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

ABSTRACT

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.