Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles

Advancedintracellular delivery of proteins has profound applicationsin both scientific investigations and therapies. However, existingstrategies relying on various chemical and physical methods have drawbackssuch as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins.Developing new delivery systems integrating the enveloping and labelingof target proteins would bring great advantages for efficient proteintransfections. Here, we enriched a high concentration (62 mg/mL) ofseveral target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property ofOMVs to deliver proteins into the cytosol of eukaryotic cells. Theresults revealed a high protein transfection efficiency from 90 to97% for different cell lines. Moreover, the free penetration of moleculesless than 600 Da across the membrane of OMVs allows direct labelingof target proteins within OMVs, facilitating the visualization oftarget proteins. Importantly, the nanobody delivered intracellularlyby OMVs retains the biological activity of binding with its target,highlighting the advantages of OMVs as an emerging tool for efficientintracellular delivery of proteins.