Research on nanomaterials in the past decades has undergone explosive growth. Although recent progress in nanomanufacturing technologies led to successfully large-scale manufacturing of a range of inorganic and organic nanostructured materials, the extension of those manufacturing processes to the production of advanced functional bio-nanoparticles (bio-NPs) is currently an unmet challenge. This is mainly due to the sensitivity of bio-NPs to the harsh conditions of current manufacturing processes. The goal of this proposal is to design an advanced biomanufacturing process to manufacture genetically engineered multi-functional bio-NPs. The local and global structure of the advanced bio-NPs will be monitored and characterized, and their functionality and utility will be examined and validated in bioimaging of brain tumor in mouse model.
Our hypothesis is that by employing genetically engineered vesicle-forming bacteria as microbial cell factories, we will reliably produce uniform bio-NPs with precisely controlled biological functionality in a continuous and scalable way. To accomplish it, recombinant DNA technology will first be used to design novel genetically engineered protein multi-functional bio-NPs for capture and detection functions in bioimaging. The bio-NPs are lipid-based outer membrane vesicles (OMVs) with a uniform diameter of ~50 nm and the outer leaflet of the bilayer is decorated with novel engineered protein fusion, endowing multi-functionality. The OMVs, co-displaying multiple copies (~50, each) of super-active NanoLuc luciferase enzyme (~150-fold more active than other luciferases), will contain (i) an antibody-binding domain (ZZ domain) for anchoring antibodies of interest, and (ii) a thermo-responsive elastin-like protein domain (ELP, soluble at room temperature and insoluble/aggregated at 42 °C) for simple purification of the OMVs via size filtration. A fermentation process integrated with two-stage size filtration will then be designed for continuous, sustainable production of multi-functional OMVs at a large quantity.