Amplifying Engineered Bacterial Outer Membrane Vesicle Production Using Functional Peptidoglycan Inhibitors

Outer membrane vesicles (OMVs) are promising candidates for biomedical applications. However, their widespread use has been limited by low productivity and suboptimal therapeutic effects. In this study, we introduce an effective chemical method using designed boosters to enhance endogenous OMV generation, achieving up to a 65-fold increase through a simple one-step coculture process. These boosters are formed by conjugating 4-(naphthalen-2-yl)-4-oxobutanoic acid (NA) with different compounds, allowing for the customizable production of either natural or functionalized OMVs. Furthermore, the resulting functionalized OMVs exhibit good photodynamic treatment potential for both in vitro and in vivo experiments. Detailed mechanism study suggests that upon the entry of boosters into bacteria, the synthesis of peptidoglycan was inhibited, leading to membrane protrusion and OMV release. Importantly, this strategy is successfully validated across five classical bacterial strains, demonstrating the versatility of the boosters. This work provides a simple, effective, and universal method to enhance the generation of OMVs, offering new insights for the development of biomanufacturing platforms in cancer therapy.