Prototyping Minimal Extracellular Vesicle Mimetics Using Cell-Free Synthesis

The many surface proteins on extracellular vesicles (EVs) allow them to target recipient cells and modulate cellular responses. Despite their importance, relating surface protein and EV function is challenging due to surface protein heterogeneity. Here, we create a bottom-up, cell-free protein-synthesis platform to engineer artificial nanovesicles (ANVs) that display different EV surface protein domains. The platform is termed VESSEL (<u>V</u>esicle <u>E</u>ngineering <u>S</u>ystems using <u>S</u>ynthetic <u>E</u>xpression and <u>L</u>oading). The surface proteins are selected based on proteomics data of native EVs from placental mesenchymal stem cells (PMSCs). To create VESSEL, we establish a protein anchor based on the bacteria membrane protein Aquaporin-Z. This anchor allows the flexible and cell-free protein synthesis of 39 different EV surface protein domains, each anchoring into more than 10⁸ ANVs per μL. Furthermore, we measure the ANVs using high-fidelity assays, including single-ANV flow cytometry, super-resolution imaging, and vesicle-based ELISA. Next, we show the impact of each EV surface protein on cellular uptake. Specifically, we find that certain EV surface protein domains govern ANV uptake into HEK293FT cells, explaining the variable observations in the field. We discovered new proteins, such as CADM1 and NPTN, that mediate high-efficiency cellular uptake. Additionally, five proteins were selected for our neuroprotection assay, where three proteins were significant in increasing SH-SY5Y neurite growth. Our work demonstrates a high-throughput cell-free synthesis platform for studying surface proteins of EVs. It enables the systematic interrogation of EV's function as "signalosomes" and facilitates the designing of well-defined EV mimetics to mediate cellular function.