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Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

BACKGROUND: Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In...

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Autores principales: Kooijmans, Sander A. A., Aleza, Clara Gómez, Roffler, Steve R., van Solinge, Wouter W., Vader, Pieter, Schiffelers, Raymond M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Co-Action Publishing 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793259/
https://www.ncbi.nlm.nih.gov/pubmed/26979463
http://dx.doi.org/10.3402/jev.v5.31053
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author Kooijmans, Sander A. A.
Aleza, Clara Gómez
Roffler, Steve R.
van Solinge, Wouter W.
Vader, Pieter
Schiffelers, Raymond M.
author_facet Kooijmans, Sander A. A.
Aleza, Clara Gómez
Roffler, Steve R.
van Solinge, Wouter W.
Vader, Pieter
Schiffelers, Raymond M.
author_sort Kooijmans, Sander A. A.
collection PubMed
description BACKGROUND: Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. METHODS: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR) nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF). EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. RESULTS: EV analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression), but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. CONCLUSIONS: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.
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spelling pubmed-47932592016-04-29 Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting Kooijmans, Sander A. A. Aleza, Clara Gómez Roffler, Steve R. van Solinge, Wouter W. Vader, Pieter Schiffelers, Raymond M. J Extracell Vesicles Short Communication BACKGROUND: Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. METHODS: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR) nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF). EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. RESULTS: EV analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression), but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. CONCLUSIONS: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules. Co-Action Publishing 2016-03-14 /pmc/articles/PMC4793259/ /pubmed/26979463 http://dx.doi.org/10.3402/jev.v5.31053 Text en © 2016 Sander A. A. Kooijmans et al. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License, permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Communication
Kooijmans, Sander A. A.
Aleza, Clara Gómez
Roffler, Steve R.
van Solinge, Wouter W.
Vader, Pieter
Schiffelers, Raymond M.
Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title_full Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title_fullStr Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title_full_unstemmed Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title_short Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
title_sort display of gpi-anchored anti-egfr nanobodies on extracellular vesicles promotes tumour cell targeting
topic Short Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4793259/
https://www.ncbi.nlm.nih.gov/pubmed/26979463
http://dx.doi.org/10.3402/jev.v5.31053
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