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

November 3, 2022

Im van der Wurff-Jacobsa, Banuja Balachandrana, Linglei Jiangb and Raymond Schiffelersc Division Imaging, UMC Utrecht, The Netherlands, Utrecht, Netherlands; Division of Clinical Chemistry and Haematology, UMC Utrecht, The Netherlands; cLaboratory of Clinical Chemistry and Hematology, University Health-related Center Utrecht, Utrecht, Netherlandsb aAstraZeneca, molndal, Sweden; bAstraZeneca, M ndal, AstraZeneca, Molndal, Sweden; dAstraZeneca, Macclesfield, UKSweden;Introduction: Cell engineering is amongst the most common approaches to modify extracellular vesicles (EVs) for therapeutic drug delivery. Engineering can be applied to optimize cell tropism, targeting, and cargo loading. Within this study, we screened several EV proteins fused with EGFP to evaluate the surface display of the EV-associated cargo. Additionally, we screened for EV proteins that could effectively website traffic cargo proteins into the lumen of EVs. We also developed a novel technology to quantify the amount of EGFP molecules per vesicle employing total internal reflection (TIRF) microscopy for single-molecule investigation. Techniques: Human Expi293F cells were transiently transfected with DNA constructs coding for EGFP fused for the N- or C-terminal of EV proteins (e.g., CD63, CD47, Syntenin-1, Lamp2b, Tspan14). 48 h just after transfection, cells had been analysed by flow cytometry and confocal microscopy for EGFP expression and EVs had been isolated by differential centrifugation followed by separation gp130/CD130 Proteins site utilizing iodixanol density gradients. EVs have been characterized by nanoparticle tracking evaluation, western blotting, and transmission electron microscopy. Single-molecule TIRF microscopy was made use of to ascertain the protein quantity per vesicle at aIntroduction: Improvement of extracellular vesicles (EVs) as nanocarriers for drug delivery relies on loading a substantial level of drug into EVs. Loading has been performed from the simplest way by co-incubating the drug with EVs or producer cells till working with physical/chemical methods (e.g. electroporation, extrusion, and EV surface functionalization). We use physical system combining gas-filled microbubbles with ultrasound generally known as sonoporation (USMB) to pre-load drug inside the producer cells, which are at some point loaded into EVs. Strategies: Cells were grown overnight in 0.01 poly-Llysine coated cell culture cassette. Before USMB, cells have been starved for 4 h. Remedy medium containing microbubbles and 250 BSA-Alexa Fluor 488 as a model drug was added for the cells grown in the cassette. Cells were exposed straight to pulsed ultrasound (ten duty cycle, 1 kHz pulse repetition frequency, and one hundred s pulse duration) with as much as 845 kPa acoustic pressure. Right after USMB, cells had been incubated for 30 min and then therapy medium was removed.ISEV2019 ABSTRACT BOOKCells were washed and incubated within the culture medium for two h. Afterward, EVs in the conditioned medium have been CD131 Proteins Purity & Documentation collected and measured. Benefits: Cells took up BSA-Alexa Fluor 488 immediately after USMB treatment as measured by flow cytometry. These cells released EVs in the conditioned medium which had been captured by anti-CD9 magnetic beads. About 5 in the CD9-positive EVs contained BSAAlexa Fluor 488. The presence of CD9-positive EVs containing BSA also had been 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 form of drugs are going to be investigated to additional optimize.