Nanobody‐Modified Outer Membrane Vesicles Attenuate Alzheimer's Disease‐Like Neurodegeneration by Modulating Mitophagy‐Associated Phenotypes and cGAS‐STING‐Related Inflammatory Signaling

This study examined anti-transferrin receptor (TfR) nanobody-functionalized bacterial outer membrane vesicles (OMVs) as an engineered platform for improving Alzheimer's disease (AD)-like neuronal injury. Escherichia coli-derived OMVs were characterized for morphology, size distribution, surface charge, protein features, nanobody loading, residual endotoxin levels, endotoxin-associated inflammatory responses, and repeated-exposure immune-safety readouts, and were evaluated in neuronal-related cells and APP/PS1 mice. Engineered OMVs showed receptor-associated uptake and preferential brain-delivery signals, increased HT-22 cell viability, reduced apoptosis, and elevated mitophagy-associated markers including LC3-II, PINK1, and Parkin. OMV treatment also reduced cytosolic mtDNA abundance, DNA/cGAS colocalization, phosphorylation of STING/TBK1/IRF3, and inflammatory-marker expression. PINK1 or Parkin knockdown partially weakened OMV-associated mitochondrial and inflammatory readouts, whereas pharmacological autophagy modulation supported a contribution of autophagy-related processes without establishing selective PINK1/Parkin-dependent mitophagy in vivo. In APP/PS1 mice, OMV administration improved Morris water maze and rotarod performance and alleviated neuroinflammatory and histological injury-related changes. Overall, anti-TfR nanobody-modified OMVs were associated with enhanced receptor-related delivery, mitophagy-linked phenotypes, reduced cytosolic mtDNA/cGAS-STING-related inflammatory signaling, and improved AD-like outcomes, supporting further delivery-system optimization and mechanistic validation.