Microfluidic Fabrication of Alendronate-Modified Lipid Nanoparticles for Bone-Targeted mRNA Delivery

Background/Objectives: Bone-targeted drug delivery systems hold great promise for treating skeletal diseases, yet the optimal strategy for functionalizing lipid nanoparticles (LNPs) with bone-homing ligands remains insufficiently explored. Herein, we compared two alendronate sodium (Alen) modification approaches (pre-conjugation and post-conjugation) for constructing bone-targeted LNPs capable of delivering mRNA to skeletal tissues. Methods: LNPs were fabricated via microfluidic mixing, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-alendronate conjugate (DSPE-PEG-Alen) required for the pre-conjugation method was synthesized. The bone-targeting ability of LNPs prepared by the two Alen modification strategies was evaluated using an in vitro hydroxyapatite (HAP) binding assay. Furthermore, the physicochemical properties, bone-targeting performance, mRNA delivery efficiency, and biosafety of the LNPs prepared by the post-conjugation method were assessed through cellular uptake, in vivo imaging, and other methods. Results: Hydroxyapatite binding assays revealed that the post-conjugation strategy afforded significantly superior bone affinity compared to the pre-conjugation approach. In addition, ex vivo bone fragment binding experiments further confirmed that the bone-targeting LNPs prepared by the post-conjugation method exhibited stronger bone-binding capability compared to unmodified LNPs. The optimized Alen-LNPs demonstrated efficient cellular uptake and functional mRNA translation in bone marrow mesenchymal stem cells with negligible cytotoxicity. In vivo studies in mice confirmed the preferential accumulation of Alen-LNPs in bone tissues, with successful green fluorescent protein (GFP) mRNA translation detected in bone tissue sections. Histopathological analysis confirmed the biosafety of the formulation. Conclusions: This study establishes the post-conjugation strategy as the superior approach for Alen functionalization of LNPs, providing a robust and reproducible platform for bone-targeted mRNA therapeutics.