Genetically Engineered Bacterial Outer Membrane Vesicles with Expressed Nanoluciferase Reporter for in Vivo Bioluminescence Kinetic Modeling through Noninvasive Imaging.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria play significant roles in the biomedical field as they can be facilely functionalized using genetic engineering tools and thus often serve as a versatile multifunctional nanoparticles for a variety of applications. In this study, we investigated the multifaceted bioluminescence kinetics of a NanoLuc luciferase-expressed outer membrane vesicle produced by E. coli . This multifunctional OMV emits strong blue luminescence at 460 nm after mixing with the substrate furimazine, which potentially can be used for bioluminescence-based optical imaging. Characterization of the vesicles was performed via dynamic light scattering and nanoparticle tracking analysis. A murine animal model was used to observe the in vivo behavior of the bioluminescence produced by outer membrane vesicles through post subcutaneous administration. The bioluminescence signal was tracked by noninvasive in vivo optical imaging, while in vitro cytotoxicity and ex vivo tissue histopathology were studied to demonstrate the biocompatibility of the engineered OMVs. A theoretical model was also developed to simulate the relevant enzyme-substrate reaction kinetics along with absorption of the in vivo system. The interplay of the reaction and absorption is in good agreement with the experimental results. The study shows a great potential of the genetically engineered vesicles as an interesting class of functional nanomaterials for imaging-related biomedical applications.