Targeted Inhibition of Intracellular Coxiella burnetii by Overcoming the Vacuolar Barrier with Autologous Exosomal Doxycycline

Abstract Coxiella burnetii, the causative agent of Q fever, is an obligate intracellular bacterium that replicates within the Coxiella-containing vacuole (CCV), a lysosome-like compartment. The acidic microenvironment within the CCV impairs the activity of weak-base antibiotics such as doxycycline, necessitating prolonged, high-dose therapy. To overcome this fundamental delivery barrier, we engineered a biomimetic nanoplatform by encapsulating doxycycline (DOX) within exosomes derived from THP-1 cell-derived macrophages (tExo). DOX@tExo nanoparticles displayed characteristic exosomal morphology and protein markers and demonstrated favorable biocompatibility both in vitro and in vivo. Importantly, DOX@tExo were efficiently internalized by macrophages, trafficked specifically to lysosomes, and accumulated within CCVs. This precise targeting resulted in an enhancement of antibacterial potency, reducing the minimal effective concentration for marked inhibition of intracellular bacterial growth to 0.015 μg/mL, more than an order of magnitude lower than that of free DOX. Beyond direct drug delivery, DOX@tExo treatment concurrently reduced autophagic cargo delivery, as indicated by decreased p62 accumulation, thereby potentially disrupting a host process coopted by the pathogen. In a murine model of pulmonary infection, DOX@tExo treatment significantly reduced bacterial burdens in multiple organs. Collectively, this work establishes a homologous exosome platform derived from THP-1 cells that combines targeted antibiotic delivery with host-directed immunomodulation, offering a therapeutic strategy against refractory intracellular bacterial infections.