Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation
Abstract Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenes...
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| Format: | Article |
| Language: | English |
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BMC
2025-08-01
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| Series: | Journal of Nanobiotechnology |
| Online Access: | https://doi.org/10.1186/s12951-025-03640-3 |
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| author | Jana Ramon Cláudio Pinheiro Charysse Vandendriessche Estefanía Lozano-Andrés Herlinde De Keersmaecker Deep Punj Juan C. Fraire Edward Geeurickx Marca H. M. Wauben Pieter Vader Roosmarijn E. Vandenbroucke An Hendrix Stephan Stremersch Stefaan C. De Smedt Koen Raemdonck Kevin Braeckmans |
| author_facet | Jana Ramon Cláudio Pinheiro Charysse Vandendriessche Estefanía Lozano-Andrés Herlinde De Keersmaecker Deep Punj Juan C. Fraire Edward Geeurickx Marca H. M. Wauben Pieter Vader Roosmarijn E. Vandenbroucke An Hendrix Stephan Stremersch Stefaan C. De Smedt Koen Raemdonck Kevin Braeckmans |
| author_sort | Jana Ramon |
| collection | DOAJ |
| description | Abstract Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenesis), offers advantages compared to post-formation loading (i.e., after EV isolation), as EV integrity and composition are minimally perturbed. Pre-formation EV loading is primarily achieved through the genetic engineering of the producer cell, which is time consuming and not very flexible regarding the types of molecules that can be incorporated into EVs. In this work, we investigated the possibility of loading cargo molecules into EVs by delivering the cargo directly into the cytosol of the producer cells, which can subsequently be encapsulated into EVs as they are formed. For the cytosolic delivery of cargo molecules, we evaluated the use of photoporation. This membrane disruption technology has been demonstrated to successfully deliver a broad range of cargo molecules into virtually any cell type, while minimally impacting the cell’s normal functioning and homeostasis. As a proof-of-concept, we delivered fluorescently labeled dextran macromolecules and anti-EGFP nanobodies into HEK293T cells genetically engineered with gag-EGFP fusion proteins, which are shuttled into EVs. Colocalization of cargo and EGFP fluorescence in secreted EVs can then serve as a convenient readout for successful EV loading. We established that photoporation had minimal impact on EV characteristics such as concentration, size, zeta potential and the enrichment of EV tetraspanin membrane surface molecules. We found that using EGFP-targeted nanobodies resulted in up to 53% loaded EVs (relative to the amount of EGFP EVs), while non-targeted dextran molecules produced on average 12% loaded EVs (relative to the amount of EGFP EVs). These results highlight the promise of photoporation for pre-formation loading of EVs. |
| format | Article |
| id | doaj-art-dcbb1d652b2a42ff902de308d8ad4e92 |
| institution | Kabale University |
| issn | 1477-3155 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Nanobiotechnology |
| spelling | doaj-art-dcbb1d652b2a42ff902de308d8ad4e922025-08-20T03:42:57ZengBMCJournal of Nanobiotechnology1477-31552025-08-0123111510.1186/s12951-025-03640-3Pre-formation loading of extracellular vesicles with exogenous molecules using photoporationJana Ramon0Cláudio Pinheiro1Charysse Vandendriessche2Estefanía Lozano-Andrés3Herlinde De Keersmaecker4Deep Punj5Juan C. Fraire6Edward Geeurickx7Marca H. M. Wauben8Pieter Vader9Roosmarijn E. Vandenbroucke10An Hendrix11Stephan Stremersch12Stefaan C. De Smedt13Koen Raemdonck14Kevin Braeckmans15Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityCancer Research Institute Ghent (CRIG)VIB Center for Inflammation ResearchDepartment of Biomolecular Health Sciences, Faculty of Veterinary Sciences, Utrecht UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of Experimental Cancer Research, Faculty of Medicine and Health Sciences, Ghent UniversityDepartment of Biomolecular Health Sciences, Faculty of Veterinary Sciences, Utrecht UniversityCDL Research, University Medical Center UtrechtCancer Research Institute Ghent (CRIG)Cancer Research Institute Ghent (CRIG)Laboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityLaboratory of General Biochemistry and Physical Pharmacy, Faculty of Pharmaceutical Sciences, Ghent UniversityAbstract Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenesis), offers advantages compared to post-formation loading (i.e., after EV isolation), as EV integrity and composition are minimally perturbed. Pre-formation EV loading is primarily achieved through the genetic engineering of the producer cell, which is time consuming and not very flexible regarding the types of molecules that can be incorporated into EVs. In this work, we investigated the possibility of loading cargo molecules into EVs by delivering the cargo directly into the cytosol of the producer cells, which can subsequently be encapsulated into EVs as they are formed. For the cytosolic delivery of cargo molecules, we evaluated the use of photoporation. This membrane disruption technology has been demonstrated to successfully deliver a broad range of cargo molecules into virtually any cell type, while minimally impacting the cell’s normal functioning and homeostasis. As a proof-of-concept, we delivered fluorescently labeled dextran macromolecules and anti-EGFP nanobodies into HEK293T cells genetically engineered with gag-EGFP fusion proteins, which are shuttled into EVs. Colocalization of cargo and EGFP fluorescence in secreted EVs can then serve as a convenient readout for successful EV loading. We established that photoporation had minimal impact on EV characteristics such as concentration, size, zeta potential and the enrichment of EV tetraspanin membrane surface molecules. We found that using EGFP-targeted nanobodies resulted in up to 53% loaded EVs (relative to the amount of EGFP EVs), while non-targeted dextran molecules produced on average 12% loaded EVs (relative to the amount of EGFP EVs). These results highlight the promise of photoporation for pre-formation loading of EVs.https://doi.org/10.1186/s12951-025-03640-3 |
| spellingShingle | Jana Ramon Cláudio Pinheiro Charysse Vandendriessche Estefanía Lozano-Andrés Herlinde De Keersmaecker Deep Punj Juan C. Fraire Edward Geeurickx Marca H. M. Wauben Pieter Vader Roosmarijn E. Vandenbroucke An Hendrix Stephan Stremersch Stefaan C. De Smedt Koen Raemdonck Kevin Braeckmans Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation Journal of Nanobiotechnology |
| title | Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation |
| title_full | Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation |
| title_fullStr | Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation |
| title_full_unstemmed | Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation |
| title_short | Pre-formation loading of extracellular vesicles with exogenous molecules using photoporation |
| title_sort | pre formation loading of extracellular vesicles with exogenous molecules using photoporation |
| url | https://doi.org/10.1186/s12951-025-03640-3 |
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