Macrophages play a vital role in controlling tissue homeostasis, yet their dysregulation can drive the pathogenesis of various diseases. In this context, P2Y2, a purinergic receptor that binds ATP, could be involved in several biological mechanisms in inflammatory, infectious, or cancer diseases. Lipid nanoparticles (LNPs) varying by the difference of lipid moiety of the polyethyleneglycol-lipid (PEG-lipid) conjugate entering their composition were prepared by microfluidic mixing technology and loaded with a siRNA targeting the P2y2 mRNA. Three PEG-lipids with varying saturated alkyl chain lengths - DMG-PEG (C14), DPPE-PEG (C16), and DSPE-PEG (C18) - were evaluated for formulation optimization. The results showed that all siRNA-LNPs were efficiently taken up by unpolarized (M0) and M2-polarized bone marrow-derived macrophages (BMDMs). Despite comparable cellular uptake across formulations, their performance in gene silencing differed significantly. While (DMG-PEG)-LNPs demonstrated the highest efficacy in both cellular models, (DSPE-PEG)-LNPs remained ineffective in knocking down the target gene. Our findings further revealed that gene silencing is more efficient in M2 BMDMs than in M0 BMDMs, emphasizing the influence of macrophage polarization. Additionally, the PEG-lipid type notably influenced the intracellular distribution patterns of lipids and siRNA, with DPPE-PEG and DSPE-PEG formulations leading to higher siRNA compartmentalization. In contrast, the DMG-PEG formulation enabled more effective siRNA delivery to the cytoplasm. This research provides valuable insights into the impact of different PEG-lipid anchors on the performance of siRNA-LNPs targeting macrophages in vitro, paving the way for developing new macrophage-based immunotherapies.
