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4. EV‐Elute: A universal platform for the enrichment of functional surface marker‐defined extracellular vesicle subpopulations.

Author

de Voogt, Willemijn S., Frunt, Rowan, Leandro, Raul M., Triesscheijn, Casper S., Monica, Bella, Paspali, Ioanna, Tielemans, Mark, François, Jerney J. J. M., Seinen, Cor W., de Jong, Olivier G., and Kooijmans, Sander A. A.

Subjects
*MONONUCLEAR leukocytes, *EXTRACELLULAR vesicles, *CELL communication, *PHOSPHATIDYLSERINES, *ENDOTHELIAL cells
Abstract

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, EV functionality likely differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non‐destructive methods to separate EV subpopulations based on their surface markers. Here, we used the Design‐of‐Experiments (DoE) methodology to optimize a protocol, which we name 'EV‐Elute', to elute intact EVs from commercially available Protein G‐coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom‐made protein binding phosphatidylserine (PS). When applying EV‐Elute, over 70% of bound EVs could be recovered from the beads in a pH‐ and incubation‐time‐dependent fashion. EV subpopulations showed intact integrity by electron microscopy and Proteinase K protection assays and showed uptake patterns similar to whole EV isolates in co‐cultures of peripheral blood mononuclear cells (PBMCs) and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy‐to‐use platform to isolate and functionally compare surface marker‐defined EV subpopulations. This platform does not require specialized equipment or reagents and is universally applicable to any capturing antibody and EV source. Hence, EV‐Elute can open new opportunities to study EV functionality at the subpopulation level. [ABSTRACT FROM AUTHOR]

Published
2024
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5. Plasmin-cleaved von Willebrand factor as a biomarker for microvascular thrombosis

Author

CDL Contact activatie, CDL Research analisten, El Otmani, Hinde, Frunt, Rowan, Smits, Simone, Barendrecht, Arjan D., de Maat, Steven, Fijnheer, Rob, Lenting, Peter J., Tersteeg, Claudia, CDL Contact activatie, CDL Research analisten, El Otmani, Hinde, Frunt, Rowan, Smits, Simone, Barendrecht, Arjan D., de Maat, Steven, Fijnheer, Rob, Lenting, Peter J., and Tersteeg, Claudia

Published
2024

6. Factor XII contact activation can be prevented by targeting 2 unique patches in its epidermal growth factor-like 1 domain with a nanobody

Author

CDL Contact activatie, CDL Research analisten, Poli Van Creveldkliniek Medisch, CDL Cluster Onderzoek en Onderwijs, Circulatory Health, Frunt, Rowan, El Otmani, Hinde, Smits, Simone, Clark, Chantal C, Maas, Coen, CDL Contact activatie, CDL Research analisten, Poli Van Creveldkliniek Medisch, CDL Cluster Onderzoek en Onderwijs, Circulatory Health, Frunt, Rowan, El Otmani, Hinde, Smits, Simone, Clark, Chantal C, and Maas, Coen

Published
2024

7. Biofluid specific protein coronas affect lipid nanoparticle behavior in vitro

Author

CDL Staf Research, CDL Nanomedicine, CDL extracellular vesicles, CDL Contact activatie, Centraal Diagnostisch Laboratorium, Cancer, Circulatory Health, van Straten, Demian, Sork, Helena, van de Schepop, Luuk, Frunt, Rowan, Ezzat, Kariem, Schiffelers, Raymond M., CDL Staf Research, CDL Nanomedicine, CDL extracellular vesicles, CDL Contact activatie, Centraal Diagnostisch Laboratorium, Cancer, Circulatory Health, van Straten, Demian, Sork, Helena, van de Schepop, Luuk, Frunt, Rowan, Ezzat, Kariem, and Schiffelers, Raymond M.

Published
2024

8. EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Author

de Voogt, Willemijn S, primary, Frunt, Rowan, additional, Leandro, Raul M, additional, Triesscheijn, Casper S, additional, Monica, Bella, additional, Paspali, Ioanna, additional, Tielemans, Mark, additional, Francois, Jerney JJM, additional, Seinen, Cor W, additional, de Jong, Olivier G, additional, and Kooijmans, Sander AA, additional

Published
2023
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10. EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Author

Voogt, Willemijn S de, Frunt, Rowan, Leandro, Raul M, Triesscheijn, Casper S, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Francois, Jerney JJM, Seinen, Cor W, de Jong, Olivier G., Kooijmans, Sander AA, Voogt, Willemijn S de, Frunt, Rowan, Leandro, Raul M, Triesscheijn, Casper S, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Francois, Jerney JJM, Seinen, Cor W, de Jong, Olivier G., and Kooijmans, Sander AA

Abstract

Intercellular communication via extracellular vesicles (EVs) has been identified as a vital component of a steadily expanding number of physiological and pathological processes. To accommodate these roles, EVs are equipped with specific proteins, lipids, and RNA molecules by EV-secreting cells. Consequently, EVs have highly heterogeneous molecular compositions. Given that surface molecules on EVs determine their interactions with their environment, it is conceivable that EV functionality differs between subpopulations with varying surface compositions. However, it has been technically challenging to examine such functional heterogeneity due to a lack of non-destructive methods to separate EV subpopulations based on their surface markers. Here, we used Design-of-Experiments methodology to rapidly optimize a protocol, which we name ‘EV-Elute’, to elute intact EVs from commercially available Protein G-coated magnetic beads. We captured EVs from various cell types on these beads using antibodies against CD9, CD63, CD81 and a custom-made protein binding phosphatidylserine (PS). When applying EV-Elute, over 70% of bound EVs could be recovered from the beads in a pH– and incubation time-dependent fashion. EV subpopulations were found to be devoid of co-isolated protein contaminants observed in whole EV isolates and showed intact morphology by electron microscopy. Proteinase K protection assays showed a mild and reversible decrease of EV membrane integrity during elution. Depending on the type of capturing antibody used, some antibodies remained EV-associated after elution. EV subpopulations showed uptake patterns similar to whole EV isolates in co-cultures of peripheral blood mononuclear cells and endothelial cells. However, in Cas9/sgRNA delivery assays, CD63+ EVs showed a lower capacity to functionally deliver cargo as compared to CD9+, CD81+ and PS+ EVs. Taken together, we developed a novel, easy-to-use platform to isolate and functionally compare surface marker-defined

Published
2023

11. EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Author

Afd Pharmaceutics, Pharmaceutics, Voogt, Willemijn S de, Frunt, Rowan, Leandro, Raul M, Triesscheijn, Casper S, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Francois, Jerney JJM, Seinen, Cor W, de Jong, Olivier G., Kooijmans, Sander AA, Afd Pharmaceutics, Pharmaceutics, Voogt, Willemijn S de, Frunt, Rowan, Leandro, Raul M, Triesscheijn, Casper S, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Francois, Jerney JJM, Seinen, Cor W, de Jong, Olivier G., and Kooijmans, Sander AA

Published
2023

12. Factor XII Explored with AlphaFold - Opportunities for Selective Drug Development

Author

CDL Contact activatie, CDL Nanomedicine, Circulatory Health, CDL Cluster Onderzoek en Onderwijs, Frunt, Rowan, El Otmani, Hinde, Gibril Kaira, Bubacarr, de Maat, Steven, Maas, Coen, CDL Contact activatie, CDL Nanomedicine, Circulatory Health, CDL Cluster Onderzoek en Onderwijs, Frunt, Rowan, El Otmani, Hinde, Gibril Kaira, Bubacarr, de Maat, Steven, and Maas, Coen

Published
2023

13. EV-Elute: a universal platform for enrichment of functional surface marker-defined extracellular vesicle subpopulations

Author

CDL Nanomedicine, Circulatory Health, CDL extracellular vesicles, CDL Contact activatie, Zorgeenheid Vaatchirurgie Medisch, CDL Research analisten, de Voogt, Willemijn, Frunt, Rowan, Leandro, Raul, Triesscheijn, Casper, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Gitz-Francois, Jerney J. J. M., Seinen, CW, de Jong, Olivier, Kooijmans, Sander, CDL Nanomedicine, Circulatory Health, CDL extracellular vesicles, CDL Contact activatie, Zorgeenheid Vaatchirurgie Medisch, CDL Research analisten, de Voogt, Willemijn, Frunt, Rowan, Leandro, Raul, Triesscheijn, Casper, Monica, Bella, Paspali, Ioanna, Tielemans, Mark, Gitz-Francois, Jerney J. J. M., Seinen, CW, de Jong, Olivier, and Kooijmans, Sander

Published
2023

18. Therapeutic outcome of early-phase clinical trials in multiple myeloma: a meta-analysis

Author

MS Hematologie, Cancer, CDL Contact activatie, Epidemiology & Health Economics, JC onderzoeksprogramma Cancer, Infection & Immunity, Regenerative Medicine and Stem Cells, van Nieuwenhuijzen, Niels, Frunt, Rowan, May, Anne M, Minnema, Monique C, MS Hematologie, Cancer, CDL Contact activatie, Epidemiology & Health Economics, JC onderzoeksprogramma Cancer, Infection & Immunity, Regenerative Medicine and Stem Cells, van Nieuwenhuijzen, Niels, Frunt, Rowan, May, Anne M, and Minnema, Monique C

Published
2021

19. FXII contact activation is dependent on binding through two unique patches in its EGF-1 domain and can be prevented by (VHH) targeting.

Author

Frunt, Rowan, El Otmani, Hinde, Smits, Simone, Clark, Chantal C., and Maas, Coen

Abstract

Factor XII (FXII) triggers contact activation by binding to foreign surfaces, with the EGF-1 domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis.

Published
2024
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