title: PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time.
abstract: Extracellular vesicles (EVs) are increasingly being recognized as candidate drug delivery systems due to their ability to functionally transfer biological cargo between cells. However, the therapeutic applicability of EVs may be limited due to a lack of cell-targeting specificity and rapid clearance of exogenous EVs from the circulation. In order to improve EV characteristics for drug delivery to tumor cells, we have developed a novel method for decorating EVs with targeting ligands conjugated to polyethylene glycol (PEG). Nanobodies specific for the epidermal growth factor receptor (EGFR) were conjugated to phospholipid (DMPE)-PEG derivatives to prepare nanobody-PEG-micelles. When micelles were mixed with EVs derived from Neuro2A cells or platelets, a temperature-dependent transfer of nanobody-PEG-lipids to the EV membranes was observed, indicative of a 'post-insertion' mechanism. This process did not affect EV morphology, size distribution, or protein composition. After introduction of PEG-conjugated control nanobodies to EVs, cellular binding was compromised due to the shielding properties of PEG. However, specific binding to EGFR-overexpressing tumor cells was dramatically increased when EGFR-specific nanobodies were employed. Moreover, whereas unmodified EVs were rapidly cleared from the circulation within 10min after intravenous injection in mice, EVs modified with nanobody-PEG-lipids were still detectable in plasma for longer than 60min post-injection. In conclusion, we propose post-insertion as a novel technique to confer targeting capacity to isolated EVs, circumventing the requirement to modify EV-secreting cells. Importantly, insertion of ligand-conjugated PEG-derivatized phospholipids in EV membranes equips EVs with improved cell specificity and prolonged circulation times, potentially increasing EV accumulation in targeted tissues and improving cargo delivery.
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: S A A
authors.initials: SAA
authors.lastname: Kooijmans
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.
authors.forename: L A L
authors.initials: LAL
authors.lastname: Fliervoet
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: R
authors.initials: R
authors.lastname: van der Meel
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M H A M
authors.initials: MHAM
authors.lastname: Fens
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: H F G
authors.initials: HFG
authors.lastname: Heijnen
authors.affiliation: Division of Cell Biology, Department of Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: P M P
authors.initials: PMP
authors.lastname: van Bergen En Henegouwen
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Vader
authors.affiliation: Department of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, The Netherlands. Electronic address: r.schiffelers@umcutrecht.nl.
authors.forename: R M
authors.initials: RM
authors.lastname: Schiffelers
mesh_terms: D061605:Administration, Intravenous; D001792:Blood Platelets / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016503:Drug Delivery Systems; D066246:ErbB Receptors / Q000008:administration & dosage; D005079:Excipients; D000067128:Extracellular Vesicles / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008823:Micelles; D053758:Nanoparticles; D010316:Particle Size; D010743:Phospholipids / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*
chemical_list: D005079:Excipients; D008024:Ligands; D008823:Micelles; D010743:Phospholipids; D011092:Polyethylene Glycols; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Circulation time; Drug delivery; Extracellular vesicles; Nanobody; Polyethylene glycol; Targeting