Intranasal Delivery of 3D-Bioprinted Mesenchymal Stem Cell-Derived Extracellular Vesicles Targeting Aβ Deposition and Neuroinflammation for Alzheimer’s Disease Therapy

Neurons and the broader central nervous system present a significant challenge for therapeutic delivery due to the presence of the blood-brain barrier. Extracellular vesicles (EVs) derived from mesenchymal stem cells have emerged as promising nanocarriers for drug delivery and intercellular communication, with inherent therapeutic potential for neurodegenerative diseases such as Alzheimer's disease (AD). The bioactivity and targeting efficiency of these EVs are critically influenced by the physiological state of their parent cells. Here, we posited that the three-dimensional (3D) bioprinting culture microenvironment could enhance the therapeutic profile of EVs derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) compared to those from conventional two-dimensional (2D) culture. We successfully generated and characterized EVs from both 2D-cultured and 3D-bioprinted hUC-MSCs (2D-EVs and 3D-EVs). Intranasal administration of 3D-EVs demonstrated superior brain targeting, achieving higher concentrations and longer retention times in mouse brain compared to intravenous delivery, with no observed organ toxicity. In APP/PS1 AD model mice, intranasal delivery of 3D-EVs significantly reduced amyloid-beta plaques, suppressed neuroinflammation by inhibiting microglial and astrocytic activation, and rescued spatial learning and memory deficits. Integrated miRNA sequencing and proteomic analysis of the EVs revealed a distinct molecular cargo, including elevated levels of miRNAs such as hsa-miR-182-5p and hsa-miR-30c-5p, and specific proteins including APP and APOE, which are implicated in the observed therapeutic effects. Therefore, our results support the argument that 3D-bioprinted EVs, owing to their enhanced biological properties and efficient non-invasive brain delivery, represent a potent and translatable therapeutic strategy for Alzheimer's disease.