Validating a Bottom-Up Strategy for the Synthesis of EV Mimetics for Drug Delivery

Abstract

Liposomes are synthetic vesicles and have been extensively researched for their use as versatile drug carriers. However, they face challenges such as limited targeting and rapid clearance by the reticuloendothelial system (RES). Alternatively, Extracellular Vesicles (EVs) are naturally occurring cell-derived nanosized vesicles that mediate intercellular communication and offer natural transportation, targeting and biocompatibility. Unfortunately, their complex inherent surface structure and core, gives rise to challenges concerning heterogeneity, like their characterization and large-scale production for drug delivery applications. To address these limitations, Extracellular Vesicle Mimetics (EV mimetics) have been proposed as a less complex synthetic alternative, containing specific functional surface modifications to emulate the characteristics of EVs. Previous research presented a scalable bottom-up strategy for the synthesis of EV mimetics, which comprised the coupled cell-free expression and insertion of the protein Cluster of Differentiation 47 (CD47) into the liposomal membrane. CD47 is cellular protein that prevents phagocytotic uptake by macrophages and monocytes upon binding to signal-regulatory protein (SIRP) α. However, due to its role within the immune system, demonstration of CD47 EV mimetic functionality could not be performed in vitro. Therefore, we aimed at assessing the intact protein functionality by the synthesis of other EV mimetics, whilst examining the versatility that the bottom-up synthesis strategy offers. In this study, CD47, Cluster of Differentiation 39 (CD39) and N-Cadherin (N-Cad) EV mimetics were successfully synthesized, purified and characterized with Western Blotting and Transmission Electron Microscopy. (TEM). Additionally, immuno-Electron Microscopy and/or membrane permeabilization revealed the proper topology of CD47 and CD39 in the liposomal membrane. Furthermore, we demonstrate the ability to co-insert multiple proteins and use variable liposome formulations. Finally, purified N-Cad EV mimetics, containing fluorescent lipids, were taken up in MDA-MB-231 cells as demonstrated by means of Fluorescence-activated Cell Sorting (FACS), whereas a D134A mutation of N-Cadherin inactivated the protein and reduced EV mimetic cell-uptake significantly. Thereby, it provides evidence for functional EV mimetics. In conclusion, we present a tuneable bottom-up strategy for the synthesis of functional EV mimetics and thereby pave the way for their clinical applicability within drug delivery.