(17e) Biocompatible Genetically-Engineered Outer Membrane Vesicles with Expressed Nanoluc Reporter: Preparation, Characterization and In Vivo Kinetic Modeling

Outer membrane vesicles produced by Gram-negative bacterial play significant part in the biomedical field as they can be functionalized by genetic engineering tools, and thus often serving as a versatile template for a variety of applications. We investigated the multifaceted bioluminescence kinetics of a novel NanoLuc® luciferase-loaded outer membrane vesicle produced by E. coli. This multi-functionally engineered vesicle can display strong blue luminescence at 460 nm after mixing with the coelenterazine substrates, which potentially can be used for bioluminescence optical imaging. Characterization of the vesicles was performed via dynamic light scattering and nanoparticle tracking analysis. For the post in vitro luminescence kinetics, a murine animal model was used to observe the in vivo behavior of the bioluminescence produced by outer membrane vesicles post subcutaneous administration. Bioluminescence signal was tracked by non-invasive in vivo optical imaging, and in vitro cytotoxicity and in vivo tissue histopathology were utilized to demonstrate the great biocompatibility of this system. A theoretical model was developed to simulate the relevant enzyme-substrate reaction parameters along with absorption kinetics of the in vivo system. The interplay of the reaction and absorption agrees well with the experimental results, showing a great potential of these genetically engineered vesicles as a new class of functional materials for abounding biomedical applications.