Lipid nanoparticles (LNPs) have emerged as a promising nonviral nucleic acid delivery platform for clinical use. To expand LNPs as a treatment option in nonhepatic-based diseases, LNPs surface coated with targeting moieties produce a precise modular delivery method that can bind to and be internalized by specific receptors expressed on target cells. This study showcases the tetrazine-trans-cyclooctene inverse electron-demand Diels-Alder click reaction and directly compares its performance with the widely employed thiol-maleimide conjugation. We also compare direct mixing and micelle mixing insertion methods under different conditions to determine the optimal formulation to produce targeted LNPs. For thiol-maleimide chemistry, monoclonal antibody cetuximab was modified with N-succinimidyl S-acetylthioacetate, followed by reaction with either 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-maleimide) directly or in preformed DSPE-PEG-maleimide: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG) micelles. For clickable tetrazine chemistry, cetuximab was modified with 2,5-dioxo-1-pyrrolidinyl 5-[4-(1,2,4,5-tetrazin-3-yl)benzylamino]-5-oxopentanoate, followed by reaction with either 1,2-Distearoyl-sn-glycero-3-PE-polyethylene glycol-2000- trans-cyclooctene (DSPE-PEG-TCO) directly or preformed DSPE-PEG-TCO:DMG-PEGmicelles. LNPs were prepared by mixing lipids in ethanol and mRNA (10 mM citrate buffer, pH 3.5) at a ratio of 3:1 (v/v). Insertion was carried out by combining either the direct conjugate solution or the micelle solution with the LNPs and mixing at 60 °C for 1 h. Only the micelle mixing method produced stable particles with mRNA encapsulation above 80%. The LNPs were found to be stable up to 3 weeks (stored at 4 °C) as indicated by the RiboGreen assay and particle sizes. Targeted LNPs displayed relatively weak and neutral zeta potential. In vitro studies revealed enhanced cellular uptake of Green Fluorescent Protein (GFP) mRNA by targeted LNPs with similar transfection rates with thiol-maleimide and tetrazine-TCO chemistries showing around 97% GFPcells, compared to less than 30% in control groups at 6 h. Both targeted LNP formulations showed a significant rise in mean fluorescence intensity, achieving at least a 3-fold increase over the controls. 2000 2000 2000 2000 2000 2000 +
