002/jev2.12520 |
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