Targeted Delivery of mGluR5 Agonists via Engineered hucMSC-Derived EVs to Enhance Macrophage Function and Mitigate Glutamate-Induced Toxicity

Extracellular vesicles (EVs) are emerging as a potential drug delivery system for neuroinflammatory diseases due to their ability to navigate natural barriers, maintain stability, and facilitate cell-cell signaling. However, their limited targeting ability often restricts their therapeutic efficacy, necessitating enhancements to enable precise targeting of crucial macrophage cells. To address this issue, we genetically modified EVs derived from human umbilical cord mesenchymal stem cells (hucMSCs) using a glycosylated pcDNA3.1-GNSTM-RGV-Lamp2b-HA plasmid to enhance targeted drug delivery to nAchR-expressing macrophages. Through the optimization of hucMSC isolation and transfection protocols, we produced high-yield, surface-modified EVs. Ζ-potential analysis confirmed the stability of these modified EVs, which retained their inherent characteristics, including size, membrane protein expression (TSG101, Flotillin), and morphology. Further, coculture experiments with macrophages evaluated the uptake of modified EVs labeled with PKH67, showing significantly enhanced macrophage uptake. To validate their use in targeted drug delivery, we loaded the EVs with (R, S)-2-chloro-5-hydroxyphenylglycine (CHPG), which is a mGluR5 agonist. We noted that CHPG-loaded EVs increased macrophage proliferation, enhanced macrophage migration, and significantly reduced wound open area in coculture assays. Furthermore, CHPG-loaded modified EVs reduced macrophage cell death and facilitated macrophage entry into the S-phase, mitigating oxidative stress-induced cell cycle arrest under glutamate toxicity. Together, these results highlight the therapeutic potential of hucMSC-derived EV-based drug delivery systems for treating neuroinflammatory and neurodegenerative diseases.