Te srl, Turin, Italyb aIntroduction: Extracellular vesicles (EVs) are particles released by cells that carry a complicated cargo of molecules and mediate intercellular communication. Not too long ago, they have raised good interest as drug delivery SIRT6 site systems and various engineering methods are at the moment beneath investigation. A lot of elements, having said that, influence the transfection yield, such as protocol variability and EV damage. Methods: The electroporation was investigated as system to directly load miRNAs in plasma-derived EVs. Different parameters (voltage and quantity of pulses) were compared for their effect on EV morphology and loading capacity of a synthetic miRNA, cel-39, like miRNA enrichment in EVs and its transfer to target cells. Subsequent, analyses have been performed to evaluated the transfection impact on EV endogenous cargo and also the exogenous miRNA protection from RNAse degradation. Then, EVs had been loaded with antitumour miRNAs and their proapoptotic impact was evaluated on a cell line of hepatocellular carcinoma, HepG2 cells.JOURNAL OF EXTRACELLULAR VESICLESResults: The comparison of various electroporation settings demonstrated the significance of selecting the far more appropriate protocol parameters to obtain an efficient EV transfection yield, understood as each molecules loading and EV harm. In particular, we observed the superiority of one electroporation protocol (employing 750 Volt and 10 pulses) that allowed essentially the most effective miRNA packaging and transfer to target cells, devoid of structurally damaging EVs. The most efficient electroporation protocol was also confirmed to enable a additional efficient miRNA loading in respect to incubation, superior guarding miRNA from enzymatic digestion. Moreover, our findings suggested that electroporation preserved the na e EV cargo, such as RNAs and proteins, and didn’t alter their uptake in cells. EVs engineered with antitumor miRNAs (miR-31 and miR-451a) effectively promoted the apoptosis of HepG2 cells, downregulating their target genes connected to apoptotic pathways. Summary/Conclusion: In conclusion, our findings indicate an efficient and functional miRNA encapsulation in plasma-derived EVs following an electroporation protocol that preserves EV integrity. Funding: Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), Unicyte AG (Switzerland)PS01.Development of a platform for exosome engineering utilizing a novel and selective scaffold protein for surface display Kevin PI3KC3 list Dooley, Ke Xu, Sonya Haupt, Shelly Martin, Russell McConnell, Nuruddeen Lewis, Christine McCoy, Chang Ling Sia, Jorge Sanchez-Salazar, Nikki Ross, Rane Harrison, Bryan Choi, Damian Houde, John Kulman and Sriram Sathyanarayanan Codiak BioSciences, Cambridge, USAfragments thereof were expressed in a cell line plus the minimum PrX domain requirements for exosomal enrichment had been determined. Leveraging PrX as a scaffold for exosome surface display, we created our engEx platform to generate engineered exosomes functionalized with a selection of pharmacologic payloads including enzymes, antibodies, variety I cytokines and TNF superfamily members. Biological activity of these engineered exosomes was assessed in an array of in vitro assays and when compared with previously described scaffolds. Outcomes: Stable expression of PrX in an exosome producing cell line resulted in 200-fold enrichment of PrX on secreted exosomes. Interestingly, overexpression of PrX structural paralogs didn’t result in similar levels of enrichment, suggesting PrX is special. Exos.