Engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery.

The low yield of extracellular vesicle (EV) secretion is a major obstacle for mass production and limits their potential for clinical applications as a drug delivery platform. Here, we mass produced engineered extracellular vesicles (eEVs) by fusing the surface composition of EVs with lipid-based materials via a membrane extrusion technique. A library of lipids (DOTAP, POPC, DPPC and POPG) was fused with EVs to form a hybrid-lipid membrane structure. Uniform lamellar vesicles with a controlled size around 100 nm were obtained in this study. Particle number characterization revealed this extrusion method allowed a 6- to 43-fold increase in numbers of vesicles post- isolation. Further, exogenous siRNA was successfully loaded into engineered vesicles with ~ 15% - 20% encapsulation efficiency using electroporation technique. These engineered extracellular vesicles sustained a 14-fold higher cellular uptake to lung cancer cells (A549) and achieved an effective gene silencing effect comparable to commercial Lipofectamine RNAiMax. Our results demonstrate the surface composition and functionality of EVs can be tuned by extrusion with lipids and suggest the engineered vesicles can be a potential substitute as gene delivery carriers while being able to be mass produced to a greater degree with retained targeting capabilities of EVs.