Intranasal Delivery of Engineered Neural Stem Cell-Derived Extracellular Vesicles Enables Targeted Delivery of Liraglutide to the Brain

Abstract

Liraglutide (LG), a glucagon-like peptide-1 (GLP-1) receptor agonist, is approved for treating type 2 diabetes mellitus and as a weight loss drug in obesity. Recently, LG has emerged as a promising neuroprotective agent in suppressing neuroinflammation by modulating disease-associated inflammatory pathways in various diseases. However, its therapeutic application in neurological disorders is hindered by the limited penetration across the blood–brain barrier (BBB), necessitating innovative strategies for efficient brain-targeted delivery. In this study, we engineered neural stem cell-derived extracellular vesicles (EVs) with liraglutide (LG-EVs) using a scalable microfluidics-based reconstruction approach for brain-targeted intranasal delivery of LG. We characterized the reconstructed EVs for their size, surface charge, and the integrated LG by NTA, zeta potential analysis, and flow cytometry. The engineered LG-EVs retained vesicle sizes within the expected nanoscale range, with an average size of 138 ± 4.9 nm, while showing a shift in surface charge relative to unmodified EVs (EV: −10.0 ± 0.48 mV; LG-EV: −12.5 ± 1.43 mV) and a detectable liraglutide-associated flow cytometry signal on EV preparations, consistent with successful engineering. In vitro results showed enhanced uptake of LG-EVs by macrophages, neural stem cells, and SH-SY5Y neuronal cells compared to unmodified EVs, indicating successful targeting effects of LG-EVs. In vivo results of the intranasal administration of LG-EVs into both noninflammatory and inflammatory mouse models showed improved accumulation of LG-EVs in the brain than bare EVs, indicating targeting effects of LG-EVs, which significantly facilitated increased uptake in the brain. Additionally, we did not notice any histopathological alterations in the major organs of mice after administration of LG-EVs, indicating the nontoxic nature of the formulation. In addition, we developed a lyophilized LG-EVs formulation with cyclodextrin-derived cryoprotectants, which showed no significant changes in size, surface charge, and particle count, suggesting preserved physicochemical stability during storage. Overall, our findings demonstrate the successful development of LG-EVs as a formulation for the intranasal delivery of LG to the brain, highlighting this as a promising platform for delivering anti-inflammatory drugs to the central nervous system (CNS) and supporting its further expansion for future therapeutic applications.