Artemizia Évora ^1,2 Gonçalo Garcia ^2,3 Ana Rubi ² Eleonora De Vitis ⁴ Ana Teresa Matos ² Ana Rita Vaz ^2,3 Francesca Gervaso ⁴ Giuseppe Gigli ^4,5 Alessandro Polini ⁴ Dora Brites ^2,3*

• ¹ University of Lisbon, Lisbon, Portugal
• ² Neuroinflammation, Signaling and Neuroregeneration Lab, Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
• ³ Department of Pharmaceutical Sciences and Medicines, Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
• ⁴ Institute of Nanotechnology, Department of Physical Sciences and Technologies of Matter, National Research Council (CNR), Lecce, Apulia, Italy
• ⁵ Dipartimento di Medicina Sperimentale, Università Del Salento, Lecce, Italy

Background: Alzheimer's disease (AD), a complex neurodegenerative disease associated to ageing, is the leading cause of dementia. Few people with early AD are eligible for the new FDA approved drug treatments. As so, new tools and early diagnosis markers are required to predict subtypes, individual stages and the most suitable personalized treatment. We previously demonstrated that the regulation of microRNA(miR)-124 is crucial for proper neuronal function and microglia reshaping in human AD cell models. Objective: The aim of this study was to develop an efficient miR-124-3p-loaded exosome strategy and validate its therapeutic potential in a multi-compartment microfluidic device of neuron-glia crosstalk recapitulating age-AD pathological features. Methods and Results: Using cortical microglia from mouse pubs, separated from glia mixed cultures and maintained for 2 days in-vitro (stressed-microglia), we tested the effects of SH-SY5Y-derived exosomes loaded with miR-124-3p mimic either by their direct transfection with Exo-Fect TM (ET124) or by their isolation from the secretome of miR-124 transfected cells (CT124). ET124 revealed the better delivery efficiency and the most potent effects in improving stress-microglia status. Tricultures of human SH-SY5Y neuroblastoma cells (SH-WT) were established in the presence of human microglia cell line (HMC3) and immortalized human astrocytes (IM-HA) in tricompartimentalized microfluidic devices. Replacement of the SH-WT cells by those transfected with APP695 (SH-SWE) in the tricultures and addition of low doses of hydrogen peroxide were used to simulate late-onset AD.The system showed to mimic AD-associated neurodegeneration and neuroinflammation processes. Notably, ET124 exhibited neuroprotective properties across the three cell types in the AD milieu by preventing neuronal apoptosis and neurite deficits, redirecting microglial profiles toward a steady state, and attenuating the inflammatory and miRNA fingerprints associated with astrocyte reactivity. Conclusion: To the best of our knowledge, this is the first study supporting the neuro-and immunoprotective properties of miR-124-engineered exosomes in a microfluidic triculture platform recapitulating age-related susceptibility to AD. Our system offers potential to develop personalized medicines in AD patient subtypes.