Im van der Wurff-Jacobsa, Banuja Balachandrana, Linglei Jiangb and Raymond Schiffelersc Division Imaging, UMC Utrecht,

Im van der Wurff-Jacobsa, Banuja Balachandrana, Linglei Jiangb and Raymond Schiffelersc Division Imaging, UMC Utrecht, The Netherlands, Utrecht, Netherlands; Department of Clinical Chemistry and Haematology, UMC Utrecht, The Netherlands; cLaboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, Netherlandsb aAstraZeneca, molndal, Sweden; bAstraZeneca, M ndal, AstraZeneca, Molndal, Sweden; dAstraZeneca, Macclesfield, UKSweden;Introduction: Cell engineering is one of the most typical techniques to modify extracellular vesicles (EVs) for therapeutic drug delivery. Engineering might be applied to optimize cell tropism, targeting, and cargo loading. In this study, we screened various EV proteins fused with EGFP to evaluate the surface show of the EV-associated cargo. Furthermore, we screened for EV proteins that could efficiently traffic cargo proteins into the lumen of EVs. We also developed a novel technology to quantify the amount of EGFP molecules per vesicle using total internal reflection (TIRF) microscopy for single-molecule investigation. Methods: Human Expi293F cells were transiently transfected with DNA constructs coding for EGFP fused towards the N- or C-terminal of EV proteins (e.g., CD63, CD47, Syntenin-1, Lamp2b, Tspan14). 48 h right after transfection, cells have been analysed by flow cytometry and confocal microscopy for EGFP expression and EVs were isolated by differential centrifugation followed by separation employing iodixanol density gradients. EVs have been characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. Single-molecule TIRF microscopy was utilized to determine the protein quantity per vesicle at aIntroduction: Improvement of extracellular vesicles (EVs) as nanocarriers for drug delivery relies on loading a substantial volume of drug into EVs. Loading has been accomplished in the simplest way by co-incubating the drug with EVs or producer cells till making use of physical/chemical approaches (e.g. electroporation, extrusion, and EV surface functionalization). We use physical technique combining gas-filled microbubbles with ultrasound referred to as sonoporation (USMB) to pre-load drug within the producer cells, that are sooner or later loaded into EVs. Solutions: Cells have been grown overnight in 0.01 poly-Llysine coated cell culture cassette. Prior to USMB, cells had been starved for 4 h. Treatment medium containing microbubbles and 250 BSA-Alexa Fluor 488 as a model drug was added towards the cells grown inside the cassette. Cells were exposed straight to pulsed ultrasound (ten duty cycle, 1 kHz pulse repetition frequency, and 100 s pulse duration) with up to 845 kPa BTNL2 Proteins Synonyms removed.ISEV2019 ABSTRACT BOOKCells were washed and incubated in the culture medium for two h. Afterward, EVs within the conditioned medium have been collected and measured. Final results: Cells took up BSA-Alexa Fluor 488 soon after USMB treatment as measured by flow cytometry. These cells released EVs within the conditioned medium which have been captured by anti-CD9 magnetic beads. About five of the CD9-positive EVs contained BSAAlexa Fluor 488. The presence of CD9-positive EVs containing BSA also were confirmed by immunogold electron microscopy. Summary/Conclusion: USMB serves as a tool to preload the model drug, BSA-Alexa Fluor 488, endogenously and to create EVs loaded with this model drug. USMB setup, incubation time, and variety of drugs will likely be investigated to further optimize.