Isolation, characterization, and cargo-loading of small extracellular vesicles.

Abstract

Small extracellular vesicles (sEVs) are natural lipid-enclosed vesicles that are secreted from living cells and act as mediators of intercellular communication. They are biocompatible nanovesicles that can transport a range of bioactive molecules, including nucleic acids, to target tissues. mRNA-loaded sEVs deliver functional expression of encoded proteins in vitro and in vivo. They are therefore emerging as promising alternatives to synthetic lipid nanoparticles for the delivery of therapeutic compounds such as mRNA. The current study aimed to isolate and characterize sEVs generated from human embryonic kidney (HEK293) cells and evaluate their potential to delivery mRNA in vitro. Vesicles were isolated from the conditioned media of HEK293 cells using a polyethylene glycol (PEG 6000) based precipitation approach and characterized using tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM) and western blotting. sEVs with a mean diameter of 119 ± 16 nm and an average concentration of 349,14 x 109 particles/ml were successfully isolated and found to have a cup-shaped morphology and contain specific sEV proteins, including CD63, TSG101, and flotillin-1. Cytotoxicity was assessed using the MTS assay to confirm lack of vesicle toxicity for subsequent mRNA delivery. Lipofectamine, a widely used transfection reagent, was used as a positive control to introduce eGFP mRNA into HEK293 cells and compare GFP expression to that following incubation and delivery with sEVs. sEVs were able to deliver mRNA as evidenced by GFP expression in recipient cells at 24-hour post-incubation; strong expression was evident until 48 hours. In conclusion, HEK293-derived sEVs represent a feasible and minimally cytotoxic mRNA delivery system. The use of PEG 6000 for sEV isolation provided a scalable and costeffective approach, enabling further exploration of sEV therapeutic potential. The characterization methods and optimized mRNA loading protocols in this study provide valuable insight for advancing sEV-based therapeutic applications. Further studies should focus on optimizing cargo loading to improve delivery efficiency and evaluating long-term in vivo safety.