Intranasal Delivery of Bacterial Extracellular Vesicles Enables RNA Cargo Entry Into the Brain

ABSTRACT Extracellular vesicles (EVs) released by bacteria are potent mediators of host–microbe interactions. They modulate immune responses, deliver functional molecules and influence disease progression. However, whether bacterial EVs can access the brain and functionally affect host cells remains unclear. In this study, we engineered Escherichia coli ‐derived EVs by electroporating Cre recombinase mRNA (Ec EV Cre ) and assessed their transport and functional delivery following intranasal administration. Using mT/mG reporter mice, we observed EV uptake in the olfactory epithelium and recombination‐driven GFP expression in a subset of neurons in the olfactory bulb, providing proof‐of‐concept for the functional delivery of bacterial EV‐associated mRNA into the brain. Single‐cell RNA sequencing and imaging analyses of the olfactory regions revealed neuronal and immune cell subsets as key EV targets. Microfluidic biochip chamber assays with cultured sensory neurons demonstrated that EVs undergo retrograde axonal transport from neurite terminals to the soma via signalling endosomes. Pharmacological inhibition significantly impaired EV uptake, supporting the involvement of endocytic pathways. In addition to neuronal entry, we discovered that phagocytic cells, including neutrophils and macrophages, can engulf EV Cre in the nasal mucosa and migrate into the brain, providing an alternative immune‐mediated route for vesicle delivery. Together, these findings indicate that bacterial EVs exploit both neuronal and phagocytic pathways to deliver functional RNA cargo into the brain, providing novel insights into microbial access to the central nervous system and its implications for neuroimmune interactions.