Bottom-Up Synthesis and Purification of Extracellular Vesicle Mimetics.

Extracellular vesicles (EVs) are nanosized particles secreted by all cell types. As EVs are naturally occurring carriers of biological cargo, they serve as a promising candidate for drug delivery applications. Potential advantages of EVs as drug delivery systems include biological stability, intrinsic targeting properties and ability to overcome natural barriers. However, limitations such as cumbersome production and isolation procedures, batch-to-batch variability, and challenges related to efficient cargo loading limit their potential for clinical applications. Here, we introduce EV mimetics, prepared by incorporating full-length membrane proteins in the lipid bilayer of liposomes, using cell-free protein synthesis. These structures mimic functional characteristics of EVs, while offering advantages in terms of ease of manufacture, controllability and potential for efficient cargo loading. To demonstrate the feasibility of producing proteoliposomes as EV mimetics, we selected EV-associated CD47, CD39 and N-Cadherin as model proteins. We show successful production and purification of CD47, CD39 and N-Cadherin containing EV mimetics. Additionally, for CD47, we show that reaction conditions can be tailored to enhance EV mimetic yield. Furthermore, proteinase K protection assays and immuno-labelling electron microscopy revealed that correct membrane protein topology is preserved for CD47 and CD39. N-Cadherin EV mimetics show enhanced uptake by N-Cadherin-expressing MDA-MB-231 cells, proving membrane protein functionality is preserved. We demonstrate the versatility of the methodology by producing EV mimetics using a wide variety of liposomal formulations. Finally, we show that two distinct membrane proteins can be inserted in the same EV mimetic, further indicating versatility and broad applicability. This study presents a modular and controllable strategy for cell-free synthesis of functional EV mimetics, which provides a meaningful step toward addressing challenges in EV-inspired drug delivery development.