Bioengineered Bacterial Outer-membrane Vesicles as Nanoantibiotics

The rise of antibiotic-resistant pathogens poses a serious threat to global health, highlighting the urgent need for alternative therapeutic strategies beyond traditional antibiotics. This dissertation explored the bioengineering avenues for cargo loading, hybridization, and surface modification of extracellular vesicles (EVs) as biomimetic nanoparticles for developing novel antibiotic delivery systems. EVs are small, membrane-bound particles released by cells that carry proteins, lipids, and nucleic acids to mediate communication and influence biochemical processes in neighboring or distant cells. In bacteria, EVs are classified and named due to their origin: membrane vesicles (MVs) and outer membrane vesicles (OMVs), which bud from the membrane and outer membrane of Gram-positive and Gram-negative bacteria, respectively. In the first working package, we explored a novel approach for combining bacteriomimetic liposomes with outer membrane vesicles (OMVs) derived from Myxobacteria—as adaptable vehicles for targeted drug delivery. Comparative assessments of their physicochemical and biological characteristics, including their size, charge, and protein content, as well as interactions with immune cells, intestinal barriers, and bacterial biofilms, demonstrate unique benefits. The hybrid nanocarriers proved particularly effective at transporting antibiotics to intracellular locations, while OMVs from the Myxobacteria strain SBSr 073 showed an ability to evade immune detection and sustain drug presence in extracellular environments. To further facilitate clinical application, the study refined antibiotic loading techniques for SBSr 073 OMVs and investigated how OMVs from the Cbfe 23 strain influence the gut microbiota in mice over short- and long-term periods. In the second working package, we explored the possibility of developing a process based on bioorthogonal chemistry, strain-promoted azide-alkyne cycloaddition and inverse electron demand Diels–Alder reactions, for surface engineering of EVs independent of their origin and composition. In the first step, we successfully confirmed the functionality of diazotransfer method using imidazole-1-sulfonyl azide hydrochloride for transforming amines from surface proteins of Myxobacteria OMVs to azide. Then, we evaluated three chemical linkers with azide-reactive tail, two with a transcyclooctene and one with a norbornene head, to react with a methyltetrazine-containing targeting moiety (either biotin or vancomycin). We observed that the norbornene-containing linker resulted in a more robust OMV surface modification compared to transcyclooctene. Finally, we compared the physicochemical characteristics of pristine and surface-engineered Sga 15 OMVs and confirmed the superior antibiotic activity of the vancomycin-decorated OMVs against a Gram-positive pathogen model in a proof-of-concept study. Together, the results emphasize the therapeutic potential of pristine and engineered Myxobacterial OMVs, as well as their synthetic hybrids, as innovative tools for targeting pathogenic bacteria.