Engineering and Fabrication of Immunomodulatory Lipid Nanoparticles (LNPs) for mRNA Delivery to Modulate Microglial Cell Activation

This study engineered hyaluronic acid (HA) modified lipid nanoparticles (LNPs) to deliver mRNA and modulate microglia activation in Alzheimer’s disease (AD). Microglia exhibit dynamic phases of homeostatic and activated. They play a vital role in clearing Alzheimer's plaques (Aß) but are difficult to target and manipulate. To address this challenge, this study employed hyaluronic acid (HA) to modify the structure of LNPs, enabling efficient mRNA delivery specifically to activated microglia cells through CD44 targeting. HA-Cholesterol ligands were synthesized via EDC/NHS activation of HA and incorporated with different mass ratios into LNPs containing DLin/MC3/DMA, DOPE, Cholesterol, DMG-PEG2000, and mRNA with N/P=8. Detailed characterization involving DLS, RiboGreen, TEM showed HA-LNPs under 300 nm with over 90% encapsulation efficiency. The HA-LNPs took on a more negative surface charge compared to unmodified LNPs. In vitro experiments done on BV2 cells. Immunostaining and transcriptome data proved that activated microglia cells expressing elevated CD44 levels compared to homeostatic microglia. Confocal imaging further affirmed the CD44 targeting capacity of HA-LNPs compared to LNPs by demonstrating cellular uptake of cy5-mRNA by activated microglia, with/out CD44 blockade by HA. The successful delivery of target mRNA to microglia cells via these HA/LNP carriers resulted in cell transfection and modulation of their activation state, preventing aggressiveness. This was confirmed through phenotype microscopy, immunocytochemistry analysis of surface markers, ELISA assay for encoded protein, and fluorescent imaging of eGFP mRNA delivery. Future in vivo studies will evaluate HA-LNP brain targeting and microglia modulation. It is expected HA-LNP administration in the AD mouse model will demonstrate targeted mRNA delivery to microglia cells in the brain, modulating their activation phenotype to remove more Aß plaque. In summary, this study demonstrates the engineering and fabrication of immunomodulatory LNPs for precise modulation of microglial cell activation. These findings hold significant promise for developing innovative therapies addressing AD.