Te srl, Turin, Italyb aIntroduction: Extracellular vesicles (EVs) are particles released by cells that carry a complex cargo of molecules and mediate intercellular communication. Recently, they’ve raised good interest as drug delivery systems and a number of engineering techniques are currently beneath investigation. Quite a few variables, even so, influence the transfection yield, which includes protocol variability and EV damage. Approaches: The electroporation was investigated as system to directly load miRNAs in plasma-derived EVs. Diverse parameters (voltage and quantity of pulses) have been compared for their impact on EV morphology and loading capacity of a synthetic miRNA, cel-39, which includes miRNA enrichment in EVs and its MCAM/CD146 Proteins custom synthesis transfer to target cells. Subsequent, analyses were performed to evaluated the transfection impact on EV endogenous cargo and the exogenous miRNA protection from RNAse degradation. Then, EVs were 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 diverse electroporation settings demonstrated the value of deciding upon the far more acceptable protocol parameters to obtain an effective EV transfection yield, understood as each molecules loading and EV damage. In certain, we observed the superiority of one particular electroporation protocol (using 750 Volt and 10 pulses) that permitted probably the most effective miRNA packaging and transfer to target cells, without having structurally damaging EVs. One of the most effective electroporation protocol was also verified to enable a far more effective miRNA loading in respect to incubation, improved guarding miRNA from enzymatic digestion. Also, our findings suggested that electroporation preserved the na e EV cargo, such as RNAs and proteins, and did not alter their uptake in cells. EVs engineered with antitumor miRNAs (miR-31 and miR-451a) successfully promoted the apoptosis of HepG2 cells, downregulating their target genes connected to apoptotic pathways. Summary/Conclusion: In conclusion, our findings indicate an effective 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 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 have been expressed inside a cell line and the minimum PrX domain specifications for exosomal enrichment have been determined. Leveraging PrX as a scaffold for exosome surface display, we developed our engEx platform to produce engineered exosomes functionalized having a variety of pharmacologic payloads like enzymes, antibodies, form I cytokines and TNF superfamily members. Biological activity of those engineered exosomes was assessed in an array of in vitro assays and compared to previously described scaffolds. Final results: Steady expression of PrX in an exosome making cell line resulted in 200-fold enrichment of PrX on secreted exosomes. Interestingly, overexpression of PrX structural paralogs did not lead to equivalent levels of enrichment, CD217 Proteins Biological Activity suggesting PrX is one of a kind. Exos.
